IL323652A - Conditionally immortalized stem cells and uses thereof - Google Patents

Description

CONDITIONALLY IMMORTALIZED STEM CELLS AND USES THEREOF CROSS REFERENCE TO RELATED APPLICATIONS id="p-1"

[0001] This application claims priority to U.S. Provisional Patent Application No. 63/493,540 filedon March 31, 2023, the entire contents of which are incorporated herein by reference.REFERENCE TO SEQUENCE LISTING [0002] The Sequence Listing associated with this application is provided in text format in lieu ofa paper copy and is hereby incorporated by reference into the specification. The name of the textfile containing the Sequence Listing is 31C7646 _ST26.xml. The text file is 102,400 bytes, wascreated on March 23, 2024, and is being submitted electronically via Patent Center. 10 FIELD OF THE DISCLOSURE id="p-3"

[0003] The current disclosure provides conditionally immortalized stem cells and uses thereof.

BACKGROUND OF THE DISCLOSURE id="p-4"

[0004] Cell therapy is a promising field for the treatment of medical disorders. For example, theuse of engineered cells for cellular immunotherapy allows for treatment of cancers or other 15diseases by leveraging various aspects of the immune system to target and destroy diseased ordamaged cells. During cell therapy, cells from various sources can be transplanted into a subjectfor treatment of a disease. If primary cells are used for transplantation, continuous access to freshtissue sources is required. Such therapies require cells in numbers sufficient for therapeuticallyrelevant doses, however, it can be difficult to collect a desired number of cells especially if the 20cells are sourced from primary tissue. Normal human somatic cells, for example, have a finiteduplication capacity and can reach cellular senescence especially when cultured in vitro. [0005] Another drawback of cell therapy is derived from the use of an allogeneic cell product. Anallogeneic cell product refers to cells that are obtained from individuals belonging to the samespecies but are genetically dissimilar. Use of these cells can result in an immune response upon 25transplantation into a subject in a process termed host versus graft rejection or the process termedgraft versus host disease. In the former, the patient’s existing immune system attacks thetransplanted cells as foreign. In the latter, it is the transplanted cells that attack the patient’s cellsas foreign. [0006] Furthermore, for cellular therapy, cell manufacture, and research purposes, it would be 30ideal to have a cell product that is pre-manufactured, well characterized, and available forimmediate use. Because stems cells can be an important precursor to generate or regenerate organs, repair tissues, prepare or deliver certain biological factors, or treat diseases or disorders,they may be a useful candidate in generating pre-manufactured cell lines for multiple purposes.

SUMMARY OF THE DISCLOSURE id="p-7"

[0007] The current disclosure provides conditionally immortalized stem cells and uses thereof. [0008] Particular embodiments utilize stem cell-derived cell populations that are modified to 5include a conditional (e.g., drug-inducible) immortalization gene (e.g., TERT and SV40 large Tantigen). In particular embodiments, the conditional immortalization gene prevents cellsenescence when a growth controlling agent is administered to the stem cells. Theseembodiments are particularly useful to provide immortalized cell populations (e.g., differentiatedcell populations) that can be maintained as immortal cell lines by the administration of a growth 10controlling agent (e.g., drug). [0009] Particular embodiments utilize stem cells modified to include a conditional immortalizationgene and factors that support use as feeder cells during cell culture. These embodiments areparticularly useful to generate immortalized feeder cells. Feeder cells are cell that provide factorsto help a cell population of interest to proliferate. Feeder cells can be adherent cells (e.g., 15mesenchymal stem cells) or suspension cells (e.g., CD34+ cells). Immortalized feeder cells canbe genetically modified to support growth of particular cell types, such as expression ofmembrane-bound IL21 and/or knock-out of MHC Class I and/or Class II to support growth ofnatural killer (NK) cells. These embodiments may also include a suicide switch to reducecontamination of cell populations of interest with feeder cells. 20 [0010] Particular embodiments utilize stem cells modified to include a conditional immortalizationgene and factors that support use as tester cells during research and development. Theseembodiments are particularly useful to generate immortalized tester cells. Examples include testercells that express a cancer antigen or a viral antigen to test efficacy of antibodies, recombinantreceptors, or similar therapeutic treatments under development. The expression of a viral antigen 25can be used as living vaccine that allows for extended antigenic presentation in a physiologicallyappropriate manner. When manufactured for in vivo use, these immortalized tester cells may alsoexpress a detectable label, such as fluorescent proteins and/or luciferase. These embodimentsmay also include a suicide switch. [0011] Particular embodiments utilize stem cells modified to include a conditional immortalization 30gene and a suicide gene. These embodiments are particularly useful to generate immortalizedcell populations (e.g., differentiated cell populations) for a therapeutic purpose. Theimmortalization gene can prevent cell senescence during cell manipulation and culture with the administration of a growth controlling agent until administration of the cell population to a subject,at which point the growth controlling agent can be withdrawn. Furthermore, the suicide geneprovides an additional safety feature by causing the apoptosis (programmed cell death) ofgenetically modified cells both during cell manufacture and/or after administration to a subject.For example, the suicide switch provides a safety feature allowing the removal of proliferating 5cells from cultured cells in vitro before use as a therapeutic cell population. Further, their effectcan be canceled after administration to a subject. If an unwanted side effect of their administrationwere to occur. In particular embodiments, a therapeutic cell can be further genetically modified toinclude factors that support use as a therapeutic cell such as proteins, antibodies, or recombinantreceptors (e.g., chimeric antigen receptors). 10 BRIEF DESCRIPTION OF THE FIGURES id="p-12"

[0012] Some of the drawings submitted herewith may be better understood in color. Applicantconsiders the color versions of the drawings as part of the original submission and reserves theright to present color images of the drawings in later proceedings. [0013] FIG. 1. Schematic of insertion of cell division essential locus and a suicide gene. 15 [0014] FIGs. 2A-2D. Epitope Line Demonstration. (2A) In vitro assay was performed in PAN3 cellline or PLSX11 cell line with major histocompatibility complex (MHC) I and II knockout. (2B)Schematic of example genetic construct for transposon-based gene editing containing anexample expression product and luciferase. (2C) Flow cytometry results of in vitro expression ofgenetic construct for CD19 and BCMA detection. The pBP plasmid successfully transduced cells. 20(2D) In vivo detection of luciferase expressing tumor cells. [0015] FIG. 3. Using Feeder Lines to Expand Adult and cord blood-derived natural killer (NK)cells. The production of activated NK cells from naïve NK cells can traditionally include theaddition of cytokines, autologous accessory cells, irradiated autologous feeder cells, and/orirradiated allogeneic feeder cells (e.g., modified K562 or EBV-LCL cells). The activated NK cells 25have increased cell number, enhanced natural cytotoxicity and antibody-dependent cell-mediatedcytotoxicity, and enhanced secretory function. [0016] FIG. 4. Characteristics of immortalized hiPSCs further edited. Morphology of parentaliPSCs (SK005.3) and the immortalized line. [0017] FIG. 5. Expression level analysis of FMC63 clonal lines by qPCR and FACS shows the 30transgene expression in clones. The successful insertion and expression of the transgenes,FMC63 and TK.007, were confirmed by FACS analysis of FMC63 protein as well as qPCR ofFMC63 and TK.007 transcripts. The transgene copy number in each clone was determined via digit-droplet PCR, ranging from 13 copies to 28 copies. The copy number correlates to both themRNA and protein level of each of the transgene. [0018] FIGs. 6A, 6B. (6A) Inducible immortalization expression vectors. The construct map anddetailed plasmid map of inducible immortalization expression vectors. (6B) Work flow of editing apreviously edited iPSC line genome to contain an inducible immortalization gene. The workflow 5of generating FMC63-IL15 chimeric antigen receptor (CAR)+ Thymidine Kinase expressingSK005.3 hiPSC and the further insertion of immortalization vectors in this edited iPSC. SK005.3hiPSC were co-transfected with FMC63 CAR plasmid and PiggyBac (PBase) transposaseplasmid using Lipo3000 transfection reagent. Clones that express high levels of transgenes weresignal cell sorted and expanded. These clones were further transfected with the immortalization 10vector using the sleeping beauty transposon system. Clones with successful immortalizationvector insertion were enriched via Neomycin drug selection and expanded. [0019] FIG. 7. Expression analysis of Doxycycline (DOX) induction of inducible immortalizationgene hTERT and SV40 LT in the previously edited iPSC lines. The successful induction of theexpression of immortalization factors via Doxycycline treatment is shown. Under the Tet-inducible 15system, in the absence of doxycycline, very low levels of hTERT and SV40 large T antigentranscripts were detected by qPCR, likely due to the leakiness of the Tet-inducible system. In thepresence of doxycycline, hTERT and SV40 transcripts levels increase significantly in comparisonto no doxycycline and in a clear dose-dependent manner. In contrast, there is no difference in thetranscript levels of rtTA and FMC63 CAR, which were not under Tet-inducible, in the presence or 20absence of doxycycline. Each data bar was an average of triplicate technical measurements. Theerror bar represents SEM. Nd: non-detectable. [0020] FIGs 8A-8F. (8A) Representative phase contrast images (4x objective) of cell morphologyfor unmodified and TetON hTERT SV40 iPSC cultures upon expansion in iPSC culture mediumfor 3 passages, in the absence or presence of 0.1 µM Doxycycline Hyclate (DOX) are shown. (8B) 25RT-PCR analysis is shown for the expression of hTERT, SV40, and rtTA transcripts in unmodifiedand hTERT SV40 iPSCs (normalized to the YWHAZ gene), after induction with 0.1µM Doxycycline Hyclate (DOX) for 12 days. DOX treatment increased the expression of induciblehTERT and SV40 transcripts in the hTERT SV40 iPSC line only, while the rtTA genes wasconstitutively expressed in the hTERT SV40 iPSC line but not in the unmodified control cell 30line. (8C) The diagram of the hematopoietic progenitor cell differentiation process is shown. iPSCare thawed and expanded before they are passaged into AggreWells to generate embryoid bodies(EBs). After 5 days of culture in AggreWells, the EBs are transferred to a 6-well plate. At day 12of the differentiation, EBs are dissociated, positively-selected for CD34 expression and phenotypically characterized for hematopoietic progenitor cells surface marker expression. (8D)Representative phase contrast images (4x objective) are shown of TetON hTERT SV40 iPSC-derived EBs in AggreWells on day 2 of the differentiation and EBs in 6-well plates on day 12 ofthe differentiation, before harvest. Doxycycline Hyclate (DOX) treatment increased the size of EBcompared to the no treatment control, suggesting the induction of immortalization genes promotes 5cell proliferation during EB formation. (8E) The viability and the viable cell yield are shown of thecell fraction obtained after enrichment through CD34-positive selection following EB dissociation,for Unmodified and TetON hTERT SV40 iPSCs, with and without 0.1 µM Doxycycline Hyclate(DOX) treatment. DOX treatment increased the viability and viable cell yield of the positively-selected cell fraction compared to the no treatment control. (8F) Flow cytometry histograms are 10shown for measurement of CD34 protein expression of cells stained with CD34-FITC antibodyafter CD34-positive selection and the percentage of CD34-positive cells for each of theUnmodified and TetON hTERT SV40 iPSC lines, with and without DOX treatment. [0021] FIG. 9. The diagram of the natural killer (NK) cell differentiation process is shown. iPS cellsare thawed and expanded before they are passaged into AggreWells to generate EBs. After 5 15days of culture in AggreWells, the EBs are transferred to a 6-well plate. At day 12 of thedifferentiation, EBs are dissociated, positively-selected for CD34 expression, phenotypicallycharacterized for hematopoietic progenitor cells surface marker expression, and seeded forLymphoid Progenitor Cell differentiation. After 14 days of culture, Lymphoid Progenitor Cells areharvested, phenotypically characterized for cell surface marker expression and seeded for NK 20Cell differentiation. [0022] FIG. 10. Generating iPSC lines with overexpression of CD19 and BCMA by PiggyBacTransposon. The workflow of generating hCD19 or BCMA transgene PAN3 and SK005.3 hIPSCline. hCD19 and BCMA were co-transfected PAN3 and SK005.3 [0023] FIG. 11. A list of CAR-T targeted antigens that could be used to make tester lines for 25evaluating CAR T cells. [0024] FIG. 12. The piggyBac and Lentivirus vector design allows insertion of other CAR-Ttargeted antigens. A schematic of the piggyBac transposon vector and Lentivirus vector areshown. [0025] FIGs. 13A, 13B. Fluorescence and Bioluminescence principle. (13A) Fluorescence 30achieved in target cells by expression of a Green Fluorescent Protein (GFP) driven by a ubiquitouspromoter (CAG). Induction of light emission requires an excitation light. (13B) Bioluminescenseachieved by expression of Luciferase driven by an ubiquitous promoter (CAG). Induction ofluminescence requires the presence of the luciferin enzyme. id="p-26"

[0026] FIGs. 14A, 14B. Differentiation of immortalized iPSC line SK005.3-hTertSV40 to MSCs.(14A) Morphology change during differentiation of the immortalized iPSC line SK005.3-hTertSV40. (14B) Flow cytometry data showing the amount of expression of CD105 and CD73for the differentiated cells. [0027] FIGs. 15A, 15B. (15A) Schematic of cell lines used (Lines A, B, and C). (15B) Genotyping 5of lines used in NK assay. [0028] FIG. 16. MSC phenotype was assessed for CD90 and CD105 expression on cell surfaceby Flow Cytometry. A limited panel of MSC markers, CD90 and CD105 were used to assess thephenotype of the cells from Lines A, B, and C. All lines were highly positive for the 2 essentialMSC markers against unstained and isotype controls. 10 [0029] FIG. 17. HLA ABC surface expression. Class I/II knockout (KO) was assessed based onexpression of HLA type ABC measured via flow cytometry. Line A was unedited and thereforewas highly positive for HLA ABC, while Lines B and C were significantly lower in HLA ABCexpression confirming the intended gene edit for Class I/II KO. [0030] FIG. 18. State of co-culture at the beginning of assay (Day 0). 15 [0031] FIG. 19. State of co-culture on Day 3 of the co-culture assay. [0032] FIG. 20. NK Activation and Expansion – Count and viabilities. Viability and count for NKcontrol (black circles), iNK only (black square), NK on MSC (white circles), iNK on MSC (whitesquare), and MSCs co-cultured with NK cells (black diamonds) are presented. [0033] FIGs. 21A, 21B. Restimulation of Activated NK cells. (21A) NK cell number over 3 days of 20re-stimulation (Day 6 of co-culture). (21B) 4x magnification images of NK cells on feeder Line Cat Day 3 before transfer on fresh feeders (left) and on Day 6 (right). The insert shows a 10xmagnification of the same image.

DETAILED DESCRIPTION id="p-34"

[0034] The current disclosure provides conditionally immortalized stem cells and uses thereof. 25The current disclosure also provides immortalized cell lines generated from conditionallyimmortalized stem cells and uses thereof. [0035] Particular embodiments utilize stem cells that are modified to include a conditional (e.g.,drug-inducible) immortalization gene (e.g., TERT and SV40 large T antigen). In particularembodiments, the conditional immortalization gene prevents cell senescence when a growth 30controlling agent is administered to the stem cells. These embodiments are particularly useful toprovide immortalized cell populations (e.g., differentiated cell populations) that can be maintainedas immortal cell lines by the administration of a growth controlling agent (e.g., drug). id="p-36"

[0036] Particular embodiments utilize stem cells modified to include a conditional immortalizationgene and sequences encoding an expression product that support use as feeder cells during cellculture. These embodiments are particularly useful to generate immortalized feeder cells. Feedercells are cell that provide factors to help a cell population of interest to proliferate. Feeder cellscan be adherent cells (e.g., mesenchymal stem cells) or suspension cells (e.g., CD34+ cells). 5Immortalized feeder cells can be genetically modified to support growth of particular cell types,such as expression of membrane-bound IL21 and/or knock-out of MHC Class I, MHC Class II, orMHC Class I and Class II to support growth of natural killer (NK) cells. These embodiments mayalso include a suicide switch to reduce contamination of cell populations of interest with feedercells. 10 [0037] Particular embodiments utilize stem cells modified to include a conditional immortalizationgene and sequences encoding an expression product that support use as tester cells duringresearch and development. These embodiments are particularly useful to generate immortalizedtester cells. Examples include tester cells that express a cancer antigen or a viral antigen to testefficacy of antibodies, recombinant receptors, or similar therapeutic treatments under 15development. The expression of a viral antigen can be used as living vaccine that allows forextended antigenic presentation in a physiologically appropriate manner. When manufactured forin vivo use, these immortalized tester cells may also express a detectable label, such asfluorescent proteins and/or luciferase. These embodiments may also include a suicide switch. [0038] Particular embodiments utilize stem cell-derived cell populations modified to include a 20conditional immortalization gene and a suicide gene. These embodiments are particularly usefulto generate immortalized cell populations (e.g., differentiated cell populations) for a therapeuticpurpose. The immortalization gene can prevent cell senescence during cell manipulation andculture with the administration of a growth controlling agent until administration of the cellpopulation to a subject, at which point the growth controlling agent can be withdrawn. 25Furthermore, the suicide gene provides an additional safety feature by causing the apoptosis(programmed cell death) of genetically modified cells both during cell manufacture and/or afteradministration to a subject. For example, the suicide switch provides a safety feature allowing theremoval of proliferating cells from cultured cells in vitro before use as a therapeutic cell population.Further, their effect can be canceled after administration to a subject. If an unwanted side effect 30of their administration were to occur. In particular embodiments, a therapeutic cell can be furthergenetically modified to include factors that support use as a therapeutic cell such as proteins,antibodies, or recombinant receptors (e.g., chimeric antigen receptors). [0039] Aspects of the current disclosure are now described with more supporting options and detail as follows: (I) Stem Cell Types; (II) Differentiation of Stem Cells; (III) Genetic Modificationof Cells; (III-A) Conditional Immortalization; (III-B) Expression Products; (III-B-1) Protein; (III-B-2)Recombinant Receptors; (III-B-3) Detectable Labels; (III-C) Major Histocompatibility Class (MHC)Molecules; (III-D) Suicide Gene; (III-E) Other Control Features; (IV) Culture and Storage of Cells;(V) Cell-based Formulations; (VI) Methods of Use; (VI-A) Feeder Cells for Cell Manufacturing; 5(VI-B) Tester Cells for Research and Development; (VI-C) Conditionally Immortal TherapeuticCell Line; (VII) Exemplary Embodiments; (VIII) Experimental Examples; and (IX) ClosingParagraphs. These headings are provided for organizational purposes only and do not limit thescope or interpretation of the disclosure. [0040] (I) Stem Cell Types. Stem cells are cells capable of differentiation into other cell types, 10including those having a particular, specialized function (e.g., tissue specific cells, parenchymalcells and progenitors thereof). There are various classes of stem cells, which can be characterizedin their ability to differentiate into a desired cell/tissue type. For example, stem cells can betotipotent, pluripotent, multipotent, or unipotent. [0041] As used herein, the term "totipotent" or "totipotency" refers to a cell's ability to divide and 15ultimately produce an entire organism including extra embryonic tissues in vivo. In one aspect,the term "totipotent" refers to the ability of the cell to progress through a series of divisions intoa blastocyst in vitro. The blastocyst includes an inner cell mass (ICM) and a trophoblast. Thecells found in the ICM give rise to pluripotent stem cells that possess the ability to proliferateindefinitely, or if properly induced, differentiate in all cell types contributing to an organism. 20Trophoblast cells generate extra-embryonic tissues, including placenta and amnion. Totipotentstem cells can include fertilized oocytes, cells of embryos at the two and four cell stages ofdevelopment, that have the ability to differentiate into any type of cell of the particular species.For example, a single totipotent stem cell could give rise to a complete animal, as well as to anyof the myriad of cell types found in the particular species (e.g., humans). 25 [0042] Totipotent stem cells are the source of pluripotent stem cells. As used herein, the term"pluripotent" refers to a cell's potential to differentiate into cells of the three germ layers:endoderm (e.g., interior stomach lining, gastrointestinal tract, the lungs), mesoderm (e.g.,muscle, bone, blood, urogenital), or ectoderm (e.g., epidermal tissues and nervous system).Pluripotent stem cells can give rise to any fetal or adult cell type including germ cells. However, 30pluripotent stem cells alone cannot develop into a fetal or adult animal when transplanted inutero because they lack the potential to contribute to extra embryonic tissue (e.g., placenta invivo or trophoblast in vitro). "Progenitor cells" can be either multipotent or pluripotent. Progenitorcells are cells that can give rise to different terminally differentiated cell types, and cells that are capable of giving rise to various progenitor cells. A standard art-accepted test of pluripotencyincludes the ability to form a teratoma in 8-12 week old SCID mice; however identification ofvarious pluripotent stem cell characteristics can also be used to detect pluripotent cells."Pluripotent stem cell characteristics" refer to characteristics of a cell that distinguish pluripotentstem cells from other cells. The ability to give rise to progeny that can undergo differentiation, 5under the appropriate conditions, into cell types that collectively demonstrate characteristicsassociated with cell lineages from all of the three germinal layers (endoderm, mesoderm, andectoderm) is a pluripotent stem cell characteristic. Expression or non-expression of certaincombinations of molecular markers are also pluripotent stem cell characteristics. For example,human pluripotent stem cells express at least some, and in some embodiments, all of the 10markers from the following list: SSEA-3, SSEA-4, TRA-1-60, TRA-1-81, TRA-2-49/6E, ALP,Sox2, E-cadherin, UTF-1, Oct4, Rex1, and Nanog. Cell morphologies associated with pluripotentstem cells are also pluripotent stem cell characteristics. In particular embodiments, pluripotencycan be verified by reviewing cell morphology, TRA1-60 live staining, performing flow cytometryfor pluripotency markers, and/or alkaline phosphatase staining. In particular embodiments, 15reviewing the morphology of the cell includes looking for colonies with well-defined borders,looking for cells with an enlarged nucleus, and/or looking for cells with a high nucleus to cytosolratio. In particular embodiments, performing flow cytometry for pluripotency markers includesperforming flow cytometry for SSEA-4, Oct4, Nanog, and Sox2. Pluripotent stem cells includeembryonic stem cells (ESC) and induced pluripotent stem cells (iPSC). 20 [0043] The term "embryonic stem cell" used in the present invention is a cell cultured byseparating and culturing the inner cell mass (inner cell mass) of the blastocyst, which is an earlystage of development after fertilization. Although pluripotent human embryonic stem cells(hESC) derived from human blastocysts are promising sources for cell-based therapies to treatdiseases and disorders such as Parkinson's disease, cardiac infarction, spinal cord injury, and 25diabetes mellitus, their clinical potential has been hampered by their immunogenicity and ethicalconcerns. [0044] Cord blood stem cells refer to a population enriched in hematopoietic stem cells, orenriched in hematopoietic stem and progenitor cells, derived from human umbilical cord bloodand/or human placental blood collected at birth. The hematopoietic stem cells, or hematopoietic 30stem and progenitor cells, can be positive for a specific marker expressed in increased levels onhematopoietic stem cells or hematopoietic stem and progenitor cells, relative to other types ofhematopoietic cells. For example, such markers can be, but are not limited to CD34, CD43,CD45RO, CD45RA, CD59, CD90, CD109, CD117, CD133, CD166, HLA DR, or a combination thereof. Also, the hematopoietic stem cells, or hematopoietic stem and progenitor cells, can benegative for an expressed marker, relative to other types of hematopoietic cells. For example,such markers can be, but are not limited to Lin, CD38, or a combination thereof. [0045] The term "de-differentiated stem cell or induced pluripotent stem cell (iPSC)" as usedherein refers to pluripotent cells induced by artificially dedifferentiating (reprogramming) the adult 5cells that have already been differentiated. The term "adult cell" as used herein refers to a cellderived from an adult that is born and alive, as opposed to an embryonic cell. As used herein,the term "differentiation" refers to a phenomenon in which structures or functions are specializedwhile cells divide and proliferate and grow, that is, a cell or tissue of an organism has a shapeor function to perform a task given to each. iPSC can be reprogrammed from adult stem cells 10using any method known in the art. In particular embodiments, an iPSC includes PLSX11. Inparticular embodiments, a method for reprogramming adult cells into iPSC includes contactingthe adult cell with an engineered expression construct (EEC) encoding a reprogramming factor(RF) operably linked to: i) a 5’ untranslated region (UTR) including a minimal promoter, a mini-enhancer, and a Kozak sequence; and/or ii) a 3’ UTR including a spacer and a stem loop 15structure. In particular embodiments, the stem loop structure includes hybridizing sequences anda loop segment. In particular embodiments, the loop segment includes 7-15 nucleotides. Inparticular embodiments, the 5’ UTR additionally includes a start codon. In particularembodiments, the 3’ UTR additionally includes a stop codon and/or a polyA tail. In particularembodiments, the RF includes Oct4, Sox2, Klf4, Nanog, Myc, SV40Tag, or Lin28. The 20components of the 5’ UTR and 3’ UTR can be any sequences known in the art including thoseprovided in Table 1.Table 1. 5’ UTR and 3’ UTR Components and Constructs. SEQ ID NO: Sequence Description N/A CAUACUCA Mini-enhancer sequenceN/A GGGAGA Minimal promoter (e.g., Mini-T7 promoter)N/A GCCRCCAUG Kozak Consensus SequenceN/A [N1-3]AUA SpacerN/A [N1-3]AAA SpacerN/A UGCAUA SpacerN/A UGCAAA SpacerN/A CCUC and GAGG Hybridizing Sequences N/A CUCC and GGAG Hybridizing SequencesUAACGGUCUU Loop segmentGGGAGACAUACUCAGCCACC 5’ UTR Construct with "A"Kozak Sequence (no startcodon)GGGAGACAUACUCAGCCGCC 5’ UTR Construct with "G"Kozak Sequence (no startcodon)GGGAGACAUACUCAGCCACCAUG 5’ UTR Construct with "A"Kozak Sequence (with startcodon)GGGAGACAUACUCAGCCGCCAUG 5’ UTR Construct with "G"Kozak Sequence (with startcodon)[N1-3]AUACCUC[N7-15]GAGG 3’ UTR Construct[N1-3]AAACCUC[N7-15]GAGG 3’ UTR Construct[N1-3]AUAGGAG[N7-15]CUCC 3’ UTR Construct[N1-3]AAAGGAG[N7-15]CUCC 3’ UTR Construct[N1-3]AUAGAGG[N7-15]CCUC 3’ UTR Construct[N1-3]AAAGAGG[N7-15]CCUC 3’ UTR ConstructStop-UGCAUACCUCUAACGGUCUUGAGG-PolyA 3’ UTR Construct 13 Stop-UGCAUAGGAGUAACGGUCUUCUCC-PolyA 3’ UTR Construct 14 Stop-UGCAAAGGAGUAACGGUCUUCUCC-PolyA 3’ UTR Construct UAAUGCAUACCUCUAACGGUCUUGAGG 3’ UTR ConstructUGAUGCAUACCUCUAACGGUCUUGAGG 3’ UTR Construct 17 UAGUGCAUACCUCUAACGGUCUUGAGG 3’ UTR Construct 18 UAAUGCAUAGAGGUAACGGUCUUCCUC 3’ UTR ConstructUGAUGCAUAGAGGUAACGGUCUUCCUC 3’ UTR ConstructUAGUGCAUAGAGGUAACGGUCUUCCUC 3’ UTR ConstructUAAUGCAAAGAGGUAACGGUCUUCCUC 3’ UTR ConstructUGAUGCAAAGAGGUAACGGUCUUCCUC 3’ UTR ConstructUAGUGCAAAGAGGUAACGGUCUUCCUC 3’ UTR Construct id="p-46"

[0046] In comparison, a multipotent stem cell is capable of differentiating into a subset of cellscompared to a pluripotent stem cell. Pluripotent stem cells give rise to multipotent stem cellsthrough spontaneous differentiation or as a result of exposure to differentiation inductionconditions in vitro. The term "multipotent" refers to a cell's potential to differentiate and give riseto a limited number of related, different cell types. These cells are characterized by their multi- 5lineage potential and the ability for self-renewal. In vivo, the pool of multipotent stem cellsreplenishes the population of mature functionally active cells in the body. Among the exemplarymultipotent stem cell types are hematopoietic, mesenchymal, or neuronal stem cells. [0047] Hematopoietic stem cells are immature cells found in the peripheral blood and bonemarrow that can develop into all types of blood cells, including white blood cells, red blood cells, 10and platelets. Mesenchymal stem cells also known as mesenchymal stromal cells or medicinalsignaling cells are multipotent stromal cells. Mesenchymal stem cells are more differentiatedthan pluripotent stem cells but retain the ability to differentiate into a variety of cell types,including osteoblasts, chondrocytes, myocytes and adipocytes. According to certainembodiments, mesenchymal stem cells are more readily reprogrammed than fully differentiated 15somatic cells. Neural stem cells (NSCs) are self-renewing, multipotent cells that generate theradial glial progenitor cells that generate the neurons and glia of the nervous system. [0048] Unipotent stem cells are stem cells that produce one cell type but have the property ofself-renewal that distinguishes stem cells from non-stem cells. Examples of unipotent stem cellsincludes germ line stem cells and epidermal stem cells. The term "precursor cell," "progenitor 20cell," and "stem cell" are used interchangeably in the art and refer either to a totipotent,pluripotent, multipotent or in some cases, a unipotent cell. [0049] (II) Differentiation of Stem Cells. In particular embodiments, stem cells are differentiated,for example, for cell manufacturing (e.g., feeder cell), research and development (e.g., tester cell),or therapeutic purposes (e.g., therapeutic cell). Where differentiation of stem cells is desired, stem 25cells (e.g., iPSC) can be exposed to one or more activation factors (e.g., growth factors,differentiation factors, and/or survival factors) that promote differentiation into a more committed cell type. A more differentiated stem cell is more committed in relation to a different stem cell typealong a development pathway. [0050] Stem cells of the present disclosure can differentiate into more specialized cell types suchas committed progenitors as well as cells further along the differentiation and/or maturationpathway that are partly or fully matured or differentiated. "Committed progenitors" give rise to a 5fully differentiated cell of a specific cell lineage. Exemplary cells include mesenchymal stem cells(MSC) or hematopoietic stem cells (HSC). Exemplary differentiated cells include pancreatic cells(e.g., alpha, beta, and delta cells), epithelial cells, cardiac cells (e.g., cardiomyocytes),endothelial cells, liver cells (e.g., hepatocytes (HCs), hepatic stellate cells (HSCs), Kupffer cells(KCs), and liver sinusoidal endothelial cells (LSECs)), endocrine cells, connective tissue cells 10(e.g., fibroblasts), muscle cells (e.g., myoblasts), brain cells (e.g., neurons), bone cells (e.g.,osteoblasts and osteoclasts), kidney cells, and immune cells (e.g., T-cells, NK cells, ormacrophages). [0051] Many activation factors and cell culture conditions that promote differentiation are knownin the art (see, e.g., U.S. Patent No. 7,399,633 at Section 5.2 and Section 5.5). For example, stem 15cell factor (SCF) can be used in combination with granulocyte-macrophage colony-stimulatingfactor (GM-CSF) or interleukin (IL)-7 to promote differentiation into myeloid stem/progenitor cellsor lymphoid stem/progenitor cells, respectively. In particular embodiments, iPSC can bedifferentiated into a lymphoid stem/progenitor cell by exposing iPSC to 100 ng/ml of each of SCFand GM-CSF or IL-7. In particular embodiments, a retinoic acid receptor (RAR) agonist, or 20preferably all trans retinoic acid (ATRA) is used to promote the differentiation of iPSC.Differentiation into natural killer cells, e.g., can be achieved by exposing cultured iPSC to RPMImedia supplemented with human serum, IL-2 at 50 U/mL and IL-15 at 500ng/mL. In additionalembodiments, RPMI media can also be supplemented L-glutamine. [0052] Mesenchymal stem cells (MSC) are multipotent adult stem cells that are able to 25differentiate into numerous cell types including cells in skeletal tissue, such as cartilage, bone andthe fat in the bone marrow. Typically, MSC are derived from the bone marrow which can lead toa lack of accessibility to MSC despite the need for them in many mainstream clinical treatments.MSC can be identified by the presence of certain markers including CD73, CD90 and CD105, butthe absence of CD14, CD20, CD34 or CD45. MSC can be generated from pluripotent stem cells 30such as ESC and iPSC by culturing on collagen type I-coated plates; forming embryoid bodies,culturing with PDGF AB, KSB-3, EGM-2MV, DMEM, or mTeSR1 medium supplemented withROCK inhibitors (e.g., Y27632), and/or inhibiting pathways including the TGF-P pathway, or thebFGF pathway (see, e.g., Zhou et al., 2021, Stem Cell Research & Therapy. 12(175)).

Mesodermal induction can also be achieved by culture with IMDM/F12 with BMP4 and activin andculture with ES-Cult methylcellulose, StemLine II and ESFM with FGF2. [0053] The CD34 molecule, belonging to the cadherin family, is a highly glycosylated single-passtransmembrane protein that is selectively expressed on the surface of human and othermammalian hematopoietic stem cells (HSC), hematopoietic progenitor cells (HPC), and vascular 5endothelial cells (ECs), and gradually diminishes to disappear as the cells mature. Many methodsfor inducing differentiation of stem cells into CD34+ cells exist, such as an Embryoid Bodies (EB)differentiation method, an adherent induced differentiation method, and differentiation by culturingcells in a combination of different cytokines and compounds. CD34+ cells have also been derivedfrom human pluripotent stem cells by inhibition of mitogen-activated protein kinase (MAPK) 10extracellular signal-regulated protein kinase (MEK)/extracellular signal-regulated kinase (ERK)signaling and activation of bone morphogenic protein-4 (BMP4) signaling (Park et al., 2010,Blood, 116(25):5762-5772). Some medium supplements that can aid in the differentiation of stemcells to CD34+ cells includes BMP activator, bFGF, VEGF, SCF, IGF, EPO, IL6, and IL11; aROCK inhibitor; a Wnt pathway activator; and/or a TGF β receptor/ALK inhibitor. In particular 15embodiments, iPSCs can be differentitated into CD34-positive hematopoietic progenitor cellsusing the StemDiff Hematopoietic Medium and Supplements (StemCell Technologies). For thedifferentiation, adherent iPSC cultures can be dissociated to single cells and plated at 3.5x10 cells/well to generate Embryoid Bodies (EBs). After culturing the EBs, EBs can be harvested anddissociated into single cells. CD34-positive cells can be isolated from the single cell suspension. 20Staining for CD34, CD45 and CD43 expression can be used to confirm that the cells are CD34positive cells. [0054] T cells are a type of white blood cell that plays an important role in the immune system. Tcells can be differentiated from stem cells by differentiation into a CD34+ cell and thendifferentiation into a T cell. T cell differentiation protocols aim to copy the development of 25lymphocytes. Hence, stimulation by SCF, FLT3l, IL7, and Notch signaling (Moore and Zlotnik,1997; Radtke et al., 1999, 2004; Politikos et al., 2015) can be useful in differentiating T cells fromCD34+ cells. Differentiation effectiveness can be assessed by surface markers. T cells shouldstop expression of CD34 and should subsequently express CD7, CD5 and finally CD4 and CD8. [0055] NK cells are important for body defense and tumor resistance, but the function of NK cells 30in tumor patients is usually damaged. Externally inputting NK cells with normal functions orenhanced functions through genetic modification, namely NK cell adoptive therapy, is a promisingcancer treatment. However, NK cell immunotherapy requires a large number of NK cells and themain sources of NK cells are currently: NK cells (PB-NK) obtained by separating autologous/allogeneic peripheral blood, NK cells (UCB-NK) obtained by separatingautologous/allogeneic umbilical cord blood, NK cells (hESC-NK/iPSC-NK) obtained bydifferentiating embryonic stem cells/inducing pluripotent stem cells and NK cell lines such as NK-92. The production of NK cells from stem cells (e.g., iPSC) could greatly aid in NK cell adoptivetherapy and can include culturing the stem cells in SPM-NK culture medium which includes 5Stempro-34 complete medium, DMEM/F12 medium, L-glutamine, ascorbic acid, ITS-X, SCF, Flt-3L, IL-3, IL-7, IL-15. In particular embodiments, iPSCs are differentiated into NK cells. In particularembodiments, iPSCs are differentiated into NK cells by first differentiating iPSCs into CD34-positive hematopoietic progenitor cells. Next, the CD34-positive cells are differentiated into CD5-positive and CD7-positive lymphoid progenitor cells, and the lymphoid progenitor cells are 10differentiated into CD56-positive cells. Staining for CD56, CD16 and CD3 expression can be usedto confirm differentiation into NK cells. [0056] Cardiomyocytes have been generated in vitro from a wide range of stem cells, includingiPSC (see, e.g., Gai, et al., 2009, Cell. Biol. Int. 33:1184-93; Kuzmenkin, et al., 2009, FASEB J.23:4168-80; Pfannkuche, et al., 2009, Cell Physiol. Biochem. 24:73-86), ESCs (see, e.g., Beqqali, 15et al., 2009, Cell. Mol. Life Sci. 66:800-13; Steel, et al., 2009, Curr. Opin. Drug Discov. Dev12:133-40), HSPC (see, e.g., Choi, et al., 2008, Biotechnol. Lett 30:835-43; Antonitsis, et al.,2008, Thorac. Cardiovasc. Surg 56:77-82; Ge, et al., 2009, Biochem. Biophys. Res. Commun.381:317-21; Gwak, et al., 2009, Cell. Biochem. Funct. 27:148-54), and cardiomyocyte progenitorcells (see, e.g., Smits, et al., 2009, Nat. Protoc. 4:232-43). Mummery, et al., 2012 July 20, Circ. 20Res. 111(3): 344-358 provides a summary of methods to differentiate iPSC and hESCs intocardiomyocytes. Methods to differentiate stem cells (e.g., iPSC) into cardiac cells are alsodescribed in, e.g., U.S. Publication No. 2015/0017718. [0057] In particular embodiments, cardiomyocyte progenitors can be generated from embryoidbodies (EBs) treated with Activin A, BMP4 or with 2+Wnt3 and bFGF. These progenitors express 25Nkx2.5, Tbx5/20, Gata-4, Mef2c and Hand1/2. Their further differentiation to functionalcardiomyocytes can be promoted with VEGF and Dkk1 (Vidarsson, et al., 2010, Stem Cell Rev.6:108-20). [0058] A protocol for generating insulin producing beta-cells involves stepwise lineage restrictiongenerating in sequence: definitive endodermal cells (Activin+Wnt3), primitive foregut endoderm 30(FGF10+KAAD-cyclopamine), posterior foregut endoderm (RA+FGF10+KAAD-cyclopamine),pancreatic endoderm and endocrine precursors (Extendin-4), and hormone producing cells(IGF1+HGF). Transcription factor profiles include: Sox17, CER, FoxA2, and the cytokine receptorCXCR4 (definitive endodermal cells), Hnf1B, Hnf4A (primitive foregut endoderm), Pdx1, Hnf6, H1xB9 (posterior foregut endoderm), and Nkx6.1, Nkx2.2, Ngn3, Pax4 (pancreatic endoderm andendocrine precursors). See, e.g., D'Amour, et al., 2006, Nat. Biotechnol. 24:1392-401; Kroon, etal., 2008, Nat. Biotechnol. 26:443-52). Another method to induce stem cells (e.g., iPSC) to committo definitive endoderm, then to pancreatic endoderm, to pancreatic endocrine/exocrine cells andfinally to more mature islet cells is described in Jiang, et al., 2007, Stem Cells 25(8): p. 1940-53. 5 [0059] Various types of retinal cells can be generated from stem cells (e.g., iPSC) (see, e.g.,Lamba, et al., 2006, Proc. Natl. Acad. Sci. USA 103:12769-74; Reh, et al., 2010, Methods Mol.Biol. 636:139-53). EBs can be produced and thereafter treated with IGF1, Noggin (BMP inhibitor)and Dkk1 (Wnt inhibitor). This treatment with IGF1, Noggin (BMP inhibitor), and Dkk1 (Wntinhibitor) can direct stem cells (e.g., iPSC) to adopt a retinal progenitor phenotype, expressing 10Pax6 and Chx10. Exposing these progenitors to N-(N-(3,5-difluorophenacetyl)-1-alanyl)-S-phenylglycine t-butyl ester (DAPT), a blocker of Notch signaling, promotes neuronal differentiation(Lamba, et al., 2010, PLoS One 5:e8763). The decision to undergo photoreceptor differentiationis under the control of the transcription factor, Blimp1 (Brzezinski, et al., 2010, Development137:619-29). 15 [0060] In particular embodiments, neuronal differentiation can be achieved by replacing a stemcell culture media with a media including basic fibroblast growth factor (bFGF) heparin, and anN2 supplement (e.g., transferrin, insulin, progesterone, putrescine, and selenite). Two days later,differentiating cells can be attached by plating them onto dishes coated with laminin orpolyornithine. After an additional 10–11 days in culture, primitive neuroepithelial cells will have 20formed. The identity of the cells can be confirmed by staining for PAX6 (paired box protein 6, atranscription factor), SOX2 (sex-determining region Y-box 2, another transcription factor), and N-cadherin (a calcium-dependent cell adhesion molecule specific to neural tissue). Neuroepithelialcells can be further differentiated into, e.g., motor neurons (see, e.g., Li, et al. 2005, Nat.Biotechnol. 23, 215–221), dopaminergic neurons (see, e.g., Yan, et al. 2005, Stem Cells 23, 781– 25790), and oligodendrocytes (Nistor, et al. 2005, Glia 49, 385–396). [0061] Additional information regarding differentiation to motor neurons includes treatment withRA (Pax6 expressing primitive neuroepithelial cells), RA+Shh (Pax6/Sox1 expressingneuroepithelial cells), which gradually start to express the motor neuron progenitor marker Olig2.Reducing RA+Shh concentration promotes the emergence of motor neurons expressing HB9 and 30Islet1. The addition of brain-derived neurotrophic factor (BDNF), glial-derived neurotrophic factor(GDNF), insulin-like growth factor-1 (IGF1), and cAMP promotes process outgrowth (see, e.g.,Hu, et al., 2009, Nat. Protoc. 4:1614-22; Hu, et al., 2010, Proc. Natl. Acad. Sci. USA; 107:4335-40). id="p-62"

[0062] Additional information regarding differentiation to dopaminergic neurons includesoverexpression of the transcription factor Nurr1 followed by exposure to Shh, FGF-8 and ascorbicacid (see, e.g., Lee, et al., 2000 June, Nat. Biotechnol. 18(6):675-9; Kriks and Studer, 2009, Adv.Exp. Med. Biol. 651:101-11; Lindvall and Kokaia, 2009 May, Trends Pharmacol. Sci. 30(5):260-7.). The combination of stromal cell-derived factor 1 (SDF-1/CXCL12), pleiotrophin (PTN), insulin- 5like growth factor 2 (IGF2), and ephrin B1 (EFNB1) can induce stem cells (e.g., iPSC) todifferentiate to TH-positive neurons in vitro, expressing midbrain specific markers, includingEngrailed 1, Nurr1, Pitx3, and dopamine transporter (DAT). These neurons are capable ofgenerating action potentials and forming functional synaptic connections (Vazin, et al., 2009,PLoS One 4:e6606). 10 [0063] A protocol to produce mature myelinating oligodendrocytes includes directing stem cells(e.g., iPSC) toward neuroectoderm differentiation in the absence of growth factors for 2 weeks.These cells express neuroectoderm transcription factors, including Pax6 and Sox1. Next stemcells (e.g., iPSC) are exposed to the caudalizing factor retinoic acid (RA) and the ventralizingmorphogen Shh for 10 days to begin expression of Olig2. To prevent the differentiation to motor 15neurons and promote the generation of oligodendrocyte precursor cells (OPC)s, cells are culturedwith FGF2 for 10 days. By day 35, the Olig2 progenitors co-express NkxX2.2 and no longer giverise to motor neurons. The co-expression of Olig2 and Nkx2.2 reflects a stage prior to humanOPCs (pre-OPCs). These pre-OPCs are finally cultured in a glia medium includingtriiodothyronine (T3), neurotrophin 3 (NT3), PDGF, cAMP, IGF-1 and biotin, which individually or 20synergistically can promote the survival and proliferation of the OPCs, for another 8 weeks togenerate OPCs. These OPCs are bipolar or multipolar, express Olig2, Nkx2.2, Sox10 andPDGFR α, become motile and are able to differentiate to competent oligodendrocytes.WO2007/066338 also describes differentiation protocols for the generation of oligodendrocyte-like cells. 25 [0064] A protocol to produce glutamatergic neurons includes use of stem cells (e.g., iPSC) toproduce cell aggregates which are then treated for 8 days with RA. This results in Pax6 expressingradial glial cells, which after additional culturing in N2 followed by "complete" medium results in95% glutamate neurons (Bibel, et al., 2007, Nat. Protoc. 2:1034-43). [0065] A protocol to produce GABAergic neurons includes exposing EBs for 3 days to all-trans- 30RA. After subsequent culture in serum-free neuronal induction medium including Neurobasalmedium supplemented with B27, bFGF and EGF, 95% GABA neurons develop (see, e.g., Chatzi,et al., 2009, Exp. Neurol. 217:407-16). [0066] U.S. Publication No. 2013/0330306 describes compositions and methods to induce differentiation and proliferation of neural precursor cells or neural stem cells into neural cells usingumbilical cord blood-derived mesenchymal stem cells; U.S. Publication No. 2007/0179092describes use of pituitary adenylate cyclase activating polypeptide (PACAP) to enhance neuralstem cell proliferation, differentiation and survival; U.S. Publication No. 2012/0329714 describesuse of prolactin to increase neural stem cell numbers; while U.S. Publication No. 2012/0308530 5describes a culture surface with amino groups that promotes neuronal differentiation into neurons,astrocytes and oligodendrocytes. U.S. Publication No. 2006/211109 describes improved methodsfor efficiently producing neuroprogenitor cells and differentiated neural cells such as dopaminergicneurons and serotonergic neurons from pluripotent stem cells, e.g., iPSCs. [0067] Thus, the fate of neural stem cells can be controlled by a variety of extracellular factors. 10Commonly used factors include amphiregulin; BMP-2 (U.S. Pat. Nos. 5,948,428 and 6,001,654);brain derived growth factor (BDNF; Shetty and Turner, 1998, J. Neurobiol. 35:395-425);neurotrophins (e.g., Neurotrophin-3 (NT-3) and Neurotrophin-4 (NT-4); Caldwell, et al., 2001, Nat.Biotechnol. 1;19:475-9); ciliary neurotrophic factor (CNTF); cyclic adenosine monophosphate;epidermal growth factor (EGF); dexamethasone (glucocorticoid hormone); fibroblast growth factor 15(bFGF; U.S. Pat. NO.5,766,948; FGF-1, FGF-2); forskolin; GDNF family receptor ligands; growthhormone; interleukins; insulin-like growth factors; isobutyl 3-methylxanthine; leukemia inhibitorygrowth factor (LIF; U.S. Patent No. 6,103,530); Notch antagonists (U.S. Patent No. 6,149,902);platelet derived growth factor (PDGF; U.S. Patent No. 5,753,506); potassium; retinoic acid (U.S.Patent No. 6,395,546); somatostatin; tetanus toxin; and transforming growth factor- α and TGF- β 20(U.S. Pat. Nos. 5,851,832 and 5,753,506). [0068] In particular embodiments, preferred proliferation-inducing neural growth factors includeBNDF, EGF and FGF-1 or FGF-2. Growth factors can be usually added to the culture medium atconcentrations ranging between 1 fg/ml of a pharmaceutically acceptable composition (including,e.g., CNS compatible carriers, excipients and/or buffers) to 1 mg/ml. Growth factor expanded 25stem cells (e.g., iPSC) can also differentiate into neurons and glia after mitogen withdrawal froma culture medium. [0069] Additionally, WO 2004/046348 describes differentiation protocols for the generation ofneural-like cells from bone marrow-derived stem cells. WO 2006/134602 describes differentiationprotocols for the generation of neurotrophic factor secreting cells. Commercial kits are also 30available from Life Technologies and include PSC Neural Induction Medium, Geltrex™ LDEV-Free hESC-qualified Reduced Growth Factor Basement Membrane Matrix, and a Human NeuralStem Cell Immunocytochemistry kit. Stem cells (e.g., iPSC) differentiated into neural cells usingthe Life Technology kits can be further terminally differentiated into neurons, astrocytes and oligodendrocytes using Life Technologies’ B-27® supplements, with N-2 supplement andNEUROBASAL® Medium. [0070] Additional methods to assist with stem cell (e.g., iPSC) differentiation protocols include,e.g., culture vessels with a portion including an oxygen permeable substrate at least partiallycoated with a synthetic matrix having an average thickness of less than 100 nm. See, e.g., U.S. 5Publication No. 2014/0370598. [0071] U.S. Publication No. 2013/0251690 describes methods to support stem cell (e.g., iPSC)differentiation in elderly populations. [0072] A number of different differentiation methods have been described. Additional methodsthat can be used within the teaching of the current disclosure can be found in the art by those with 10ordinary skill. Furthermore, and as indicated, differentiation of stem cells (e.g., iPSC) can beconfirmed by measuring cellular markers expressed by the desired differentiated cell. [0073] The foregoing discussion describes in vitro or ex vivo differentiation methods. Modifiedstem cells (e.g., iPSC) disclosed herein can also differentiate in vivo following administration, asdescribed elsewhere herein. 15 [0074] (III) Genetic Modification of Cells. Desired genes disclosed herein can be introduced intoor deleted from cells by any method known in the art. Methods of modifying a desired gene caninclude transfection, electroporation, microinjection, lipofection, calcium phosphate mediatedtransfection, infection with a viral or bacteriophage vector including the gene sequences, cellfusion, chromosome-mediated gene transfer, microcell-mediated gene transfer, spheroplast 20fusion, in vivo nanoparticle-mediated delivery, etc. Numerous techniques are known in the artfor the modification genes in cells (see e.g., Loeffler and Behr, 1993, Meth. Enzymol. 217:599-618; Cohen, et al., 1993, Meth. Enzymol. 217:618-644; Cline, 1985, Pharmac. Ther. 29:69-92)and may be used, provided that the necessary developmental and physiological functions of therecipient cells are not unduly disrupted. The technique can provide for the stable transfer of the 25gene to the cell, so that the gene is expressible by the cell and, in certain instances, preferablyheritable and expressible by its cell progeny. The technique can also provide the stable deletionof the gene from the cell and its cell progeny. [0075] The term "gene" refers to a nucleic acid sequence (used interchangeably withpolynucleotide or nucleotide sequence) that encodes a desired gene as described herein. This 30definition includes various sequence polymorphisms, mutations, and/or sequence variantswherein such alterations do not substantially affect the function of the encoded gene. The term"gene" may include not only coding sequences but also regulatory regions such as promoters,enhancers, and termination regions. The term further can include all introns and other DNA sequences spliced from an mRNA transcript, along with variants resulting from alternative splicesites. Gene sequences encoding the molecule can be DNA or RNA that directs the expression ofa protein encoded by the gene. These nucleic acid sequences may be a DNA strand sequencethat is transcribed into RNA or an RNA sequence that is translated into protein. The nucleic acidsequences include both the full-length nucleic acid sequences as well as non-full-length 5sequences derived from the full-length protein. The sequences can also include degeneratecodons of the native sequence or sequences that may be introduced to provide codon preferencein a specific cell type. Portions of complete gene sequences are referenced throughout thedisclosure as is understood by one of ordinary skill in the art. [0076] Genes described herein can be readily prepared by synthetic or recombinant methods. In 10embodiments, the gene sequence encoding any of these sequences can also have one or morerestriction enzyme sites at the 5' and/or 3' ends of the coding sequence in order to provide foreasy excision and replacement of the gene sequence encoding the sequence with another genesequence encoding a different sequence. In embodiments, the gene sequence encoding thesequences can be codon optimized for expression in mammalian cells. 15 [0077] "Encoding" refers to the property of specific sequences of nucleotides in a gene, such asa cDNA, or an mRNA, to serve as templates for synthesis of other macromolecules such as adefined sequence of amino acids. Thus, a gene codes for a protein if transcription and translationof mRNA corresponding to that gene produces the protein in a cell or other biological system. A"gene sequence encoding a protein" includes all nucleotide sequences that are degenerate 20versions of each other and that code for the same amino acid sequence or amino acid sequencesof substantially similar form and function. [0078] Polynucleotide gene sequences encoding more than one desired gene can include desiredgenes operably linked to each other and further include relevant regulatory sequences. Forexample, there can be a functional linkage between a regulatory sequence and an exogenous 25nucleic acid sequence resulting in expression of the latter. For another example, a first nucleicacid sequence can be operably linked with a second nucleic acid sequence when the first nucleicacid sequence is placed in a functional relationship with the second nucleic acid sequence. Forinstance, a promoter is operably linked to a coding sequence if the promoter affects thetranscription or expression of the coding sequence. Generally, operably linked DNA sequences 30are contiguous and, where necessary or helpful, join coding regions, into the same reading frame. [0079] Targeted genetic engineering approaches can be used to insert, modify, and in somecases, delete targeted genes. Targeted genetic engineering approaches include transposon-based systems, the CRISPR/Cas nuclease system, zinc finger nucleases, and transcription activator like effector nucleases can be used to incorporate desired genes into stem cells. [0080] Particular embodiments can use transposon-based systems as gene editing agents tomediate the integration of a desired gene into a cell. Generally, such methods will involveintroducing into cells (i) a first vector encoding a transposase (or a transposase polypeptide) and(ii) a second vector encoding a desired genetic element that is flanked by transposon repeats. 5 [0081] A transposase refers to an enzyme that is a component of a functional nucleic acid-proteincomplex capable of transposition and which is mediating transposition. Transposase also refersto integrases from retrotransposons or of retroviral origin. A transposition reaction includes atransposase and a transposase or an integrase enzyme. In particular embodiments, the efficiencyof integration, the size of the DNA sequence that can be integrated, and the number of copies of 10a DNA sequence that can be integrated into a genome can be improved by using suchtransposable elements. Transposons include a short nucleic acid sequence with terminal repeatsequences upstream and downstream of a larger segment of DNA. Transposases bind theterminal repeat sequences and catalyze the movement of the transposon to another portion ofthe genome. 15 [0082] Several transposon/transposase systems have been adapted for genetic insertions ofheterologous DNA sequences. Examples of such transposases include sleeping beauty ("SB",e.g., derived from the genome of salmonid fish); SB100X; piggyBac™ (e.g., derived fromlepidopteran cells and/or the Myotis lucifugus); mariner (e.g., derived from Drosophila); frog prince(e.g., derived from Rana pipiens); Tol1; Tol2 (e.g., derived from medaka fish); TcBuster™ (e.g., 20derived from the red flour beetle Tribolium castaneum), Helraiser, Himar1, Passport, Minos,Ac/Ds, PIF, Harbinger, Harbinger3-DR, HSmar1, and spinON." Transposases and transposonsystems are further described in U.S. Pat. Nos. 6,489,458; 7,148,203; 8,227,432; and 9,228,180.In particular embodiments, transposon-based systems are beneficial because they allow largepayloads to be delivered allowing for dual or triple expression products. 25 [0083] The CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)/Cas(CRISPR-associated protein) nuclease system is an engineered nuclease system used forgenetic engineering that is based on a bacterial system. Information regarding CRISPR-Cassystems and components thereof are described in, for example, US8697359, US8771945,US8795965, US8865406, US8871445, US8889356, US8889418, US8895308, US8906616, 30US8932814, US8945839, US8993233 and US8999641 and applications related thereto; andWO2014/018423, WO2014/093595, WO2014/093622, WO2014/093635, WO2014/093655,WO2014/093661, WO2014/093694, WO2014/093701, WO2014/093709, WO2014/093712,WO2014/093718, WO2014/145599, WO2014/204723, WO2014/204724, WO2014/204725, WO2014/204726, WO2014/204727, WO2014/204728, WO2014/204729, WO2015/065964,WO2015/089351, WO2015/089354, WO2015/089364, WO2015/089419, WO2015/089427,WO2015/089462, WO2015/089465, WO2015/089473 and WO2015/089486, WO2016205711,WO2017/106657, WO2017/127807 and applications related thereto. [0084] Particular embodiments utilize zinc finger nucleases (ZFNs) as gene editing agents. ZFNs 5are a class of site-specific nucleases engineered to bind and cleave DNA at specific positions.ZFNs are used to introduce double stranded breaks (DSBs) at a specific site in a DNA sequencewhich enables the ZFNs to target unique sequences within a genome in a variety of different cells.A zinc finger is a domain of 30 amino acids within the zinc finger binding domain whose structureis stabilized through coordination of a zinc ion. Examples of zinc fingers include C2H2 zinc fingers, 10C3H zinc fingers, and C4 zinc fingers. A designed zinc finger domain is a domain not occurring innature whose design/composition results principally from rational criteria, e.g., application ofsubstitution rules and computerized algorithms for processing information in a database storinginformation of existing ZFP designs and binding data. A well-known example of a ZFN is a fusionof the FokI nuclease with a zinc finger DNA binding domain. For additional information regarding 15ZFNs and ZFNs useful within the teachings of the current disclosure, see, e.g., US 6,534,261; US6,607,882; US 6,746,838; US 6,794,136; US 6,824,978; 6,866,997; US 6,933,113; 6,979,539; US7,013,219; US 7,030,215; US 7,220,719; US 7,241,573; US 7,241,574; US 7,585,849; US7,595,376; US 6,903,185; US 6,479,626; US 2003/0232410 and US 2009/0203140 as well asGaj et al., Nat Methods, 2012, 9(8):805-7; Ramirez et al., Nucl Acids Res, 2012, 40(12):5560-8; 20Kim et al., Genome Res, 2012, 22(7): 1327-33; Urnov et al., Nature Reviews Genetics, 2010, 11:636-646; Miller, et al. Nature biotechnology 25, 778-785 (2007); Bibikova, et al. Science 300, 764(2003); Bibikova, et al. Genetics 161, 1169-1175 (2002); Wolfe, et al. Annual review of biophysicsand biomolecular structure 29, 183-212 (2000); Kim, et al. Proceedings of the National Academyof Sciences of the United States of America 93, 1156-1160 (1996); and Miller, et al. The EMBO 25journal 4, 1609-1614 (1985). [0085] Particular embodiments can use transcription activator like effector nucleases (TALENs)as gene editing agents. TALENs refer to fusion proteins including a transcription activator-likeeffector (TALE) DNA binding protein and a DNA cleavage domain. TALENs are used to edit genesand genomes by inducing double DSBs in the DNA, which induce repair mechanisms in cells. 30Generally, two TALENs must bind and flank each side of the target DNA site for the DNA cleavagedomain to dimerize and induce a DSB. For additional information regarding TALENs, see US8,440,431; US 8,440,432; US 8,450,471; US 8,586,363; and US 8,697,853; as well as Joung andSander, Nat Rev Mol Cell Biol, 2013, 14(l):49-55; Beurdeley et al., Nat Commun, 2013, 4: 1762; Scharenberg et al., Curr Gene Ther, 2013, 13(4):291-303; Gaj et al., Nat Methods, 2012,9(8):805-7; Miller, et al. Nature biotechnology 29, 143-148 (2011); Christian, et al. Genetics 186,757-761 (2010); Boch, et al. Science 326, 1509-1512 (2009); and Moscou, & Bogdanove, Science326, 1501 (2009). [0086] Particular embodiments can utilize MegaTALs as gene editing agents. MegaTALs have a 5sc rare-cleaving nuclease structure in which a TALE is fused with the DNA cleavage domain of ameganuclease. Meganucleases, also known as homing endonucleases, are single peptide chainsthat have both DNA recognition and nuclease function in the same domain. In contrast to theTALEN, the megaTAL only requires the delivery of a single peptide chain for functional activity. [0087] Nanoparticles that result in selective in vivo genetic modification of targeted cell types have 10been described and can be used to deliver desired genes to a stem cell. In particularembodiments, the nanoparticles can be those described in WO2014153114, WO2017181110,and WO201822672. [0088] Vectors and viral vectors can also be used to deliver desired genes to a stem cell. A"vector" is a nucleic acid molecule that is capable of transporting another nucleic acid. Vectors 15may be, e.g., plasmids (DNA plasmids or RNA plasmids), transposon-based systems, cosmids,bacterial artificial chromosomes, viruses, or phage. An "expression vector" is a vector that iscapable of directing the expression of a protein encoded by one or more genes carried by thevector when it is present in the appropriate environment. [0089] "Lentivirus" refers to a genus of retroviruses that are capable of infecting dividing and non- 20dividing cells. Several examples of lentiviruses include HIV (human immunodeficiency virus:including HIV type 1, and HIV type 2); equine infectious anemia virus; feline immunodeficiencyvirus (FIV); bovine immune deficiency virus (BIV); and simian immunodeficiency virus (SIV). [0090] A lentiviral vector is a vector derived from at least a portion of a lentivirus genome,including especially a self-inactivating lentiviral vector as provided in Milone et ah, Mol. Ther. 2517(8): 1453-1464 (2009). Other examples of lentivirus vectors that may be used in the clinic,include: the LENTIVECTOR® gene delivery technology from Oxford BioMedica, the LENTIMAX™vector system from Lentigen and the like. Nonclinical types of lentiviral vectors are also availableand would be known to one skilled in the art. In particular embodiments, cells are geneticallyengineered to express desired genes using a lentivirus or lentiviral vector. 30 [0091] "Retroviruses" are viruses having an RNA genome. "Gammaretrovirus" refers to a genusof the retroviridae family. Exemplary gammaretroviruses include mouse stem cell virus, murineleukemia virus, feline leukemia virus, feline sarcoma virus, and avian reticuloendotheliosisviruses. id="p-92"

[0092] Retroviral vectors (see Miller, et al., 1993, Meth. Enzymol. 217:581-599) can be used. Insuch embodiments, the gene to be expressed is cloned into the retroviral vector for its deliveryinto cells. In particular embodiments, a retroviral vector includes all of the cis-acting sequencesnecessary for the packaging and integration of the viral genome, i.e., (a) a long terminal repeat(LTR), or portions thereof, at each end of the vector; (b) primer binding sites for negative and 5positive strand DNA synthesis; and (c) a packaging signal, necessary for the incorporation ofgenomic RNA into virions. More detail about retroviral vectors can be found in Boesen, et al.,1994, Biotherapy 6:291-302; Clowes, et al., 1994, J. Clin. Invest. 93:644-651; Kiem, et al., 1994,Blood 83:1467-1473; Salmons and Gunzberg, 1993, Human Gene Therapy 4:129-141; andGrossman and Wilson, 1993, Curr. Opin. in Genetics and Devel. 3:110-114. Adenoviruses, 10adeno-associated viruses (AAV) and alphaviruses can also be used. See Kozarsky and Wilson,1993, Current Opinion in Genetics and Development 3:499-503, Rosenfeld, et al., 1991, Science252:431-434; Rosenfeld, et al., 1992, Cell 68:143-155; Mastrangeli, et al., 1993, J. Clin. Invest.91:225-234; Walsh, et al., 1993, Proc. Soc. Exp. Bioi. Med. 204:289-300; and Lundstrom, 1999,J. Recept. Signal Transduct. Res. 19: 673-686. Other methods of gene delivery include use of 15mammalian artificial chromosomes (Vos, 1998, Curr. Op. Genet. Dev. 8:351-359); liposomes(Tarahovsky and Ivanitsky, 1998, Biochemistry (Mosc) 63:607-618); ribozymes (Branch andKlotman, 1998, Exp. Nephrol. 6:78-83); and triplex DNA (Chan and Glazer, 1997, J. Mol. Med.75:267-282). [0093] There are a large number of available, suitable viral vectors including those identified for 20human gene therapy applications (see Pfeifer and Verma, 2001, Ann. Rev. Genomics Hum.Genet. 2:177). Methods of using retroviral and lentiviral viral vectors and packaging cells fortransducing mammalian host cells with desired genes are described in, e.g., US 8,119,772;Walchli, et al., 2011, PLoS One 6:327930; Zhao, et al., 2005, J. Immunol. 174:4415; Engels, etal., 2003, Hum. Gene Ther. 14:1155; Frecha, et al., 2010, Mol. Ther. 18:1748; and Verhoeyen, et 25al., 2009, Methods Mol. Biol. 506:97. Retroviral and lentiviral vector constructs and expressionsystems are also commercially available. [0094] In particular embodiments, the transposon-based system is used to genetically modifycells to include a gene. In particular embodiments, the CRISPR/Cas system is used to geneticallymodify cells to knockout a gene. 30 [0095] (III-A) Conditional Immortalization. Genetic tools have been created that not onlyimmortalize a cell but offer regulatory control to switch cell proliferation on and off using externalfactors as controlled by the operator through a growth controlling agent. Herein, cellimmortalization refers to the modification of a cell such that the cell can be cultured indefinitely and does not undergo cell senescence. Cell senescence refers to the process by which cellseventually stop multiplying or dividing. Cell senescence is thought to be an antitumor mechanismand typically occurs in response to various cell stressors, such as telomere erosion, DNAdamage, oxidative stress, and oncogenic activation. Several methods exist for immortalizingcells in culture including expression of viral genes (e.g., SV40 large T antigen), expression of 5telomerase reverse transcriptase protein (TERT), and other methods for the conditionalexpression of telomerase. [0096] Viral genes, including Epstein-Barr virus (EBV), Simian virus 40 (SV40) T antigen,adenovirus E1A and E1B, and human papilloma virus (HPV) E6 and E7 can induceimmortalization by a process known as viral transformation. Generally, these viral genes achieve 10immortalization of the cell by inactivating the tumor suppressor genes that put cells into areplicative senescent state. Occasionally, these cells may become genetically unstable(aneuploid) and lose the properties of the primary cell. In particular embodiments, it is desirablethat such viral-induced immortalization does not also result in transformation of the cells into atumor cell phenotype. 15 [0097] SV40 large T antigen (Simian Vacuolating Virus 40 Tag) is a hexamer protein that is anoncogene derived from the polyomavirus SV40 which is capable of transforming a variety of celltypes. The transforming activity of SV40 large T antigen is due in large part to its perturbation ofthe retinoblastoma (pRB) and p53 tumor suppressor proteins. In addition, SV40 large T antigenbinds to several other cellular factors, including the transcriptional co-activators p300 and CBP, 20which may contribute to its transformation function. [0098] Cells can also be immortalized by expression of the telomerase reverse transcriptaseprotein (TERT), particularly in those cells most affected by telomere length (e.g., human cells).The term "TERT" as used herein refers to a polypeptide sequence possessing telomerasecatalytic activity. Telomerase is an enzyme that adds specific DNA sequence repeats 25("TTAGGG" in all vertebrates) to the 3 ′ end of DNA strands in the telomere regions, which arefound at the ends of eukaryotic chromosomes. The telomeres contain condensed DNA material,giving stability to the chromosomes. The enzyme is a reverse transcriptase that carries its ownRNA molecule, which is used as a template when it elongates telomeres, which are shortenedafter each replication cycle. It includes two molecules each of telomerase catalytic subunit also 30referred to as Telomerase Reverse Transcriptase (TERT); Telomerase RNA (hTR or TERC);and dyskerin. TERT is a reverse transcriptase, which creates single-stranded DNA using single-stranded RNA as a template. This protein is inactive in most somatic cells, but when TERT isexogenously expressed, the cells are able to maintain telomere lengths sufficient to avoid replicative senescence. Analysis of several telomerase-immortalized cell lines has verified thatthe cells maintain a stable genotype and retain critical phenotypic markers. [0099] Other methods to conditionally immortalize cells include conditional expression oftelomerase using the pHUSH vector system, the transposon-based gene trap system, and/orconditional gene expression using tamoxifen-dependent Cre recombinase-loxP site-mediated 5recombination. Skilled artisans are familiar with such techniques. For example, in the expressionof telomerase, lentiviral vectors containing the drug-controllable expression of polymerase (Pol)II promoter-driven expression of transgenes (i.e. telomerase) or Pol III promoter-controlledsequences encoding small inhibitory hairpin RNAs (shRNAs) are suitable methodologies forcreating immortalized cells (Szulc, J., et al., Nature Methods 2006 3(2):109-116). The pHUSH 10vector system can be used to conditionally immortalize cells. This inducible expression vectorsystem is used for regulated expression of shRNA, miRNA or cDNA cassettes on a single viralvector (Gray, D. C., et al., BMC Biotechnology 2007, 7:61). The transposon-based gene trapsystem incorporates the doxycycline-repressive Tet-Off (tTA) system that is capable of activatingthe expression of a gene (for example telomerase) which is under control of a Tet response 15element (TRE) promoter (Geurts, A. M., et al., BMC Biotechnology 2006, 6:30). Tamoxifen-dependent Cre recombinase-loxP site-mediated recombination and bicistronic gene-trapexpression vectors allow for transgene (i.e. telomerase) expression from endogenous promoters(Vallier, L., et al., PNAS 2001 98(5):2467-2472). [0100] In particular embodiments, cells are conditionally immortalized by expression of TERT 20and/or SV40 large T antigen. In particular embodiments, the conditional immortalization genecan be turned on by administration of a growth controlling agent (e.g., drug) and can be turnedoff by stopping administration of the growth controlling agent. A "growth controlling agent" asdescribed herein, refers to Inducible immortalization of cells allows for the control of proliferationof modified cells responsive to an inducing agent or drug. Exemplary non-limiting examples of 25such inducible systems are the Tet-on/off systems which utilize tetracycline/doxycycline as theinducing agent. Other inducible systems are also contemplated for carrying out the methodsdescribed herein. Examples of non-Tet inducible systems include the coumermycin inducibleexpression system, the RheoSwitch® (RheoGene, Inc., Noristown, PA) Mammalian InducibleExpression system, estrogen receptor inducible systems, cumate-inducible systems, and Cre- 30Lox recombinase systems. In some cases, cell lines are generated that have stably incorporatedthe inducible systems or constructs described herein. Alternatively, cells can be modulated totransiently express the inducible systems or constructs described herein (e.g., via transienttransfection of at least one construct). id="p-101"

[0101] A Tet-on or Tet-off system typically utilizes a tetracycline transactivator protein. TetOsequences are typically positioned upstream of any open reading frame (ORF) whoseexpression is sought to be controlled using the Tet system. A promoter and the TetOsequence(s) can make up a tetracycline response element (TRE). In some cases, the TREincludes TetO sequence(s) and is placed upstream of a promoter and the ORF(s) for one or 5more genes of interest. In the Tet-on system, the transactivator protein has a strong bindingaffinity for TetO operator sequence(s) when it is not bound by tetracycline (or a derivative suchas doxycycline). In the absence of tetracycline, the transactivator protein does not bind to thetetracycline response element (TRE). When tetracycline is added, it binds to the transactivatorprotein and causes the transactivator protein to bind to the TRE to induce expression of 10downstream ORF(s). In a Tet-off system, the transactivator protein has a strong binding affinityfor TetO operator sequence(s) only when it is not bound by tetracycline. In the absence oftetracycline, the transactivator protein binds the TetO sequences and promotes expression ofthe downstream ORF(s). Added tetracycline binds to the transactivation protein causing aconformational change that results in decreased or loss of binding to the TRE, resulting in 15reduced expression of the downstream ORF(s). In particular embodiments, the drug includestetracycline or doxycycline. In particular embodiments, doxycycline includes doxycyclinehyclate.In particular embodiments, media is supplemented with the drug at 0.01 μM to 5 μM. Inparticular embodiments, media is supplemented with the drug at 0.1 μM to 1 μM. In particularembodiments, media is supplemented with the drug at 0.1 μM, 0.3 μM, 0.6 μM, or 1 μM. In 20particular embodiments, the drug is added to the culture and differentiation medium throughoutthe culture and/or differentiation. In particular embodiments, the drug is added to thedifferentiation medium at the initiation of the differentiation stage. [0102] (III-B) Expression Products. In particular embodiments, it is useful for a cell to express aspecific molecule or factor that is useful to support its desired function as a feeder cell, tester 25cell, or therapeutic cell. As referred to herein, an expression product is a molecule expressed bya cell that supports the cells use for a desired function (e.g., as a feeder cell, tester cell, ortherapeutic cell). An expression product can include a protein (e.g., an antibody, an antigen, adetectable label, and/or a recombinant receptor), DNA, or RNA (e.g., mRNA). The expressionproduct can be secreted by the cell into the extracellular matrix or can be expressed on the 30surface of the cell. In particular embodiments, the expression product is directed to the cellsurface or directed for secretion by a signal peptide which is encoded by a signal sequence. [0103] The term "signal peptide" or "signal peptide sequence" is defined herein as a peptidesequence usually present at the N-terminal end of newly synthesized secretory or membrane polypeptide which directs the polypeptide across or into a cell membrane of the cell (the plasmamembrane in prokaryotes and the endoplasmic reticulum membrane in eukaryotes). It is usuallysubsequently removed. In particular, said signal peptide may be capable of directing thepolypeptide into a cell's secretory pathway. The signal sequence can be foreign or native. Anative signal sequence is naturally present in relation to the encoded protein. A foreign signal 5sequence is a signal peptide that is not native to the encoded protein, i.e. it originates fromanother gene than the encoded protein. Example membrane-spanning signal peptides includeglycoprotein C signal peptide, foamy virus Env signal peptide, CD8 signal peptide, orgranulocyte-macrophage colony-stimulating factor (GM-CSF) signal peptide. An examplesecretory signal peptide includes mouse mammary tumor virus (MMTV) envelope protein signal 10peptide. [0104] Depending on the use of the cell, the cell may be genetically modified to express aspecific expression product. For example, in a feeder cell, expression products could includeproteins (e.g., antigens, or antibodies that stimulate the activation and expansion of a desiredcell type). In a tester cell, expression products could include antigens (e.g., cancer antigens) 15such that the tester cell can be used to test a new therapeutic treatment. In a therapeutic cell,expression products could include antibodies or recombinant receptors such that the therapeuticcell can target and kill an undesired cell t ype. Alternatively, a therapeutic cell could express aprotein or antibody such that the therapeutic cell can be used to replace a deficient protein orantibody within a subject. 20 [0105] Any useful protein (e.g., recombinant receptor or detectable label) or can be used. [00106] (III-B-1) Protein. A protein is molecule made of one or more chains of amino acids. Aprotein can include a peptide, an antigen, an antibody, an enzyme, etc. The protein can be asecreted protein a non-secreted protein, or a membrane-bound protein. An antigen is a type ofprotein and refers to any substance that specifically binds to a selected antibody. The term 25"antibody" includes (in addition to antibodies having two full-length heavy chains and two full-length light chains as described above) variants, derivatives, and fragments thereof. Furthermore,unless explicitly excluded, antibodies can include monoclonal antibodies, human or humanizedantibodies, bispecific antibodies, trispecific antibodies, tetraspecific antibodies, multi-specificantibodies, polyclonal antibodies, linear antibodies, minibodies, domain antibodies, synthetic 30antibodies, chimeric antibodies, antibody fusions, and fragments thereof, respectively. Inparticular embodiments, antibodies can include oligomers or multiplexed versions of antibodies. [0107] Any protein useful for cell manufacture, research and development, or therapeutictreatment can be used. For example, proteins useful in cell differentiation or cell proliferation can be expressed. For example, in the culture of NK cells, membrane-bound IL21 is important topromote cell maturation, proliferation, increase cytotoxic effect, and enhance anti-tumor activity.For the culture of NK cells, the IL21 can be made membrane-bound by expressing it on anexosome, membrane lysate, bead, or by engineering cells to express IL21 on its surface. [0108] In tumor immunotherapy, activation of cytotoxicity of NK cells and CD8 + T cells is key, 5and many studies have shown that IL21 plays an important role in this procedure. IL21 promotesmaturation of NK cells to produce IFN- γ and perforin, and induces NK cell-mediated anti-tumorcytotoxicity to target NKG2D ligands on the surface of tumor cells, and increasing the lethality ofNK cells via the antibody-dependent cell-mediated cytotoxicity (ADCC) (see literature: "Spolski,R. & Leonard, W J Interleukin-21: a double-edged sword with therapeutic potential. Nat. Rev. 10Drug Discov. 13, 379-395 (2014)."). Secondly, IL21 can induce the proliferation of CD8+ T cells,induce the generation of memory T cells, and promote the secretion of IFNy/granzyme toenhance the killing of tumors by CD8 + T cells and contribute to the memory immune responseto recurrent tumor cells. [0109] In particular embodiments, IL21 includes the sequence: 15MRSSPGNMERIVICLMVIFLGTLVHKSSSQGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQLKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPKEFLERFKSLLQKMIHQHLSSRTHGSEDS (SEQ ID NO: 24). [0110] In particular embodiments, stem cells can be engineered to express a cancer cell antigen.Cancer cell antigens are molecules preferentially expressed by cancer cells. "Preferentially 20expressed" means that a cancer cell antigen is found at higher levels on cancer cells ascompared to other cell types. In some instances, the cancer antigen is expressed on the cancercell types at least 25%, 35%, 45%, 55%, 65%, 75%, 85%, 95%, 96%, 97%, 98%, 99%, or 100%more than on non-cancer cells. [0111] The following provides examples of cancer antigens that are more likely to be co- 25expressed in particular cancers: BCMA, CD4, CD5, CD7, CD19, CD20, CD22, CD33, CD73,CD123, CD133, CD138, CD244, CD276, CS1, EGFR, EGFRVIII, EpCAM, FLT3, GD2, GPA7,GPC3, HER2, Mesothelin, MUC1, NKG2D, PSMA, PSCA, or TF. [0112] The following table provides examples of cancer antigens that are more likely to be co-expressed in particular cancer types: 30Cancer Antigens Likely to be Co-Expressed Cancer TypeCD138, CS1, B-cell maturation antigen (BCMA), PD-L1, EFGR Multiple MyelomaCD22, CD19, CD4 LymphomaCD7, CD19, CD5, FLT3 Acute lymphocyticleukemia (ALL) CD33, CD19, CD4, CD123 Acute myelocytic leukemia(AML)CD19 Chronic lymphocyticleukemia (CLL)PSMA, WT1, Prostate Stem Cell antigen (PSCA), SV40 T, PD-L1, EFGRProstate Cancer HER2, EpCAM, TF, ERBB2, ROR1, PD-L1, EFGR, MUC16,folate receptor (FOLR), CEABreast Cancer CD133, PD-L1, EFGR Stem Cell CancerHER1, L1-CAM, MUC16, FOLR, Lewis Y, ROR1, mesothelin,WT-1, PD-L1, EFGR, CD56Ovarian Cancer HER2 Renal cell carcinoma(RCC)EGFR, EGFRVIII, CD73, HER2 Glioblastomamesothelin, PD-L1, EFGR Mesotheliomacarboxy-anhydrase-IX (CAIX); PD-L1, EFGR Renal Cell CarcinomaGPA7, GD2, PD-L1, EFGR Melanomamesothelin, CEA, CD24, ROR1, PD-L1, EFGR, MUC16 Pancreatic CancerROR1, PD-L1, EFGR, mesothelin, MUC16, FOLR, CEA, CD56 Lung Cancermesothelin, PD-L1, EFGR CholangiocarcinomaMUC16, PD-L1, EFGR, Bladder CancerGD2, CD244, CD276, ROR1, glypican-2, CD56,disialoganglioside, PD-L1, EFGR,Neuroblastoma HER2, EpCAM, NKG2D, CEA, PD-L1, EFGR Colorectal CancerGD2 Ewing sarcomaGPC3 Hepatocellular cancer(HCC)CD56, PD-L1, EFGR, Merkel Cell Carcinoma [0113] In particular embodiments, the protein can be a viral, bacterial, fungal, or parasiticantigen. In particular embodiments, viral antigens include viral entry proteins. Examples of viralentry proteins include [virus (entry protein)]: Chikungunya (E1 Env and E2 Env); Ebolaglycoprotein (EBOV GP); Hendra (F glycoprotein and G glycoprotein); hepatitis B (large (L),middle (M), and small (S)); hepatitis C (glycoprotein E1 and glycoprotein E2); HIV envelope 5(Env); influenza hemagglutinin (HA); Lassa virus envelope glycoprotein (GPC); measles(hemagglutinin glycoprotein (H) and fusion glycoprotein F0 (F)); MERS-CoV (Spike (S)); Nipah(fusion glycoprotein F0 (F) and glycoprotein G); Rabies virus glycoprotein (RABV G); RSV(fusion glycoprotein F0 (F) and glycoprotein G); and SARS-CoV (Spike (S)); among many others. [0114] Additional HIV proteins include gene products of the gag, pol, and env genes such as 10HIV gp32, HIV gp41, HIV gp120, HIV gp160, HIV P17/24, HIV P24, HIV P55 GAG, HIV P66POL, and HIV GP36. Other HIV proteins of interest include the Nef protein and other accessoryproteins such as Vpr, Vpu, Tat, and Rev. Very particular examples of specific viral proteins andstrains include BF520.W14.C2; BG505.W6M.C2.T332N; BG505 SOSIP Env trimer; BL035.W6M.ENV.C1; SF162; ZM109F.PB4; C2-94UG114; HIV-BAL, HIV-LAI, SIV/mac239; MNgp41 monomer; ectodomain ZA.1197/MB; Q23; QA013.70I.Env.H1; QA013.385M.Env.R3 677;QB850.73P.C14; QB850.632P.B10; Q461.D1; and QC406.F3. Numerous additionalproteins/strains are known to one of ordinary skill in the art. [0115] As further particular examples of viral antigens, cytomegaloviral antigens include 5envelope glycoprotein B and CMV pp65; Epstein-Barr antigens include EBV EBNAI, EBV P18,and EBV P23; hepatitis antigens include the S, M, and L proteins of hepatitis B virus, the pre-Santigen of hepatitis B virus, HBCAG DELTA, HBV HBE, hepatitis C viral RNA, HCV NS3 andHCV NS4; herpes simplex viral antigens include immediate early proteins and glycoprotein D;influenza antigens include hemagglutinin and neuraminidase; Japanese encephalitis viral 10antigens include proteins E, M-E, M-E-NS1, NS1, NS1-NS2A and 80% E; measles antigensinclude the measles virus fusion protein; rabies antigens include rabies glycoprotein and rabiesnucleoprotein; respiratory syncytial viral antigens include the RSV fusion protein and the M2protein; rotaviral antigens include VP7sc; rubella antigens include proteins E1 and E2; andvaricella zoster viral antigens include gpl and gpll. 15 [0116] Bacterial antigen can include: anthrax antigens include anthrax protective antigen; gram-negative bacilli antigens include lipopolysaccharides; diptheria antigens include diptheria toxin;Mycobacterium tuberculosis antigens include mycolic acid, heat shock protein 65 (HSP65), thekDa major secreted protein and antigen 85A; pertussis toxin antigens include hemagglutinin,pertactin, FIM2, FIM3 and adenylate cyclase; pneumococcal antigens include pneumolysin and 20pneumococcal capsular polysaccharides; rickettsiae antigens include rompA; streptococcalantigens include M proteins; and tetanus antigens include tetanus toxin. [0117] Fungal antigens can include: coccidiodes antigens include spherule antigens;cryptococcal antigens include capsular polysaccharides; histoplasma antigens include heatshock protein 60 (HSP60); leishmania antigens include gp63 and lipophosphoglycan; 25plasmodium falciparum antigens include merozoite surface antigens, sporozoite surfaceantigens, circumsporozoite antigens, gametocyte/gamete surface antigens, protozoal and otherparasitic antigens including the blood-stage antigen pf 155/RESA; schistosomae antigensinclude glutathione-S-transferase and paramyosin; tinea fungal antigens include trichophytin;toxoplasma antigens include SAG-1 and p30; and Trypanosoma cruzi antigens include the 75- 30kDa antigen and the 56 kDa antigen. [0118] In particular embodiments, the protein can include an enzyme or protein useful for atherapeutic treatment. For example, there are several proteins that are deficient in certaindiseases that could be replaced by administering cells that can produce said protein. For example, insulin can be useful for the treatment of diabetes; factor VIII, factor IX, or factor XI forthe treatment of clotting disorders; alpha-1 antitrypsin (A1AT) for the treatment of chronicobstructive pulmonary disease (COPD) and liver disorders; and glucocerebrosidase (GC), acidsphingomyelinase, mucopolysaccharides, acid alpha-glucosidase, aspartylglucosaminidase,alpha-galactosidase A, palmitoyl protein thioesterase, tripeptidyl peptidase, lysosomal 5transmembrane protein, cysteine transporter, acid ceramidase, acid alpha-L-fucosidase,cathepsin A, acid beta-glucosidase, acid beta-galactosidase, iduronate-2-sulfatase, alpha-L-iduronidase, galactocerebrosidase, acid alpha-mannosidase, acid beta-mannosidase,arylsulfatase B, arylsulfatase A, N-acetylgalactosamine-6-sulfate, N-acetylglucosamine-1-phosphotransferase, acid sphingomyelinase, NPC-1, alpha-glucosidase, beta-hexosaminidase 10B, heparan N-sulfatase, alpha-N-acetylglucosaminidase, acetyl-CoA: alpha-glucosaminide, N-acetylglucosamine-6-sulfate, alpha-N-acetylgalactosaminidase, alpha-neuramidase, beta-glucuronidase, beta-hexosaminidase A, and/or acid lipase can be useful for the treatment oflysosomal storage diseases. [0119] (III-B-2) Recombinant Receptors. Cells described herein can be genetically modified to 15express a recombinant receptor. In particular embodiments, a recombinant receptor includes achimeric antigen receptor (CAR) and/or an engineered T cell receptor (eTCR). [0120] CAR include several distinct subcomponents that allow genetically modified cells (e.g.,regulatory T cells) to recognize and kill cells expressing an antigen (e.g., a cancer antigen). Thesubcomponents include at least an extracellular component and an intracellular component. The 20extracellular component includes a binding domain that specifically binds an antigen epitope thatis preferentially present on the surface of cells or in the area thereof. When the binding domainbinds such epitopes, the intracellular component activates the cell to destroy the bound cell.CAR additionally include a transmembrane domain that directly or indirectly links theextracellular component to the intracellular component, and other subcomponents that can 25increase the CAR’s function. For example, the inclusion of a spacer region and/or one or morelinker sequences can allow the CAR to have additional conformational flexibility, often increasingthe binding domain’s ability to bind the targeted epitope. [0121] A TCR is a heterodimeric fusion protein that typically includes an α and β chain. Eachchain includes a variable region (V α and V β) and a constant region (C α and C β). In particular 30embodiments, an eTCR does not include the native TCR variable region but does include thenative TCR constant region. In particular embodiments, the eTCR includes a binding domain(e.g., antibody) as the variable region of the α and/or β chain. In particular embodiments, eTCRinclude a C α and/or C β chain sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least99.5%, or 100% identical to an amino acid sequence of a known or identified TCR C α or C β. [0122] The binding domains of CAR and eTCR include a molecule that binds an antigen ofinterest. In many cases, the antigen of interest is a cancer antigen (see description of cancerantigens elsewhere herein). Other antigens of interest can include viral antigens, bacterial 5antigens, fungal antigens, etc. Antibodies are one example of binding domains and include wholeantibodies or binding fragments of an antibody, e.g., Fv, Fab, Fab', F(ab')2, and single chain (sc)forms and fragments thereof that specifically bind a cellular marker. Antibodies or antigen bindingfragments can include all or a portion of polyclonal antibodies, monoclonal antibodies, humanantibodies, humanized antibodies, synthetic antibodies, non-human antibodies, recombinant 10antibodies, chimeric antibodies, bispecific antibodies, mini bodies, and linear antibodies. Otherbinding fragments, such as Fv, Fab, Fab', F(ab')2, can also be used within a CAR. Additionalexamples of antibody-based binding domain formats for use in a CAR include scFv-basedgrababodies and soluble VH domain antibodies. These antibodies form binding regions usingonly heavy chain variable regions. See, for example, Jespers et al., Nat. Biotechnol. 22:1161, 152004; Cortez-Retamozo et al., Cancer Res. 64:2853, 2004; Baral et al., Nature Med. 12:580,2006; and Barthelemy et al., J. Biol. Chem. 283:3639, 2008. [0123] In addition to binding domains, CAR and eTCR can additionally include transmembranedomains, intracellular effector domains, spacer regions, transduction markers, and tags. [0124] Transmembrane domains typically have a three-dimensional structure that is 20thermodynamically stable in a cell membrane, and generally ranges in length from 15 to 30amino acids. The structure of a transmembrane domain can include an α helix, a β barrel, a βsheet, a β helix, or any combination thereof. Transmembrane domains can include at least thetransmembrane region(s) of the α, β or ζ chain of a T-cell receptor, CD28, CD27, CD3, CD45,CD4, CD5, CD8, CD9, CD16, CD22; CD45, CD37, CD64, CD80, CD86, CD134, CD137 and 25CD154. [0125] A transmembrane domain can include one or more additional amino acids adjacent tothe transmembrane region, e.g., one or more amino acid within the extracellular region of theexpressed protein (e.g., up to 15 amino acids of the extracellular region) and/or one or moreadditional amino acids within the intracellular region of the expressed protein (e.g., up to 15 30amino acids of the intracellular components). [0126] Intracellular effector domains activate the expressing cell when the binding domain bindsantigen. The term "effector domain" is thus meant to include any portion of the intracellulardomain sufficient to transduce an activation signal. id="p-127"

[0127] An effector domain can include one, two, three or more intracellular signaling components(e.g., receptor signaling domains, cytoplasmic signaling sequences), co-stimulatory domains, orcombinations thereof. Exemplary effector domains include signaling and stimulatory domainsselected from: 4-1BB (CD137), CD3 γ, CD3 δ, CD3 ε, CD3 ζ, CD27, CD28, DAP10, ICOS, LAG3,NKG2D, NOTCH1, OX40, ROR2, SLAMF1, TCR α, TCRβ, TRIM, Wnt, Zap70, or any 5combination thereof. In particular embodiments, exemplary effector domains include signalingand co-stimulatory domains selected from: CD86, Fc γRIIa, DAP12, CD30, CD40, PD-1,lymphocyte function-associated antigen-1 (LFA-1), LIGHT, NKG2C, B7-H3, a ligand thatspecifically binds with CD83, CDS, ICAM-1, GITR, BAFFR, SLAMF7, NKp80 (KLRF1), CD127,CD19, CD4, CD8 α, CD8β, IL2R β, IL2R γ, IL7R α, ITGA4, VLA1, CD49a, IA4, CD49D, ITGA6, 10VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, ITGAM, CD11b, ITGAX, CD11c,ITGB1, CD29, ITGB2, CD18, ITGB7, TNFR2, TRANCE/RANKL, DNAM1 (CD226), SLAMF4(CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), PSGL1, CD100(SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (CD150, IPO-3), BLAME (SLAMF8),SELPLG (CD162), LTBR, GADS, PAG/Cbp, NKp44, NKp30, or NKp46. 15 [0128] Intracellular signaling component sequences that act in a stimulatory manner may includeiTAMs. Examples of iTAMs including primary cytoplasmic signaling sequences include thosederived from CD3 γ, CD3 δ, CD3 ε, CD3 ζ, CD5, CD22, CD66d, CD79a, CD79b, and commonFcR γ (FCER1G), Fc γRlla, FcR β (Fc ε Rib), DAP10, and DAP12. In particular embodiments,variants of CD3 ζ retain at least one, two, three, or all ITAM regions. 20 [0129] A co-stimulatory domain is a domain whose activation can be required for an efficientlymphocyte response to cellular marker binding. Some molecules are interchangeable asintracellular signaling components or co-stimulatory domains. Examples of costimulatorydomains include CD27, CD28, 4-1BB (CD137), OX40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), NKG2C, and a ligand that specifically binds with CD83. 25 [0130] Spacer regions are used to create appropriate distances and/or flexibility between sub-components of a protein. Spacer regions typically include 10 to 250 amino acids, 10 to 200 aminoacids, 10 to 150 amino acids, 10 to 100 amino acids, 10 to 50 amino acids, or 10 to 25 aminoacids. Exemplary spacer regions include all or a portion of an immunoglobulin hinge region. [0131] Transduction markers and tags can be helpful in identifying and isolating cells that have 30been successfully modified. Additional details about transduction makers and tags can be foundelsewhere herein. [0132] (III-B-3) Detectable Labels. In particular embodiments, stem cells are genetically modifiedto express a detectable label. Detectable labels can include any suitable label or detectable group detectable by, for example, optical, spectroscopic, photochemical, biochemical,immunochemical, electrical, optical or chemical means. Such detectable labels includefluorescent proteins, radiolabels, radioacoustic labels, enzyme labels, chemiluminescencelabels, fluorescence labels, and biotin (with labeled avidin or streptavidin). [0133] Fluorescent proteins can be particularly useful in cell staining, identification, and isolation 5uses. Exemplary fluorescent proteins include luciferase; blue fluorescent proteins (e.g. eBFP,eBFP2, Azurite, mKalama1, GFPuv, Sapphire, T-sapphire); cyan fluorescent proteins (e.g. eCFP,Cerulean, CyPet, AmCyanl, Midoriishi-Cyan, mTurquoise); green fluorescent proteins (e.g. GFP,GFP-2, tagGFP, turboGFP, EGFP, Emerald, Azami Green, Monomeric Azami Green(mAzamigreen)), CopGFP, AceGFP, avGFP, ZsGreenl, Oregon Green™(Thermo Fisher 10Scientific)); orange fluorescent proteins (mOrange, mKO, Kusabira-Orange, MonomericKusabira-Orange, mTangerine, tdTomato); red fluorescent proteins (mKate, mKate2, mPlum,DsRed monomer, mCherry, mRuby, mRFP1, DsRed-Express, DsRed2, DsRed-Monomer,HcRed-Tandem, HcRedl, AsRed2, eqFP611, mRaspberry, mStrawberry, Jred, Texas Red™(Thermo Fisher Scientific)); far red fluorescent proteins (e.g., mPlum and mNeptune); yellow 15fluorescent proteins (e.g., YFP, eYFP, Citrine, SYFP2, Venus, YPet, PhiYFP, ZsYellowl); andtandem conjugates. In particular embodiments, stem cells are genetically modified to expressluciferase. [0134] Radioisotopes can be used as a type of detectable label called a radiolabel. In particularembodiments, a radioisotope includes131I,Y, and/or211At. In particular embodiments, a 20radioisotope is selected that includes a half-life (t1/2) that enables high-yield radiolabeling and drugdelivery. In particular embodiments, a radioisotope is selected that includes a half-life (t 1/2) of 7.2hours. In particular embodiments, a radioisotope is selected that does not emit daughterradionuclides that cause organ toxicity. Exemplary radiolabels include228Ac,111Ag,124Am,As,211At,209At,194Au,128Ba,Be,206Bi,245Bk,246Bk,Br,C,C,Ca,254Cf,242Cm,Cr,Cu,153Dy, 25157Dy,159Dy,165Dy,166Dy,171Er,250Es,254Es,147Eu,157Eu,Fe,Fe,251Fm,252Fm,253Fm,Ga,Ga,146Gd,153Gd,Ge,H,170Hf,171Hf,193Hg,193mHg,160mHo,130I, 135I,114mIn,185Ir,K,K,Kr,Kr,mKr,132La,262Lr,169Lu,174mLu,176mLu,257Md,260Md,Mg,Mn,Mo,Na,Nb,138Nd,Ni,Ni,234Np,O,182Os,189mOs,191Os,P,201Pb,101Pd,143Pr,191Pt,243Pu,225Ra,Rb,188Re,105Rh,211Rn,103Ru,S,Sc,Se,153Sm,125Sn,Sr,173Ta,154Tb,127Te,234Th,Ti,166Tm,230U, 30237U,240U,V,178W,181W,188W,125Xe,127Xe,133Xe,133mXe,135Xe,mY,Y,Y,Y,169Yb,175Yb,Zn,mZn,Zr,Zr, and/orZr. [0135] Exemplary enzyme labels include horseradish peroxidase, hydrolases, and alkalinephosphatase. Exemplary fluorescence labels include rhodamine, phycoerythrin, and fluorescein. id="p-136"

[0136] (III-C) Major Histocompatibility Class (MHC) Molecules. In particular embodiments, cellsare genetically modified to knockout MHC. TCR ligands can be divided into two classes majorhistocompatibility complex class I (MHC I) and MHC class II (MHC II). Human MHC Is arecomplexes of human leukocyte antigens (HLAs: HLA-A, HLA-B, and HLA-C) and β2-microglobulinwhile MHC IIs are heterodimers of several HLAs (HLA-DP, HLA-DQ, and HLA-DR). Antigen 5peptide-bound MHC I (pMHC-I) molecules can be presented on any nucleated cells recognizedby CD8+ T cells. On the other hand, CD4+ T cells recognize antigen peptide-bound MHC II(pMHC-II) molecules that are presented on the antigen-presenting cells (APCs), such as B cells,macrophages, and dendritic cells (Wieczorek et al. Front. Immunol. 8, 292, 2017). Studies haveshown that CD8 and CD4 molecules may play a role during the development of T cells by helping 10the TCR complex select a different class of MHC molecules (Tikhonova, et al. Immunity 36, 79–91, 2012). [0137] T cells, through their T cell receptor (TCR) may recognize the T cell epitope in the contextof an MHC class I molecule. MHC class I proteins can be expressed in all nucleated cells of highervertebrates. The MHC class I molecule is a heterodimer composed of a 46-kDa heavy chain 15which is non-covalently associated with the 12-kDa light chain β2-microglobulin (or P-2 -microglobulin or B2M). In the human genome, B2M is encoded by the b2m gene located onchromosome 15, while other MHC genes are present as gene clusters on chromosome 6. Thehuman β2-microglobulin protein has 119 amino acids (see UniProt database number P61769). Ina model of mice lacking B2M, it can be demonstrated that B2M is essential for the presentation 20on the cell surface and the stability of the polypeptide binding groove of MHC class I molecules.Mismatches in MHC can cause immune rejection, resulting in graft destruction. Removal of MHCclass I molecules on the cell surface by knocking out B2M genes can prevent mismatches. [0138] The human MHC is also called the human leukocyte antigen (HLA) complex. In humans,there are several MHC class I alleles, such as, for example, HLA-A2, HLA-A1, HLA-A3, HLA-A24, 25HLA-A28, HLA-A31, HLA-A33, HLA-A34, HLA-B7, HLA-B45 and HLA-Cw8. In some cases, therecan be differences in the frequency of subtypes between different populations. [0139] In some embodiments, the TCR may recognize the T cell epitope in the context ofan MHC class I or class II molecule. MHC class II proteins can be expressed in a subset of APCs.In humans, there are several MHC class II alleles, such as, for example, DR1, DR3, DR4, DR7, 30DR52, DQ1, DQ2, DQ4, DQ8 and DPI. In some embodiments, the MHC class II allele is an HLA-DRB 1*0101, an HLA-DRB*0301, an HLA-DRB*0701, an HLA-DRB*0401 or an HLA- DQB1*0201 gene product. [0140] MHC class II expression depends on CIITA and RFX, two transcription factors that are highly selective for MHC class II genes. RFX is expressed ubiquitously, while CIITA expressionis cell-specific and finely regulated. Hence, the pattern of MHC class II expression replicatesfaithfully the expression pattern of the gene encoding CIITA (MHC2TA). MHC2TA is expressedthrough a set of three cell-specific promoters, referred to as promoters I, III and IV. Promoters Iand III are constitutively active in professional antigen-presenting cells, while in most other cell 5types CIITA expression is inducible with interferon gamma (IFN γ) through promoter IV.Expression of the MHC class II transactivator, CIITA, closely parallels that of class II MHC geneexpression. It has also been shown that CIITA is induced by gamma interferon, and thattransfection of CIITA alone into cells is sufficient to activate class II MHC. [0141] The N-terminal of CIITA contains an acidic domain (amino acids 30-160), followed by 10domains rich in proline (amino acids 163-195), serine (amino acids 209-257), and threonine(amino acids 260-322). An acidic domain has been found in many transcription factors and hasbeen shown to interact with basal transcriptional machinery in vitro and in vivo. However, it islikely that the acidic domain alone is not sufficient to activate the class II MHC promoter in CIITA,and that the acidic domains of other transcription factors behave differently from the CIITA acidic 15domain. H. Analysis of the primary amino acid sequence of CIITA does not show any homologyto known conserved DNA-binding motif of transcription factors, and in vitro translated CIITAapparently does not interact with DNA. [0142] Multiple HLA class I and class II proteins must be matched for histocompatibility inallogeneic recipients to avoid allogeneic rejection problems and immune responses. Provided 20herein are conditionally immortalized stem cells and cells differentiated therefrom with eliminatedor substantially reduced expression of both HLA class I and HLA class II proteins. HLA class Ideficiency can be achieved by functional deletion of any region of the HLA class I locus(chromosome 6p21), or deletion or reducing the expression level of HLA class-I associated genesincluding beta-2 microglobulin (B2M) gene, TAP1 gene, TAP2 gene and Tapasin. For example, 25the B2M gene encodes a common subunit essential for cell surface expression of all HLA class Iheterodimers. B2M null cells are HLA-I deficient. [0143] HLA class II deficiency can be achieved by functional deletion or reduction of HLA-IIassociated genes including RFXANK, CIITA, RFXS and RFXAP. CIITA is a transcriptionalcoactivator, functioning through activation of the transcription factor RFXS required for class II 30protein expression. CIITA null cells are HLA-II deficient. [0144] In particular embodiments, undesirable immune responses to a cell is overcome bygenetically modify or knocking out MHC genes. Various gene editing methods can be used toinactivate a gene encoding an MHC molecule or a subunit thereof, or inactivate a gene regulating the expression of the gene encoding an MHC molecule or a subunit thereof. In particularembodiments, to genetically modify or knockout an MHC Class I molecule, the gene encoding thealpha chain and/or gene encoding B2M of the MHC Class I molecule can be targeted for geneticmodification or knockout. In particular embodiments, the gene encoding B2M is knocked out. Inparticular embodiments, to genetically modify or knockout an MHC Class II molecule, the gene 5encoding CIITA is knocked out. In particular embodiments, an MHC null (HLA-I and HLA-IIdeficient) cell is produced by knocking out the gene encoding B2M and the gene encoding CIITA. [0145] In particular embodiments, B2M is knocked out using B2M guide RNA (gRNA). Inparticular embodiments, CIITA is knocked out using CIITA gRNA. Example gRNA sequences arepresented in Table 2. 10Table 2. gRNA targeting B2M and CIITA. Target Sequence SEQ ID NO: CIITA in mouse CATTGCAGCTGGATACCAG 25CIITA in mouse ACTGAGACAGTAACGGCCA 26CIITA in mouse ATAGAGATCCCTGTAGAAGC 27CIITA in human TCAACTGCGACCAGTTCAGC 28CIITA in human CATCGCTGTTAAGAAGCTCC 29CIITA in human TTCCTACACAATGCGTTGCC 30CIITA in human GCCCCTAGAAGGTGGCTACC 31CIITA in human GATATTGGCATAAGCCTCCC 32CIITA in human TCCTACCTGTCAGAGCCCCA 33B2M in mouse AGTATACTCACGCCACCCAC 34B2M in mouse AGTCGTCAGCATGGCTCGCT 35B2M in mouse CGTATGTATCAGTCTCAGTG 36B2M in human GAGTAGCGCGAGCACAGCTA 37B2M in human GGCCGAGATGTCTCGCTCCG 38B2M in human CAGTAAGTCAACTTCAATGT 39B2M in human CGTGAGTAAACCTGAATCTT 40B2M in human ACCCAGACACATAGCAATTC 41B2M in human ACTCACGCTGGATAGCCTCC 42 id="p-146"

[0146] (III-D) Suicide Gene. Suicide genes can be used for the elimination of geneticallyengineered cells. Suicide genes are nucleic acid constructs encoding a protein (also referred toas a kill switch) that inducibly causes cell death or stops cell proliferation. The suicide gene is inserted at a defined, specific target locus in the genome of an engineered cell, usually at bothalleles of the target locus. The suicide gene is activated by contacting cells with an effective doseof a clinically acceptable orthologous activating agent. In particular embodiments, theorthologous activating agent can be a molecule (e.g., drugs, antibodies, small molecules), light,radiation, ultrasound, temperature, pH, or other means that causes activation of the kill switch. 5When activated, the kill switch causes the cell to stop proliferation, and in some embodimentsactivates apoptosis of the cell. [0147] The suicide gene is inserted at a targeted site of the genome, where it is operably linkedto the promoter of a gene of interest, without disrupting expression of the gene of interest. Insome embodiments the suicide gene is integrated to replace the stop codon of the gene of 10interest. The kill switch protein in this embodiment may be flanked by self-cleaving peptidesequences to provide for cleavage of the gene of interest protein and the kill switch protein [0148] In some embodiments, the protein encoded by the suicide gene is a protein that inducesapoptosis upon dimerization. In some embodiments the protein is a human caspase protein,e.g., caspase 1, caspase 2, caspase 3, caspase 4, caspase 5, caspase 6, caspase 7, caspase 158, caspase 9, caspase 10, caspase 14, etc. In certain embodiments the protein is humancaspase 9. The caspase protein is fused to a sequence that provides for chemically induceddimerization (CID), in which dimerization occurs only in the presence of the orthologousactivating agent. [0149] A popular kill switch for drug-induced cell apoptosis uses a modified human caspase 9 to 20the FK506 binding protein (FKBP) that dimerizes in the presence of a small-molecule drug. Thiskill switch is referred to as inducible caspase 9 or iCasp9 (Straathof et al., Blood. 2005,105(11):4247-4254). This kill switch has shown efficacy in both preclinical and clinical contexts(Diaconu et al., Mol Ther. 2017, 25(3):580-592; and Stasi et al., N Engl J Med. 2011,365(18):1673-1683). In particular embodiments, FDA-approved small molecules such as 25rapamycin can be used control iCasp9 kill switches (Stavrou et al., mBio. 2018, 9(3):e00923-18). [0150] A suicide gene can be prepared by transcriptionally linking a cell division locus (CDL) anda sequence encoding a negative selectable marker. This allows a user to inducibly killproliferating host cells including the suicide gene or inhibit the host cell's proliferation by killing 30at least a portion of proliferating cells by exposing the modified cells to an inducer of the negativeselectable marker. A cell modified to include the suicide gene can be treated with an inducer(e.g., a drug) of the negative selectable marker in order to ablate proliferating cells or to inhibitcell proliferation by killing at least a portion of proliferating cells. id="p-151"

[0151] Example CDLs include CDK1, TOP2A, CENPA, BIRC5, and EEF2. Example negativeselectable markers include Herpes Simplex Virus type 1 (HSV) thymidine kinase/ganciclovir(TK/GCV), deaminase/5-fluorocytosine (CD/5-FC), and carboxyl esterase/irinotecan (CE/CPT-11). [0152] Host cells modified with the HSV-TK/GCV negative selectable marker will produce 5thymidine kinase (TK) and the TK protein will convert GCV into GCV monophosphate, which isthen converted into GCV triphosphate by cellular kinases. GCV triphosphate incorporates intothe replicating DNA during S phase, which leads to the termination of DNA elongation and cellapoptosis (Halloran and Fenton, 1998, Cancer Res. 58(17): 3855-65). [0153] The CD/5-FC negative selectable marker system is a widely used suicide gene system. 10Cytosine deaminase (CD) is a non-mammalian enzyme that may be obtained from bacteria oryeast (e.g., from Escherichia coli or Saccharomyces cerevisiae, respectively) (Ramnaraine etal., 2003). CD catalyzes conversion of cytosine into uracil and is an important member of thepyrimidine salvage pathway in prokaryotes and fungi, but it does not exist in mammalian cells.5-fluorocytosine (5-FC) is an antifungal prodrug that causes a low level of cytotoxicity in humans 15(Denny, 2003, J Biomed Biotechnol). CD catalyzes conversion of 5-FC into the genotoxic agent5-FU, which has a high level of toxicity in humans (Ireton et al., 2002, J Molec. Biol. 315(4):687-697). [0154] The CE/CPT-11 system is based on the carboxyl esterase enzyme, which is a serineesterase found in a different tissues of mammalian species (Humerickhouse et al., 2000, Cancer 20Res. 60(5):1189-92). The anti-cancer agent CPT-11 is a prodrug that is activated by CE togenerate an active referred to as 7-ethyl-10-hydroxycamptothecin (SN-38), which is a strongmammalian topoisomerase I inhibitor (Wierdl et al., 2001). SN-38 induces accumulation ofdouble-strand DNA breaks in dividing cells (Kojima et al., 1998, Anal Chem. 70(13:2446-53). [0155] In particular embodiments, the suicide gene includes a CDK1 cell division locus 25transcriptionally linked to an HSV-TK/GCV negative selectable marker (CDK1/HSV-KT/GCV). Inparticular embodiments, the suicide gene includes a TOP2A cell division locus transcriptionallylinked to an HSV-TK/GCV negative selectable marker (TOP2A/HSV-KT/GCV). In particularembodiments, the suicide gene includes CDK1/HSV-TK/GCV. In particular embodiments, thesuicide gene includes TOP2A/HSV-KT/GCV. 30 [0156] (III-E) Other Control Features. In particular embodiments, cells can be geneticallymodified to include other control features. In particular embodiments, other control featuresinclude tag cassettes and transduction markers. In particular embodiments, genetic constructsfor insertion into stem cells and described herein can include one or more tag cassettes and/or transduction markers. Tag cassettes and transduction markers can be used to activate, promoteproliferation of, detect, enrich for, isolate, track, deplete and/or eliminate genetically modifiedcells in vitro, in vivo and/or ex vivo. "Tag cassette" refers to a unique synthetic peptide sequenceaffixed to, fused to, or that is part of a genetic construct, to which a cognate binding molecule(e.g., ligand, antibody, or other binding partner) is capable of specifically binding where the 5binding property can be used to activate, promote proliferation of, detect, enrich for, isolate,track, deplete and/or eliminate the tagged protein and/or cells expressing the tagged protein.Transduction markers can serve the same purposes but are derived from naturally occurringmolecules and are often expressed using a skipping element that separates the transductionmarker from the rest of the genetic construct molecule. 10 [0157] Tag cassettes that bind cognate binding molecules include, for example, His tag(HHHHHH; SEQ ID NO: 43), Flag tag (DYKDDDDK; SEQ ID NO: 44), Xpress tag (DLYDDDDK;SEQ ID NO: 45), Avi tag (GLNDIFEAQKIEWHE; SEQ ID NO: 46), Calmodulin tag(KRRWKKNFIAVSAANRFKKISSSGAL; SEQ ID NO: 47), Polyglutamate tag, HA tag(YPYDVPDYA; SEQ ID NO: 48), Myc tag (EQKLISEEDL; SEQ ID NO: 49), Strep tag (which 15refers the original STREP® tag (WRHPQFGG; SEQ ID NO: 50), STREP® tag II (WSHPQFEKSEQ ID NO: 51 (IBA Institut fur Bioanalytik, Germany); see, e.g., US 7,981,632), Softag 1(SLAELLNAGLGGS; SEQ ID NO: 52), Softag 3 (TQDPSRVG; SEQ ID NO: 53), and V5 tag(GKPIPNPLLGLDST; SEQ ID NO: 54). [0158] Conjugate binding molecules that specifically bind tag cassette sequences disclosed 20herein are commercially available. For example, His tag antibodies are commercially availablefrom suppliers including Life Technologies, Pierce Antibodies, and GenScript.Flag tag antibodiesare commercially available from suppliers including Pierce Antibodies, GenScript, and Sigma-Aldrich. Xpress tag antibodies are commercially available from suppliers including PierceAntibodies, Life Technologies and GenScript. Avi tag antibodies are commercially available from 25suppliers including Pierce Antibodies, IsBio, and Genecopoeia. Calmodulin tag antibodies arecommercially available from suppliers including Santa Cruz Biotechnology, Abcam, and PierceAntibodies. HA tag antibodies are commercially available from suppliers including PierceAntibodies, Cell Signal and Abcam. Myc tag antibodies are commercially available from suppliersincluding Santa Cruz Biotechnology, Abcam, and Cell Signal. Strep tag antibodies are 30commercially available from suppliers including Abcam, Iba, and Qiagen. [0159] Transduction markers may be selected from at least one of a truncated CD19 (tCD19;see Budde et al., Blood 122: 1660, 2013); a truncated human EGFR (tEGFR; see Wang et al.,Blood 118: 1255, 2011); an extracellular domain of human CD34; and/or RQR8 which combines target epitopes from CD34 (see Fehse et al, Mol. Therapy 1( 5 Pt 1); 448–456, 2000) and CD20antigens (see Philip et al, Blood 124: 1277–1278). [0160] Selection cassettes can be used for the selection of transformed cells. A selectioncassette includes a selective marker gene. Selective marker genes are used to selecttransformed cells. Such selective markers may, for example, confer resistance to antibiotics, 5such as G418, hygromycin, blasticidin, neomycin, or puromycin. In other embodiments of theinvention, the selective marker is operably linked to the inducible promoter, and the expressionof the selective marker is toxic to the cell. Examples of such selective markers include xanthine/ guanine phosphoribosyltransferase (gpt), hypoxanthine-guanine phosphoribosyltransferase(HGPRT) or thymidine kinase of the herpes simplex virus (HSV-TK). Polynucleotides encoding 10selective markers are functionally linked to the promoter active in the cell. In particularembodiments, the selection cassette includes a gene encoding neomycin resistance. Inparticular embodiments, the selection cassette includes a gene encoding puromycin resistance. [0161] Control features may be present in multiple copies in a genetic construct or can beexpressed as distinct molecules with the use of a skipping element. Exemplary skipping 15elements include a self-cleaving polypeptide or IRES. In particular embodiments, a self-cleavingpolypeptide includes a 2A peptide from porcine teschovirus-1 (P2A), Thosea asigna virus (T2A),equine rhinitis A virus (E2A), foot-and-mouth disease virus (F2A), or variants thereof. Furtherexemplary nucleic acid and amino acid sequences of 2A peptides are set forth in, for example,Kim et al. (PLOS One 6:e18556 (2011). 20 [0162] For example, a genetic construct can have one, two, three, four, or five tag cassettesand/or one, two, three, four, or five transduction markers could also be expressed. Exemplarytransduction markers and cognate pairs are described in US 13/463,247. [0163] One advantage of including at least one control feature in a genetic construct is that cellsexpressing the genetic construct administered to a subject can be depleted using the cognate 25binding molecule to a tag cassette. In certain embodiments, the present disclosure provides amethod for depleting a modified cell expressing a genetic construct by using an antibody specificfor the tag cassette, using a cognate binding molecule specific for the control feature, or by usinga second modified cell expressing an antibody or chimeric antigen receptor having specificity forthe control feature. Elimination of modified cells may be accomplished using depletion agents 30specific for a control feature. For example, if tEGFR is used, then an anti-tEGFR binding domain(e.g., antibody, scFv) fused to or conjugated to a cell-toxic reagent (such as a toxin, radiometal)may be used, or an anti-tEGFR /anti-CD3 bispecific scFv, or an anti-tEGFR CAR T cell may beused. id="p-164"

[0164] In certain embodiments, modified cells may be detected or tracked in vivo by usingantibodies that bind with specificity to a control feature (e.g., anti-Tag antibodies), or by othercognate binding molecules that specifically bind the control feature, which binding partners forthe control feature are conjugated to a fluorescent dye, radio-tracer, iron-oxide nanoparticle orother imaging agent known in the art for detection by X-ray, CT scan, MRI-scan, PET-scan, 5ultrasound, flow-cytometry, near infrared imaging systems, or other imaging modalities (see,e.g., Yu, et al., Theranostics 2:3, 2012). [0165] Thus, modified cells expressing at least one control feature can be, e.g., more readilyidentified, isolated, sorted, induced to proliferate, tracked, and/or eliminated as compared to amodified cell without a tag cassette. 10 [0166] (IV) Culture and Storage of Cells. In another embodiment, the disclosure providesmethods of establishing and/or maintaining populations of cells (e.g., stem cells, engineeredcells, and/or differentiated cells), or the progeny thereof, as well as mixed populations includingboth stem cells (e.g., iPSC) and progeny cells, and the populations of cells so produced. Oncea culture of cells or a mixed culture of cells is established, the population of cells is mitotically 15expanded in vitro by passage to fresh medium as cell density dictates under conditionsconducive to cell proliferation, with or without tissue formation. Such culturing methods for stemcells can include, for example, passaging the cells in culture medium lacking particular growthfactors that induce differentiation (e.g., IGF, EGF, FGF, VEGF, and/or other growth factor), inthe presence of an agent that stimulates (e.g., an agonist) of Klf, Oct4, Sox, Myc, SV40Tag, 20Nanog, Lin28 or any combination thereof, in the presence of Klf, Oct4, Sox, Myc, SV40Tag,Nanog, Lin28 or any combination thereof, or any combination of the foregoing. Mixed culturesor cultures containing differentiated and/or engineered cells can be cultured in fresh mediumcontaining particular growth factors and supplements to aid in their maintenance and growth.Accordingly, appropriate passaging techniques can be used to control contact inhibition and 25quiescence. Thus, in one embodiment, for example, transferring a portion of the cells to a newculture vessel with fresh medium. Such removal or transfer can be done in any culture vessel. [0167] In particular embodiments, the disclosure provides cell lines. As used herein a "cell line"means a culture of cells of the disclosure, or progeny cells thereof, that can be reproduced foran extended period of time, preferably indefinitely, and which term includes, for example, cells 30that are cultured, cryopreserved and re-cultured following cryopreservation. As used herein a"culture" means a population of cells grown in a medium and optionally passaged accordingly.A cell culture may be a primary culture (e.g., a culture that has not been passaged) or may be asecondary or subsequent culture (e.g., a population of cells which have been subcultured or passaged one or more times). [0168] Once the cells have been established in culture, as described above, they may bemaintained or stored in cell "banks" including either continuous in vitro cultures of cells requiringregular transfer or cells which have been cryopreserved. In some embodiments, the banked cellsare used for autologous treatment of a subject. 5 [0169] Cryopreservation of cells may be carried out according to known methods, such as thosedescribed in Doyle et al., (eds.), 1995, Cell & Tissue Culture: Laboratory Procedures, John Wiley& Sons, Chichester. For example, cells may be suspended in a "freeze medium" such as, forexample, culture medium further including 15-20% fetal bovine serum (FBS) and 10%dimethylsulfoxide (DMSO), with or without 5-10% glycerol, at a density, for example, of 4-10×1010cells/ml. The cells are dispensed into glass or plastic vials which are then sealed and transferredto a freezing chamber of a programmable or passive freezer. The optimal rate of freezing maybe determined empirically. For example, a freezing program that gives a change in temperatureof −1° C/min through the heat of fusion may be used. Once vials containing the cells havereached −80° C, they are transferred to a liquid nitrogen storage area. Cryopreserved cells can 15be stored for a period of years, though they should be checked at least every 5 years formaintenance of viability. [0170] The cryopreserved cells of the disclosure constitute a bank of cells, portions of which canbe withdrawn by thawing and then used to produce a cell culture as needed. Thawing shouldgenerally be carried out rapidly, for example, by transferring a vial from liquid nitrogen to a 37° 20C. water bath. The thawed contents of the vial should be immediately transferred under sterileconditions to a culture vessel containing an appropriate medium. It is advisable that the cells inthe culture medium be adjusted to an initial density of 1-3×10 cells/ml. Once in culture, the cellsmay be examined daily, for example, with an inverted microscope to detect cell proliferation, andsubcultured as soon as they reach an appropriate density. 25 [0171] The cells of the disclosure may be withdrawn from a cell bank as needed, and used forthe production of new cells, either in vitro, or in vivo, for example, by direct administration of cellsto the subject. [0172] Once established, a culture of cells may be used to produce progeny cells. Differentiationof stem cells (e.g., iPSC) to other cell types, followed by the production of tissue therefrom, can 30be triggered by specific exogenous growth factors or by changing the culture conditions (e.g.,the density) of a stem cell (e.g., iPSC) culture. The cells can be used to reconstitute an irradiatedsubject and/or a subject treated with chemotherapy; or as a source of cells for specific lineages,by providing for their maturation, proliferation and differentiation into one or more selected lineages. Examples of factors that can be used to induce differentiation include erythropoietin,colony stimulating factors, e.g., GM-CSF, G-CSF, or M-CSF, interleukins, e.g., IL-1, -2, -3, -4, -5, -6, -7, -8, and the like, Leukemia Inhibitory Factory (LIF), Steel Factor (Stl), or the like,coculture with tissue committed cells, or other lineage committed cells types to induce the stemcells (e.g., iPSC) into becoming committed to a particular lineage. Additional methods of 5differentiation are described in more detail elsewhere herein. [0173] (V) Cell-based Formulations. In particular embodiments, stem cells, engineered cells,and/or differentiated cells of the present disclosure can be harvested from a culture medium andwashed and concentrated into a carrier in a therapeutically-effective amount. Exemplary carriersinclude saline, buffered saline, physiological saline, water, Hanks' solution, Ringer's solution, 10Normosol-R (Abbott Labs), PLASMA-LYTE A® (Baxter Laboratories, Inc., Morton Grove, IL), andcombinations thereof. [0174] In particular embodiments, carriers can be supplemented with human serum albumin(HSA) or other human serum components or fetal bovine serum. In particular embodiments, acarrier for infusion includes buffered saline with 5% HSA or dextrose. Additional isotonic agents 15include polyhydric sugar alcohols including trihydric or higher sugar alcohols, such as glycerin,erythritol, arabitol, xylitol, sorbitol, or mannitol. [0175] Carriers can include buffering agents, such as citrate buffers, succinate buffers, tartratebuffers, fumarate buffers, gluconate buffers, oxalate buffers, lactate buffers, acetate buffers,phosphate buffers, histidine buffers, and/or trimethylamine salts. 20 [0176] Stabilizers refer to a broad category of excipients which can range in function from abulking agent to an additive which helps to prevent cell adherence to container walls. Typicalstabilizers can include polyhydric sugar alcohols; amino acids, such as arginine, lysine, glycine,glutamine, asparagine, histidine, alanine, ornithine, L-leucine, 2-phenylalanine, glutamic acid, andthreonine; organic sugars or sugar alcohols, such as lactose, trehalose, stachyose, mannitol, 25sorbitol, xylitol, ribitol, myoinisitol, galactitol, glycerol, and cyclitols, such as inositol; PEG; aminoacid polymers; sulfur-containing reducing agents, such as urea, glutathione, thioctic acid, sodiumthioglycolate, thioglycerol, alpha-monothioglycerol, and sodium thiosulfate; low molecular weightpolypeptides (i.e., <10 residues); proteins such as HSA, bovine serum albumin, gelatin orimmunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; monosaccharides such as 30xylose, mannose, fructose and glucose; disaccharides such as lactose, maltose and sucrose;trisaccharides such as raffinose, and polysaccharides such as dextran. [0177] Where necessary or beneficial, formulations can include a local anesthetic such aslidocaine to ease pain at a site of injection. id="p-178"

[0178] Exemplary preservatives include phenol, benzyl alcohol, meta-cresol, methyl paraben,propyl paraben, octadecyldimethylbenzyl ammonium chloride, benzalkonium halides,hexamethonium chloride, alkyl parabens such as methyl or propyl paraben, catechol, resorcinol,cyclohexanol, and 3-pentanol. [0179] Therapeutically effective amounts of cells within formulations can be greater than 10 cells, 5greater than 10 cells, greater than 10 cells, greater than 10 cells, greater than 10 cells, greaterthan 10 cells, greater than 10 cells, greater than 10 cells, greater than 10 cells, or greaterthan 10. [0180] In formulations disclosed herein, cells are generally in a volume of a liter or less, 500 mlor less, 250 ml or less or 100 ml or less. Hence the density of administered cells is typically greater 10than 10 cells/ml, 10 cells/ml or 10 cells/ml. [0181] The cell-based formulations disclosed herein can be prepared for administration by, e.g.,injection, infusion, perfusion, or lavage. The formulations can further be formulated for bonemarrow, intravenous, intradermal, intraarterial, intranodal, intralymphatic, intraperitoneal,intralesional, intraprostatic, intravaginal, intrarectal, intrathecal, intratumoral, intramuscular, 15intravesicular, and/or subcutaneous injection. In particular embodiments, cell-based formulationsdisclosed herein can be cryopreserved for later use. In particular embodiments, cell-basedformulation can be used for research and/or cell manufacturing protocols. [0182] (VI) Methods of Use. The methods and formulations disclosed herein can be used for cellmanufacturing, research, and therapeutic purposes. The methods and formulations disclosed 20herein can be used in vitro or in vivo. [0183] (VI-A) Feeder Cells for Cell Manufacturing. Coculture with feeder cells can greatlyenhance the proliferation and activation of some cell types such as T cells and NK cells. Forexample, in some cases, T cells do not receive a strong enough activation signal when thebinding domain of an extracellular component binds a targeted cell marker, resulting in a failure 25to kill the bound cell. Further, administered T cell populations often do not proliferate sufficientlyor persist in vivo for sufficient periods of time following administration to maintain on-going anti-target effects. [0184] An engineered feeder cell is ideally designed to include characteristics necessary orbeneficial for the activation and expansion of the cell it is designed to be cocultured with. A 30cocultured cell refers to the cell from a population of cells that is activated and expanded withthe addition of an appropriate feeder cell. Considerations for a feeder cell include expressionproducts to be expressed by the feeder cell, whether the feeder cell should be adherent orcultured in suspension, the MHC background of the feeder, and what differentiated cell type the feeder cell should be. [0185] T-cells can further be classified into helper cells (CD4+ T-cells) and cytotoxic T-cells(CTLs, CD8+ T-cells), which include cytolytic T-cells. T helper cells assist other white blood cellsin immunologic processes, including maturation of B cells into plasma cells and activation ofcytotoxic T-cells and macrophages, among other functions. These cells are also known as CD4+ 5T-cells because they express the CD4 protein on their surface. Helper T-cells become activatedwhen they are presented with peptide antigens by MHC class II molecules that are expressedon the surface of antigen presenting cells (APCs). Once activated, they divide rapidly andsecrete small proteins called cytokines that regulate or assist in the active immune response. [0186] T cell activation can occur in the presence of trimeric CD70, αCD3, CD28, 4-1BB and/or 10other activation molecules. In one embodiment, expansion refers to a period following activationwherein CD3, CD27 or CD28 binding molecules are no longer present. In particularembodiments, expansion refers to a period following activation wherein activating αCD3molecules and CD27 binding molecules are no longer present. This expansion period can lastuntil the end of culture when, for example, the cells are formulated for administration. In certain 15embodiments, expansion may take place in the presence of αCD3 molecules, CD27 bindingmolecules (CD70 molecules), other co-stimulatory molecules, and/or cytokines. In someembodiments, T cell activation occurs using a CD28/CD3 stimulating molecule (e.g.,DynaBeads). [0187] NK cells also benefit from coculture with feeder cells. NK cells sense and kill target cells 20that lack major histocompatibility complex (MHC)-class I molecules. NK cell activating receptorsinclude, among others, the natural cytotoxicity receptors (NKp30, NKp44 and NKp46), and lectin-like receptors NKG2D and DNAM-1. Their ligands are expressed on stressed, transformed, orinfected cells but not on normal cells, making normal cells resistant to NK cell killing (Bottino,Castriconi et al. 2005; Gasser, Orsulic et al. 2005; Lanier 2005). NK cell activation is negatively 25regulated via inhibitory receptors, such as killer immunoglobin (Ig)-like receptors (KIRs),NKG2A/CD94, and leukocyte Ig-like receptor-1 (LIR-1). Engagement of one inhibitory receptormay be sufficient to prevent target lysis (Bryceson, Ljunggren et al. 2009). Hence NK cellsefficiently target cells that express many stress-induced ligands, and few MHC class I ligands. [0188] NK cell effector agents can be a cytokine, an adhesion molecule, or an NK cell activating 30agent. In particular embodiments, the NK cell effector agent can be IL-15, IL-21, IL-2, 41BBL,IL-12, IL-18, MICA, 2B4, LFA-1, and BCM1/SLAMF2. Some cancer cell lines have been usedas feeder cells such as genetically modified K562 cells (artificial antigen-presenting cells withmembrane-bound MICA, 4-1BBL, membrane-bound IL-15 and IL-21) and Epstein-Barr virus- transformed lymphoblastoid cell lines. Even though these methods allow for large-scale NK cellexpansion, they have safety issues because of concerns of using cancer cell-based feeder cellsin therapeutic methods. [0189] The engineered feeder cell can be an adherent cell or a cell grown in suspension. Thiswill also affect the specialized type of cell chosen or differentiated into to prepare the feeder cell. 5In particular embodiments, a conditionally immortalized stem cell is differentiated into anadherent, differentiated feeder cell. In particular embodiments, a conditionally immortalized stemcell is differentiated into suspended, differentiated feeder cell. In particular embodiments, whendesigning feeder cells for T cells, the engineered feeder cells can be adherent cells such asmesenchymal stem cells. In particular embodiments, when designing feeder cells for NK cells, 10the engineered feeder cells can be cells in suspension such as CD34+ cells. [0190] The feeder cell can be engineered to express a similar MHC genetic background as thecocultured cell or can be engineered to be MHC null. In particular embodiments, the feeder cellcan be engineered to be conditionally immortalized. In particular embodiments, the feeder cellcan be engineered to include a suicide gene. In particular embodiments, the feeder cell can be 15engineered to express an expression product for the activation and/or expansion of thecocultured cell. For example, an engineered feeder cell for T cells can express CD70, αCD3,CD28, and/or 4-1BB. In particular embodiments, an engineered feeder cell for NK cells mightexpress MICA, 4-1BBL, membrane-bound IL-15 and/or membrane-bound IL21. [0191] In particular embodiments, a feeder cell is used in coculture with a cocultured cell for 20activation and/or expansion of the cocultured cell. In particular embodiments, a feeder cellincludes a conditional immortalization gene. In particular embodiments, a feeder cell includes aconditional immortalization gene and a sequence encoding an expression product. In particularembodiments, the conditional immortalization gene includes a sequence encoding SV40 largeT antigen and/or TERT. In particular embodiments, the expression product is important for cell 25activation and/or expansion. In particular embodiments, the expression product includes CD70,αCD3, CD28, and/or 4-1BB. In particular embodiments, the expression product includes MICA,4-1BBL, membrane-bound IL-15 and/or membrane-bound IL21. [0192] In particular embodiments, a feeder cell includes a conditional immortalization gene, asequence encoding an expression product, and a suicide gene. In particular embodiments, the 30suicide gene includes CDK1/HSV-TK/GCV. In particular embodiments, the suicide geneincludes TOP2A/HSV-TK/GCV. [0193] In particular embodiments, a feeder cell includes a conditional immortalization gene anda sequence encoding an expression product, wherein the feeder cell is genetically modified to knockout MHC expression. In particular embodiments, MHC expression includes B2M, CITA,and/or CIITA expression. [0194] In particular embodiments, a feeder cell includes a conditional immortalization gene, asequence encoding an expression product, and a suicide gene wherein the feeder cell isgenetically modified to knockout MHC expression. 5 [0195] In particular embodiments, a feeder cell used in coculture with a T cell includes aconditional immortalization gene and a sequence encoding CD70, αCD3, CD28, and/or 4-1BB.In particular embodiments, a feeder cell used in coculture with a T cell includes a conditionalimmortalization gene; a sequence encoding CD70, αCD3, CD28, and/or 4-1BB; and a suicidegene. In particular embodiments, a feeder cell used in coculture with a T cell includes a 10conditional immortalization gene and a sequence encoding CD70, αCD3, CD28, and/or 4-1BB;wherein the feeder cell is genetically modified to knockout B2M and/or CIITA. In particularembodiments, a feeder cell used in coculture with a T cell includes a conditional immortalizationgene; a sequence encoding CD70, αCD3, CD28, and/or 4-1BB; and a suicide gene; wherein thefeeder cell is genetically modified to knockout B2M and/or CIITA (e.g., B2M and CIITA). In 15particular embodiments, the conditional immortalization gene includes a sequence encodingSV40 large T antigen and/or TERT. In particular embodiments, the suicide gene includesCDK1/HSV-TK/GCV. In particular embodiments, the suicide gene includes TOP2A/HSV-TK/GCV. [0196] In particular embodiments, a feeder cell used in coculture with an NK cell includes a 20conditional immortalization gene and a sequence encoding MICA, 4-1BBL, membrane-boundIL-15 and/or membrane-bound IL21. In particular embodiments, a feeder cell used in coculturewith an NK cell includes a conditional immortalization gene and a sequence encodingmembrane-bound IL21. In particular embodiments, a feeder cell used in coculture with an NKcell includes a conditional immortalization gene; a sequence encoding membrane-bound IL21; 25and a suicide gene. In particular embodiments, a feeder cell used in coculture with an NK cellincludes a conditional immortalization gene and a sequence encoding membrane-bound IL21;wherein the feeder cell is genetically modified to knockout B2M and/or CIITA (e.g., B2M andCIITA). In particular embodiments, a feeder cell used in coculture with an NK cell includes aconditional immortalization gene; a sequence encoding membrane-bound IL21; and a suicide 30gene; wherein the feeder cell is genetically modified to knockout B2M and/or CIITA. In particularembodiments, the conditional immortalization gene includes a sequence encoding SV40 largeT antigen and/or TERT. In particular embodiments, the suicide gene includes CDK1/HSV-TK/GCV. In particular embodiments, the suicide gene includes TOP2A/HSV-TK/GCV. id="p-197"

[0197] In particular embodiments, a method of preparing a feeder cell includes differentiating aconditionally immortalized stem cell into a specialized cell type. In particular embodiments, themethod further includes genetically modifying the stem cell or feeder cell to express anexpression product. In particular embodiments, the expression product is important for cellactivation and/or expansion. In particular embodiments, the expression product includes CD70, 5αCD3, CD28, 4-1BB, MICA, 4-1BBL, membrane-bound IL-15, and/or membrane-bound IL21. Inparticular embodiments, the method further includes genetically modifying the feeder cell or stemcell to knock-out MHC expression. In particular embodiments, the method further includesgenetically modifying the feeder cell or stem cell to include a suicide gene. [0198] (VI-B) Tester Cells for Research and Development. Methods and formulations described 10herein can also be used to produce immortalized cell lines for research and developmentpurposes. These immortalized cell lines are referred to herein as tester cells. In addition toimmortalized cell lines for feeder cells, it can be beneficial to have a standardized, pre-manufactured, well-characterized cell line for use in research. Cellular disease models for in vitroor in vivo use can be very helpful in assessing the success or outcomes of potential therapeutics, 15however variations in the cells used for the disease models can lead to significant variation fromexperiment to experiment. The present disclosure describes engineered cells that can be usedas tester cells. These tester cells can be differentiated into the desired cell type and can beengineered to be conditionally immortal, express desired expression products (e.g., proteins),be MHC null, and/or include a suicide gene. 20 [0199] In particular embodiments, a conditionally immortalized stem cell is differentiated into atester cell. In particular embodiments, stem cell or tester cell is genetically modified to include aa sequence encoding an expression product. In particular embodiments, a stem cell or testercell is genetically modified to include a sequence encoding an expression product; andgenetically modified to knockout MHC expression. In particular embodiments, a stem cell or 25tester cell is genetically modified to include a sequence encoding an expression product, and asuicide gene. In particular embodiments, a stem cell or tester cell is genetically modified toinclude a sequence encoding an expression product, and a suicide gene; and geneticallymodified to knockout MHC expression. [0200] In particular embodiments, the conditionally immortalized stem cell is an iPSC. In 30particular embodiments, the conditionally immortalized stem cell can be differentiated into anyuseful cell type. In particular embodiments, the conditionally immortalized stem cell isdifferentiated into a pancreatic cell (e.g., alpha, beta, and delta cell), epithelial cell, cardiac cell(e.g., cardiomyocyte), endothelial cell, liver cell (e.g., hepatocyte, hepatic stellate cell, Kupffer cell (KC), and liver sinusoidal endothelial cell (LSEC)), endocrine cell, connective tissue cell(e.g., fibroblast), muscle cell (e.g., myoblast), brain cell (e.g., neuron), bone cell (e.g., osteoblastand osteoclast), kidney cell, cartilage cell (e.g., chondrocyte), immune cell (e.g., T-cell, NK cell,or macrophage), other stem cell (e.g., mesenchymal stem cell, hematopoietic stem cell, CD34+cell, neural stem cell), or any other useful cell. In particular embodiments, the conditional 5immortalization gene includes a sequence encoding SV40 large T antigen and/or TERT. Inparticular embodiments, the expression product includes a detectable label and/or a cancerantigen. In particular embodiments, the detectable label includes fluorescent protein, aradioisotope, an enzyme label, or a fluorescent label. In particular embodiments, the fluorescentprotein includes luciferase. In particular embodiments, the cancer antigen includes BCMA, CD4, 10CD5, CD7, CD19, CD20, CD22, CD33, CD73, CD123, CD133, CD138, CD244, CD276, CS1,EGFR, EGFRVIII, EpCAM, FLT3, GD2, GPA7, GPC3, HER2, Mesothelin, MUC1, NKG2D,PSMA, PSCA, or TF. In particular embodiments, the tester cell is genetically modified toknockout MHC expression by knocking out B2M, CITA, and/or CIITA (e.g., B2M and CIITA). Inparticular embodiments, the suicide gene includes CDK1/HSV-TK/GCV, TOP2A/HSV-TK/GCV, 15or iCasp9. [0201] In particular embodiments, a method of preparing a tester cell includes differentiating aconditionally immortalized stem cell into a tester cell. A method of preparing a tester cell includesdifferentiating a conditionally immortalized stem cell into a tester cell. In particular embodiments,a method of preparing a tester cell includes differentiating a conditionally immortalized stem cell 20into a tester cell; and genetically modifying the stem cell or tester cell to encode an expressionproduct; and genetically modifying the stem cell or tester cell to knockout MHC expression. Inparticular embodiments, a method of preparing a tester cell includes differentiating aconditionally immortalized stem cell into a tester cell; and genetically modifying the stem cell ortester cell to include a sequence encoding an expression product and a suicide gene. In 25particular embodiments, a method of preparing a tester cell includes differentiating aconditionally immortalized stem cell into a tester cell; genetically modifying the stem cell or testercell to encode an expression product, and a suicide gene; and genetically modifying the testercell to knockout MHC expression. [0202] (VI-C) Conditionally Immortal Therapeutic Cell Line. Another use of the methods and 30compositions disclosed herein includes the production of immortalized therapeutic cells for usein cell therapy. Cell therapy refers to the use of cells to replace or kill damaged or diseased cells.Herein, stem cells can be differentiated into therapeutic cells and can be further engineered toexpress desirable characteristics. For example, differentiated therapeutic cells can be conditionally immortalized, express an expression product, and or include a suicide gene. [0203] Therapeutic uses of the methods and formulations disclosed herein include treatingsubjects (humans, non-human primates, veterinary animals (dogs, cats, reptiles, birds, etc.)livestock (horses, cattle, goats, pigs, chickens, etc.) and research animals (monkeys, rats, mice,fish, etc.)) with formulations disclosed herein. Treating subjects includes delivering 5therapeutically effective amounts. Therapeutically effective amounts include those that provideeffective amounts, prophylactic treatments and/or therapeutic treatments. [0204] An "effective amount" is the amount of a formulation necessary to result in a desiredphysiological change in the subject. For example, an effective amount can provide an anti-cancer,anti-infection, anti-diabetic, or healing effect. Effective amounts are often administered for 10research purposes. Effective amounts disclosed herein can cause a statistically significant effectin an animal model or in vitro assay relevant to the assessment of a disease, disorder, or injury’sdevelopment or progression. [0205] A "prophylactic treatment" includes a treatment administered to a subject who does notdisplay signs or symptoms of a disease, disorder, or injury or displays only early signs or 15symptoms of a disease, disorder, or injury such that treatment is administered for the purpose ofdiminishing or decreasing the risk of developing the disease, disorder, or injury further. Thus, aprophylactic treatment functions as a preventative treatment against a disease, disorder, or injury.In particular embodiments, prophylactic treatments reduce, delay, or prevent disease, disorder,or injury. 20 [0206] A "therapeutic treatment" includes a treatment administered to a subject who displayssymptoms or signs of a disease, disorder, or injury and is administered to the subject for thepurpose of diminishing or eliminating those signs or symptoms of the disease, disorder, or injury.The therapeutic treatment can reduce, control, or eliminate the presence or activity of the disease,disorder, or injury and/or reduce control or eliminate side effects of the disease, disorder, or injury. 25 [0207] Function as an effective amount, prophylactic treatment or therapeutic treatment are notmutually exclusive, and in particular embodiments, administered dosages may accomplish morethan one treatment type. [0208] Uses of the conditionally immortalized stem cell populations, progeny, or engineeredprogeny thereof include administration into subjects to treat a variety of pathological states 30including diseases and disorders resulting from cancers, neoplasms, injury, viral infections,diabetes and the like. Cells are introduced into a subject in need of such cells or in need of amolecule encoded or produced by the genetically altered cell. [0209] The cells of the disclosure can be used in a variety of applications. These include: transplantation or implantation of the cells either in a differentiated form, an undifferentiated form,a de-differentiated form. Such cells and tissues serve to repair, replace or augment tissue thathas been damaged due to disease or trauma, or that failed to develop normally. [0210] In one embodiment, a formulation including the cells of the disclosure is prepared forinjection directly to the site where the production of new tissue is desired. For example, the cells 5of the disclosure may be suspended in a hydrogel solution for injection. Alternatively, thehydrogel solution containing the cells may be allowed to harden, for instance in a mold to forma matrix having cells dispersed therein prior to implantation. Once the matrix has hardened, thecell formations may be cultured so that the cells are mitotically expanded prior to implantation.A hydrogel is an organic polymer (natural or synthetic) which is cross-linked via covalent, ionic, 10or hydrogen bonds to create a three-dimensional open-lattice structure, which entraps watermolecules to form a gel. Examples of materials which can be used to form a hydrogel includepolysaccharides such as alginate and salts thereof, polyphosphazines, and polyacrylates, whichare cross-linked ionically, polyethylene oxide-polypropylene glycol block copolymers which arecross-linked by temperature or pH, respectively. Methods of synthesis of the hydrogel materials, 15as well as methods for preparing such hydrogels, are known in the art. [0211] Such cell formulations may further include one or more other components, includingselected extracellular matrix components, such as one or more types of collagen known in theart, and/or growth factors and drugs. Growth factors which may be usefully incorporated into thecell formulation include one or more tissue growth factors known in the art such as: any member 20of the transforming growth factor (TGF)- β family, insulin-like growth factor (IGF)-1 and -2, growthhormone, bone morphogenetic proteins (BMPs) such as BMP-13, and the like. Alternatively, thecells of the disclosure may be genetically engineered to express and produce growth factorssuch as BMP-13 or TGF- β. Other components may also be included in the formulation include,for example, buffers to provide appropriate pH and isotonicity, lubricants, viscous materials to 25retain the cells at or near the site of administration, (e.g., alginates, agars and plant gums) andother cell types that may produce a desired effect at the site of administration (e.g., enhancementor modification of the formation of tissue or its physicochemical characteristics, support for theviability of the cells, or inhibition of inflammation or rejection). The cells can be covered by anappropriate wound covering to prevent cells from leaving the site. Such wound coverings are 30known to those of skill in the art. [0212] Alternatively, the formulations of the disclosure may be seeded onto a three-dimensionalframework or scaffold and cultured to allow the cells to differentiate, grow and fill the matrix orimmediately implanted in vivo, where the seeded cells will proliferate on the surface of the framework and form a replacement tissue in vivo in cooperation with the cells of the subject.Such a framework can be implanted in combination with any one or more growth factors, drugs,additional cell types, or other components that stimulate formation or otherwise enhance orimprove the practice of the disclosure. [0213] The cells may be introduced directly into the peripheral blood or deposited within other 5locations throughout the body, e.g., a desired tissue, or on microcarrier beads in the peritoneum. [0214] The cells of the disclosure may be used to treat subjects requiring the repair orreplacement of tissue resulting from disease or trauma. Treatment may entail the use of the cellsof the disclosure to produce new tissue, and the use of the tissue thus produced, according toany method presently known in the art or to be developed in the future. In one embodiment, 10administration includes the administration of genetically modified stem cells (e.g., iPSC). Inparticular embodiments, the administration includes the administration of differentiated stemcells. In particular embodiments, the administration includes the administration of differentiated,genetically modified stem cells. [0215] In yet another embodiment, the formulations of the disclosure can be used in conjunction 15with a three-dimensional culture system in a "bioreactor" to produce tissue constructs whichpossess critical biochemical, physical and structural properties of native human tissue byculturing the cells and resulting tissue under environmental conditions which are typicallyexperienced by native tissue. The bioreactor may include a number of designs. Typically, theculture conditions will include placing a physiological stress on the construct containing cells 20similar to what will be encountered in vivo. [0216] For example, in one embodiment, the formulation can be administered to cancer patientswho have undergone chemotherapy that have killed, reduced, or damaged stem cells or othercells of a subject, wherein the formulations replace the damaged or dead cells. Methods andcompositions can provide stem cell bioreactors for the production of a desired polypeptide or 25may be used for gene delivery or gene therapy. In this embodiment, the cell-based formulationmay be implanted or administered to a subject, or may be further differentiated to a desired celltype and implanted and delivered to the subject. [0217] Formulations (e.g., therapeutic cells) which express a gene product of interest, or tissueproduced in vitro therefrom, can be implanted into a subject who is otherwise deficient in that 30gene product. For example, genes that express products capable of preventing or amelioratingsymptoms of various types of vascular diseases or disorders, or that prevent inflammatorydisorders are of particular interest. In one embodiment, the cells of the disclosure are geneticallyengineered to express an anti-inflammatory gene product that would serve to reduce the risk of failure of implantation or further degenerative change in tissue due to inflammatory reaction. Forexample, formulations of the disclosure can be genetically engineered to express one or moreanti-inflammatory gene products including, for example, peptides or polypeptides correspondingto the idiotype of antibodies that neutralize granulocyte-macrophage colony stimulating factor(GM-CSF), tumor necrosis factor (TNF), IL-1, IL-2, or other inflammatory cytokines. IL-1 has 5been shown to decrease the synthesis of proteoglycans and collagens type II, IX, and XI (Tyleret al., 1985, Biochem. J. 227:69-878; Tyler et al., 1988, Coll. Relat. Res. 82:393-405; Goldringet al., 1988, J. Clin. Invest. 82:2026-2037; and Lefebvre et al., 1990, Biophys. Acta. 1052:366-72). TNF also inhibits synthesis of proteoglycans and type II collagen, although it is much lesspotent than IL-1 (Yaron, I., et al., 1989, Arthritis Rheum. 32:173-80; Ikebe, T., et al., 1988, J. 10Immunol. 140:827-31; and Saklatvala, J., 1986, Nature 322:547-49). Also, for example, the cellsof the disclosure may be engineered to express the gene encoding the human complementregulatory protein that prevents rejection of a graft by the host. See, for example, McCurry et al.,1995, Nature Medicine 1:423-27. [0218] It has been previously demonstrated that transplantation of beta islet cells provides 15therapy for patients with diabetes (Shapiro et al., N. Engl. J. Med. 343:230-238, 2000). Theformulations provide an alternative source of islet cells to prevent or treat diabetes. For example,stem cells of the disclosure can be generated, isolated and differentiated to a pancreatic celltype, genetically modified, and delivered to a subject. Alternatively, the conditionallyimmortalized stem cells can be genetically modified and delivered to the pancreas of the subject 20and differentiated to islet cells in vivo. Accordingly, the cells are useful for transplantation in orderto prevent or treat the occurrence of diabetes. [0219] In another embodiment, the formulations are genetically engineered to express genes forspecific types of growth factors for successful and/or improved differentiation to fibroblasts, otherstromal cells, or parenchymal cells and/or turnover either pre- or post-implantation. 25 [0220] The disclosure contemplates that the in vitro methods described herein can be used fornon-autologous transplantations. The disclosure contemplates that the in vitro methodsdescribed herein can be used for autologous transplantation of stem cells or differentiated cells.In any of the foregoing embodiments, the disclosure contemplates that stem cells can beexpanded in culture and stored for later retrieval and use. Similarly, the disclosure contemplates 30that differentiated cells can be expanded in culture and stored for later retrieval and use. [0221] For administration, therapeutically effective amounts (also referred to herein as doses) canbe initially estimated based on results from in vitro assays and/or animal model studies. Suchinformation can be used to more accurately determine useful doses in subjects of interest. The actual dose amount administered to a particular subject can be determined by a physician,veterinarian or researcher taking into account parameters such as physical and physiologicalfactors including target, body weight, severity of condition, type of disease or injury, previous orconcurrent therapeutic interventions, idiopathy of the subject and route of administration. [0222] Therapeutically effective amounts of cell-based formulations can include 10 to 10 cells/kg 5body weight, or 10 to 10 cells/kg body weight. Therapeutically effective amounts to administercan include greater than 10 cells, greater than 10 cells, greater than 10 cells, greater than 10cells, greater than 10 cells, greater than 10 cells, greater than 10 cells, greater than 10 cells,greater than 10 cells, or greater than 10. [0223] Therapeutically effective amounts can be achieved by administering single or multiple 10doses during the course of a treatment regimen (e.g., daily, every other day, every 3 days, everydays, every 5 days, every 6 days, weekly, every 2 weeks, every 3 weeks, monthly, every 2months, every 3 months, every 4 months, every 5 months, every 6 months, every 7 months, everymonths, every 9 months, every 10 months, every 11 months or yearly). In particularembodiments, the treatment protocol may be dictated by a clinical trial protocol or an FDA- 15approved treatment protocol. [0224] Therapeutically effective amounts can be administered by, e.g., injection, infusion,perfusion, or lavage. Routes of administration can include bolus intravenous, intradermal,intraarterial, intraparenteral, intranodal, intralymphatic, intraperitoneal, intralesional,intraprostatic, intravaginal, intrarectal, topical, intrathecal, intratumoral, intramuscular, 20intravesicular, and/or subcutaneous administration. [0225] In certain embodiments, formulations are administered to a patient in conjunction with(e.g., before, simultaneously or following) any number of relevant treatment modalities. Inparticular embodiments, cells may be used in combination with chemotherapy, radiation,immunosuppressive agents, such as cyclosporin, azathioprine, methotrexate, mycophenolate, 25and FK506, antibodies, or other immunoablative agents such as CAM PATH, anti-CD3 antibodiesor other antibody therapies, cytoxin, fludaribine, cyclosporin, FK506, rapamycin, mycoplienolicacid, steroids, FR901228, cytokines, and irradiation. [0226] Therapeutic cells can include any cell type that is useful in cell therapy includingpancreatic cell (e.g., alpha, beta, and delta cell), epithelial cell, cardiac cell (e.g., cardiomyocyte), 30endothelial cell, liver cell (e.g., hepatocyte, hepatic stellate cell, Kupffer cell (KC), and liversinusoidal endothelial cell (LSEC)), endocrine cell, connective tissue cell (e.g., fibroblast),muscle cell (e.g., myoblast), brain cell (e.g., neuron), bone cell (e.g., osteoblast and osteoclast),kidney cell, cartilage cell (e.g., chondrocyte), immune cell (e.g., T-cell, NK cell, or macrophage), or other stem cell (e.g., mesenchymal stem cell, hematopoietic stem cell, CD34+ cell, neuralstem cells, or any other useful cell. In particular embodiments, immune cells includes a T cell, aB cell, a natural killer (NK) cell, an NK-T cell, a monocyte/macrophage, a hematopoietic stemcells (HSC), or a hematopoietic progenitor cell (HPC). [0227] In particular embodiments, a conditionally immortalized stem cell is differentiated into a 5therapeutic cell. In particular embodiments, the stem cell or therapeutic cell is geneticallymodified to express an expression product. In particular embodiments, the expression productcan include a protein (e.g., an antigen, an antibody, a recombinant receptor, and/or a detectablelabel). In particular embodiments, the recombinant receptor includes a CAR or an eTCR. Inparticular embodiments, the detectable label is a fluorescent protein and/or luciferase. 10 [0228] In particular embodiments, a therapeutic cell is genetically modified to include a suicidegene. In particular embodiments, the suicide gene includes CDK1/HSV-TK/GCV, TOP2A/HSV-TK/GCV, or iCasp9. Although growth of the therapeutic cell can be controlled because of theconditional immortalization gene, a suicide gene provides an extra layer of safety by removingor killing the genetically modified cells. 15 [0229] In particular embodiments, a conditionally immortalized stem cell is differentiated into aT cell. In particular embodiments, the stem cell or T cell is genetically modified to express a CAR.In particular embodiments, the stem cell or T cell is genetically modified to include a suicidegene. In particular embodiments, the stem cell or T cell is genetically modified to encode asuicide gene and a CAR. 20 [0230] In particular embodiments, a conditionally immortalized stem cell is differentiated into anNK cell. In particular embodiments, the stem cell or NK cell is genetically modified to include asequence encoding an expression product. In particular embodiments, the stem cell or NK cellis genetically modified to include a suicide gene. In particular embodiments, the stem cell or NKcell is genetically modified to include a a suicide gene and a sequence encoding an expression 25product. [0231] In particular embodiments, a conditionally immortalized stem cell is differentiated into aliver cell. In particular embodiments, the stem cell or liver cell is genetically modified to includea sequence encoding an expression product. In particular embodiments, the stem cell or livercell is genetically modified to include a suicide gene. In particular embodiments, the stem cell or 30liver cell is genetically modified to include a suicide gene, and a sequence encoding anexpression product. [0232] In particular embodiments, a conditionally immortalized stem cell is differentiated into apancreatic beta cell. In particular embodiments, the stem cell or pancreatic beta cell is genetically modified to include a sequence encoding insulin. In particular embodiments, the stem cell orpancreatic beta cell is genetically modified to include a suicide gene. In particular embodiments,the stem cell or pancreatic beta cell is genetically modified to include a suicide gene, and asequence encoding insulin. [0233] The Exemplary Embodiments and Experimental Examples below are included to 5demonstrate particular embodiments of the disclosure. Those of ordinary skill in the art shouldrecognize in light of the present disclosure that many changes can be made to the specificembodiments disclosed herein and still obtain a like or similar result without departing from thespirit and scope of the disclosure. [0234] (VII) Exemplary Embodiments. 101. A stem cell including a conditional immortalization gene.2. The stem cell of embodiment 1, wherein the conditional immortalization gene encodes TERT.3. The stem cell of embodiments 1 or 2, wherein the conditional immortalization gene encodesSV40 large T antigen.4. The stem cell of any of embodiments 1-3, wherein the conditional immortalization gene 15includes TERT and SV40 large T antigen.5. The stem cell of any of embodiments 1-4, wherein the conditional immortalization gene isinduced by a drug.6. The stem cell of embodiment 5, wherein the drug includes tetracycline and/or doxycycline.7. The stem cell of any of embodiments 1-6, wherein the stem cell is a totipotent stem cell, a 20pluripotent stem cell, a multipotent stem cell, or a unipotent stem cell.8. The stem cell of any of embodiments 1-7, wherein the pluripotent stem cell is an embryonicstem cell, a cord blood stem cell, or an induced pluripotent stem cells (iPSC).9. The stem cell of any of embodiments 1-8, wherein the multipotent stem cell is a hematopoieticstem cell, a mesenchymal stem cell, or a neuronal stem cell. 2510. The stem cell of any of embodiments 1-9, further including an exogenous sequence thatencodes an expression product.11. The stem cell of embodiment 10, wherein the expression product is a protein.12. The stem cell of embodiment 11, wherein the protein includes a recombinant receptor, adetectable label, an antigen, an antibody, and/or an enzyme. 3013. The stem cell of embodiments 11 or 12, wherein the protein includes CD70, αCD3, CD28, 4-1BB, MICA, 4-1BBL, membrane-bound interleukin (IL)-15, and/or membrane-bound IL2114. The stem cell of any of embodiments 11-13, wherein the protein includes membrane-boundIL21.

. The stem cell of any of embodiments 11-14, wherein the protein includes a cancer antigen.16. The stem cell of embodiment 15, wherein the cancer includes multiple myeloma, lymphoma,acute lymphocytic leukemia (ALL), acute myelocytic leukemia (AML), chronic lymphocyticleukemia (CLL), breast cancer, colorectal cancer, ovarian cancer, renal cell carcinoma (RCC),glioblastoma, prostate cancer, neuroblastoma, melanoma, Ewing sarcoma, and/or 5hepatocellular cancer (HCC).17. The stem cell of embodiments 15 or 16, wherein the cancer antigen includes BCMA, CD4,CD5, CD7, CD19, CD20, CD22, CD33, CD73, CD123, CD133, CD138, CD244, CD276, CS1,EGFR, EGFRVIII, EpCAM, FLT3, GD2, GPA7, GPC3, HER2, Mesothelin, MUC1, NKG2D,PSMA, PSCA, and/or TF. 1018. The stem cell of any of embodiments 15-17, wherein the cancer antigen includes BCMA,CD19, CD20, CD33, CD133, CD138, CS1, EGFR, EGFRVII, EpCAM, GD2, GPA7, HER2,NKG2D, MUC1, and/or PSCA.19. The stem cell of any of embodiments 11-18, wherein the protein includes a viral, bacterial,fungal, and/or parasitic antigen. 1520. The stem cell of any of embodiments 11-19, wherein the protein includes insulin, factor VIII,factor IX, factor XI, alpha-1 antitrypsin (A1AT), glucocerebrosidase (GC), acidsphingomyelinase, mucopolysaccharides, acid alpha-glucosidase, aspartylglucosaminidase,alpha-galactosidase A, palmitoyl protein thioesterase, tripeptidyl peptidase, lysosomaltransmembrane protein, cysteine transporter, acid ceramidase, acid alpha-L-fucosidase, 20cathepsin A, acid beta-glucosidase, acid beta-galactosidase, iduronate-2-sulfatase, alpha-L-iduronidase, galactocerebrosidase, acid alpha-mannosidase, acid beta-mannosidase,arylsulfatase B, arylsulfatase A, N-acetylgalactosamine-6-sulfate, N-acetylglucosamine-1-phosphotransferase, acid sphingomyelinase, NPC-1, alpha-glucosidase, beta-hexosaminidase B, heparan N-sulfatase, alpha-N-acetylglucosaminidase, acetyl-CoA: alpha- 25glucosaminide, N-acetylglucosamine-6-sulfate, alpha-N-acetylgalactosaminidase, alpha-neuramidase, beta-glucuronidase, beta-hexosaminidase A, and/or acid lipase.21. The stem cell of any of embodiments 12-20, wherein the recombinant receptor includes anextracellular component including a binding domain; an intracellular component including aneffector domain; and a transmembrane domain linking the extracellular component to the 30intracellular component.22. The stem cell of any of embodiments 12-21, wherein the recombinant receptor includes achimeric antigen receptor and/or an engineered T cell receptor.23. The stem cell of embodiments 21 or 22, wherein the binding domain of the recombinant receptor binds a cancer antigen, a viral antigen, a bacterial antigen, and/or a fungal antigen.24. The stem cell of embodiment 23, wherein the cancer antigen includes BCMA, CD4, CD5,CD7, CD19, CD20, CD22, CD33, CD73, CD123, CD133, CD138, CD244, CD276, CS1,EGFR, EGFRVIII, EpCAM, FLT3, GD2, GPA7, GPC3, HER2, Mesothelin, MUC1, NKG2D,PSMA, PSCA, and/or TF. 525. The stem cell of embodiments 23 or 24, wherein the cancer antigen includes BCMA, CD19,CD20, CD33, CD133, CD138, CS1, EGFR, EGFRVII, EpCAM, GD2, GPA7, HER2, NKG2D,MUC1, and/or PSCA.26. The stem cell of any of embodiments 21-25, wherein the effector domain includes all or aportion of the signaling domain of CD3 ζ and/or 4-1BB. 1027. The stem cell of any of embodiments 21-26, wherein the transmembrane domain includes aCD28 transmembrane domain.28. The stem cell of any of embodiments 12-27, wherein the recombinant receptor includes aCD19 binding domain.29. The stem cell of any of embodiments 12-28, wherein the recombinant receptor includes a 15BCMA binding domain.30. The stem cell of any of embodiments 12-29, wherein the detectable label includes afluorescent protein, a radioisotope, an enzyme label, and/or a fluorescent label.31. The stem cell of embodiment 30, wherein the fluorescent protein includes luciferase.32. The stem cell of any of embodiments 1-31, wherein the stem cell is genetically modified to 20knockout a major histocompatibility complex (MHC).33. The stem cell of any of embodiments 1-32, wherein the stem cell is genetically modified toknockout β2-microglobulin (B2M).34. The stem cell of any of embodiments 1-33, wherein the stem cell is genetically modified toknockout Class I Major Histocompatibility Complex Transactivator and/or Class II Major 25Histocompatibility Complex Transactivator.35. The stem cell of any of embodiments 1-34, wherein the stem cell is genetically modified toknockout B2M, CIITA, or B2M and CIITA.36. The stem cell of embodiments 33 or 35, wherein B2M is knocked out with the gRNA sequenceas set forth in SEQ ID NOs: 34-42. 3037. The stem cell of embodiments 34 or 35, wherein CIITA is knocked out with the gRNAsequence as set forth in SEQ ID NOs: 25-33.38. The stem cell of any of embodiments 1-37, wherein the stem cell further includes a suicidegene. 39. The stem cell of embodiment 38, wherein the suicide gene includes CDK1 linked Herpessimplex virus-thymidine kinase/ganciclovir (CDK1/HSV-TK/GCV), TOP2A/HSV-TK/GCV,and/or inducible Casp9.40. The stem cell of embodiments 38 or 39, wherein the suicide gene includes CDK1/HSV-TK/GCV. 541. The stem cell of any of embodiments 1-40, wherein the stem cell further includes a sequenceencoding a tag cassette, a transduction marker, selection cassette, and/or a skipping element.42. A cell line differentiated from the stem cell of any of embodiments 1-41.43. The cell line of embodiment 42, wherein the cell line includes more differentiated stem cellsthan the stem cell of any of embodiments 1-41. 1044. The cell line of embodiment 43, wherein the more differentiated stem cells are CD34+hematopoietic stem cells or mesenchymal stem cells.45. The cell line of any of embodiments 42-44, wherein the cell line includes pancreatic cells,epithelial cells, cardiac cells, endothelial cells, liver cells, endocrine cells, connective tissuecells, muscle cells, brain cells, bone cells, kidney cells, cartilage cells, or immune cells. 1546. The cell line of embodiment 45, wherein the pancreatic cells include alpha cells, beta cells, ordelta cells.47. The cell line of embodiments 45 or 46, wherein the cardiac cells include cardiomyocytes.48. The cell line of any of embodiments 45-47, wherein the liver cells include hepatocytes, hepaticstellate cells (HSCs), Kupffer cells (KCs), or liver sinusoidal endothelial cells (LSECs). 2049. The cell line of any of embodiments 45-48, wherein the connective tissue cells includefibroblasts.50. The cell line of any of embodiments 45-49, wherein the muscle cells include myoblasts.51. The cell line of any of embodiments 45-50, wherein the brain cells include neurons.52. The cell line of any of embodiments 45-51, wherein the bone cells include osteoblasts or 25osteoclasts.53. The cell line of any of embodiments 45-52, wherein the cartilage cells include chondrocytes.54. The cell line of any of embodiments 45-53, wherein the immune cells include T-cells, NK cells,or macrophages.55. The cell line of any of embodiments 42-54, wherein cells within the cell line are genetically 30modified to express an expression product.56. The cell line of embodiment 55, wherein the expression product is a protein.57. The cell line of embodiment 56, wherein the protein includes a recombinant receptor, adetectable label, an antigen, an antibody, and/or an enzyme. 58. The cell line of embodiments 56 or 57, wherein the protein includes CD70, αCD3, CD28, 4-1BB, MICA, 4-1BBL, membrane-bound interleukin (IL)-15, and/or membrane-bound IL2159. The cell line of any of embodiments 56-58, wherein the protein includes membrane-boundIL21.60. The cell line of any of embodiments 56-59, wherein the protein includes a cancer antigen. 561. The cell line of embodiment 60, wherein the cancer includes multiple myeloma, lymphoma,acute lymphocytic leukemia (ALL), acute myelocytic leukemia (AML), chronic lymphocyticleukemia (CLL), breast cancer, colorectal cancer, ovarian cancer, renal cell carcinoma (RCC),glioblastoma, prostate cancer, neuroblastoma, melanoma, Ewing sarcoma, and/orhepatocellular cancer (HCC). 1062. The cell line of embodiments 60 or 61, wherein the cancer antigen includes BCMA, CD4, CD5,CD7, CD19, CD20, CD22, CD33, CD73, CD123, CD133, CD138, CD244, CD276, CS1,EGFR, EGFRVIII, EpCAM, FLT3, GD2, GPA7, GPC3, HER2, Mesothelin, MUC1, NKG2D,PSMA, PSCA, and/or TF.63. The cell line of any of embodiments 60-62, wherein the cancer antigen includes BCMA, CD19, 15CD20, CD33, CD133, CD138, CS1, EGFR, EGFRVII, EpCAM, GD2, GPA7, HER2, NKG2D,MUC1, and/or PSCA.64. The cell line of any of embodiments 56-63, wherein the protein includes a viral, bacterial,fungal, and/or parasitic antigen.65. The cell line of any of embodiments 56-64, wherein the protein includes insulin, factor VIII, 20factor IX, factor XI, alpha-1 antitrypsin (A1AT), glucocerebrosidase (GC), acidsphingomyelinase, mucopolysaccharides, acid alpha-glucosidase, aspartylglucosaminidase,alpha-galactosidase A, palmitoyl protein thioesterase, tripeptidyl peptidase, lysosomaltransmembrane protein, cysteine transporter, acid ceramidase, acid alpha-L-fucosidase,cathepsin A, acid beta-glucosidase, acid beta-galactosidase, iduronate-2-sulfatase, alpha-L- 25iduronidase, galactocerebrosidase, acid alpha-mannosidase, acid beta-mannosidase,arylsulfatase B, arylsulfatase A, N-acetylgalactosamine-6-sulfate, N-acetylglucosamine-1-phosphotransferase, acid sphingomyelinase, NPC-1, alpha-glucosidase, beta-hexosaminidase B, heparan N-sulfatase, alpha-N-acetylglucosaminidase, acetyl-CoA: alpha-glucosaminide, N-acetylglucosamine-6-sulfate, alpha-N-acetylgalactosaminidase, alpha- 30neuramidase, beta-glucuronidase, beta-hexosaminidase A, and/or acid lipase.66. The cell line of any of embodiments 57-65, wherein the recombinant receptor includes anextracellular component including a binding domain; an intracellular component including aneffector domain; and a transmembrane domain linking the extracellular component to the intracellular component.67. The cell line of any of embodiments 57-66, wherein the recombinant receptor includes achimeric antigen receptor and/or an engineered T cell receptor.68. The cell line of embodiments 66 or 67, wherein the binding domain of the recombinantreceptor binds a cancer antigen, a viral antigen, a bacterial antigen, and/or a fungal antigen. 569. The cell line of embodiment 68, wherein the cancer antigen includes BCMA, CD4, CD5, CD7,CD19, CD20, CD22, CD33, CD73, CD123, CD133, CD138, CD244, CD276, CS1, EGFR,EGFRVIII, EpCAM, FLT3, GD2, GPA7, GPC3, HER2, Mesothelin, MUC1, NKG2D, PSMA,PSCA, and/or TF.70. The cell line of embodiments 68 or 69, wherein the cancer antigen includes BCMA, CD19, 10CD20, CD33, CD133, CD138, CS1, EGFR, EGFRVII, EpCAM, GD2, GPA7, HER2, NKG2D,MUC1, and/or PSCA.71. The cell line of any of embodiments 66-70, wherein the effector domain includes all or aportion of the signaling domain of CD3 ζ and/or 4-1BB.72. The cell line of any of embodiments 66-71, wherein the transmembrane domain includes a 15CD28 transmembrane domain.73. The cell line of any of embodiments 57-72, wherein the recombinant receptor includes a CD19binding domain.74. The cell line of any of embodiments 57-73, wherein the recombinant receptor includes aBCMA binding domain. 2075. The cell line of any of embodiments 57-74, wherein the detectable label includes a fluorescentprotein, a radioisotope, an enzyme label, and/or a fluorescent label.76. The cell line of embodiment 75, wherein the fluorescent protein includes luciferase.77. The cell line of any of embodiments 42-76, wherein cells within the cell line are geneticallymodified to knockout a major histocompatibility complex (MHC). 2578. The cell line of any of embodiments 42-77, wherein cells within the cell line are geneticallymodified to knockout β2-microglobulin (B2M).79. The cell line of any of embodiments 42-78, wherein cells within the cell line are geneticallymodified to knockout Class I Major Histocompatibility Complex Transactivator and/or Class IIMajor Histocompatibility Complex Transactivator. 3080. The cell line of any of embodiments 42-79, wherein cells within the cell line are geneticallymodified to B2M, CIITA, or B2M and CIITA.81. The cell line of any of embodiments 78 - 80, wherein B2M is knocked out with the gRNAsequence as set forth in SEQ ID NOs: 34-42. 82. The cell line of embodiments 79 or 80, wherein CIITA is knocked out with the gRNA sequenceas set forth in SEQ ID NOs: 25-33.83. The cell line of any of embodiments 42-82, wherein cells within the cell line further include asuicide gene.84. The cell line of embodiment 83, wherein the suicide gene includes CDK1 linked Herpes 5simplex virus-thymidine kinase/ganciclovir (CDK1/HSV-TK/GCV), TOP2A/HSV-TK/GCV,and/or inducible Casp9.85. The cell line of embodiments 83 or 84, wherein the suicide gene includes CDK1/HSV-TK/GCV.86. A method including genetically modifying a stem cell to include a conditional immortalization 10gene.87. The method of embodiment 86, wherein the genetically modifying includes transfecting a stemcell with the conditional immortalization gene using the Tet inducible system.88. The method of embodiments 86 or 87, wherein the conditional immortalization gene encodesTERT. 1589. The method of any of embodiments 86-88, wherein the conditional immortalization geneencodes SV40 large T antigen.90. The method of any of embodiments 86-89, wherein the conditional immortalization geneincludes TERT and SV40 large T antigen.91. The method of any of embodiments 86-90, wherein the conditional immortalization gene is 20induced by a drug.92. The method of embodiment 91, wherein the drug includes tetracycline and/or doxycycline.93. The method of any of embodiments 86-92, wherein the stem cell is a totipotent stem cell, apluripotent stem cell, a multipotent stem cell, or a unipotent stem cell.94. The method of embodiment 93, wherein the pluripotent stem cell is an embryonic stem cell, a 25cord blood stem cell, or an induced pluripotent stem cells (iPSC).95. The method of embodiments 93 or 94, wherein the multipotent stem cell is a hematopoieticstem cell, a mesenchymal stem cell, or a neuronal stem cell.96. The method of any of embodiments 86-95, further genetically modifying the stem cell toinclude an exogenous sequence that encodes an expression product. 3097. The method of embodiment 96, wherein the genetically modifying the stem cell to include anexogenous sequence includes transfecting the stem cell with an expression construct using atransposon-based system and/or a lentivirus system.98. The method of embodiments 96 or 97, wherein the genetically modifying the stem cell to include an exogenous sequence includes transfecting the stem cell with an expressionconstruct using a transposon-based system.99. The method of any of embodiments 96-98, wherein the expression product is a protein.100. The method of embodiment 99, wherein the protein includes a recombinant receptor, adetectable label, an antigen, an antibody, and/or an enzyme. 5101. The method of embodiment 99 or 100, wherein the protein includes CD70, αCD3, CD28,4-1BB, MICA, 4-1BBL, membrane-bound interleukin (IL)-15, and/or membrane-bound IL21102. The method of any of embodiments 99-101, wherein the protein includes membrane-bound IL21.103. The method of any of embodiments 99-102, wherein the protein includes a cancer antigen. 10104. The method of embodiment 103, wherein the cancer includes multiple myeloma,lymphoma, acute lymphocytic leukemia (ALL), acute myelocytic leukemia (AML), chroniclymphocytic leukemia (CLL), breast cancer, colorectal cancer, ovarian cancer, renal cellcarcinoma (RCC), glioblastoma, prostate cancer, neuroblastoma, melanoma, Ewing sarcoma,and/or hepatocellular cancer (HCC). 15105. The method of embodiments 103 or 104, wherein the cancer antigen includes BCMA,CD4, CD5, CD7, CD19, CD20, CD22, CD33, CD73, CD123, CD133, CD138, CD244, CD276,CS1, EGFR, EGFRVIII, EpCAM, FLT3, GD2, GPA7, GPC3, HER2, Mesothelin, MUC1,NKG2D, PSMA, PSCA, and/or TF.106. The method of any of embodiments 103-105, wherein the cancer antigen includes BCMA, 20CD19, CD20, CD33, CD133, CD138, CS1, EGFR, EGFRVII, EpCAM, GD2, GPA7, HER2,NKG2D, MUC1, and/or PSCA.107. The method of any of embodiments 99-106, wherein the protein includes a viral, bacterial,fungal, and/or parasitic antigen.108. The method of any of embodiments 99-107, wherein the protein includes insulin, factor 25VIII, factor IX, factor XI, alpha-1 antitrypsin (A1AT), glucocerebrosidase (GC), acidsphingomyelinase, mucopolysaccharides, acid alpha-glucosidase, aspartylglucosaminidase,alpha-galactosidase A, palmitoyl protein thioesterase, tripeptidyl peptidase, lysosomaltransmembrane protein, cysteine transporter, acid ceramidase, acid alpha-L-fucosidase,cathepsin A, acid beta-glucosidase, acid beta-galactosidase, iduronate-2-sulfatase, alpha-L- 30iduronidase, galactocerebrosidase, acid alpha-mannosidase, acid beta-mannosidase,arylsulfatase B, arylsulfatase A, N-acetylgalactosamine-6-sulfate, N-acetylglucosamine-1-phosphotransferase, acid sphingomyelinase, NPC-1, alpha-glucosidase, beta-hexosaminidase B, heparan N-sulfatase, alpha-N-acetylglucosaminidase, acetyl-CoA: alpha- glucosaminide, N-acetylglucosamine-6-sulfate, alpha-N-acetylgalactosaminidase, alpha-neuramidase, beta-glucuronidase, beta-hexosaminidase A, and/or acid lipase.109. The method of any of embodiments 100-108, wherein the recombinant receptor includesan extracellular component including a binding domain; an intracellular component includingan effector domain; and a transmembrane domain linking the extracellular component to the 5intracellular component.110. The method of any of embodiments 100-109, wherein the recombinant receptor includesa chimeric antigen receptor and/or an engineered T cell receptor.111. The method of embodiments 109 or 110, wherein the binding domain of the recombinantreceptor binds a cancer antigen, a viral antigen, a bacterial antigen, and/or a fungal antigen. 10112. The method of embodiment 111, wherein the cancer antigen includes BCMA, CD4, CD5,CD7, CD19, CD20, CD22, CD33, CD73, CD123, CD133, CD138, CD244, CD276, CS1,EGFR, EGFRVIII, EpCAM, FLT3, GD2, GPA7, GPC3, HER2, Mesothelin, MUC1, NKG2D,PSMA, PSCA, and/or TF.113. The method of embodiments 111 or 112, wherein the cancer antigen includes BCMA, 15CD19, CD20, CD33, CD133, CD138, CS1, EGFR, EGFRVII, EpCAM, GD2, GPA7, HER2,NKG2D, MUC1, and/or PSCA.114. The method of any of embodiments 109-113, wherein the effector domain includes all ora portion of the signaling domain of CD3 ζ and/or 4-1BB.115. The method of any of embodiments 109-114, wherein the transmembrane domain 20includes a CD28 transmembrane domain.116. The method of any of embodiments 100-115, wherein the recombinant receptor includesa CD19 binding domain.117. The method of any of embodiments 100-116, wherein the recombinant receptor includesa BCMA binding domain. 25118. The method of any of embodiments 100-117, wherein the detectable label includes afluorescent protein, a radioisotope, an enzyme label, and/or a fluorescent label.119. The method of embodiment 118, wherein the fluorescent protein includes luciferase.120. The method of any of embodiments 86-119, wherein the stem cells are geneticallymodified to knockout a major histocompatibility complex (MHC). 30121. The method of any of embodiments 86-120, wherein the stem cells are geneticallymodified to knockout β2-microglobulin (B2M).122. The method of any of embodiments 86-121, wherein the stem cells are geneticallymodified to knockout Class I Major Histocompatibility Complex Transactivator and/or Class II Major Histocompatibility Complex Transactivator.123. The method of any of embodiments 86-122, wherein the stem cells are geneticallymodified to knockout B2M, CIITA, or B2M and CIITA.124. The method of any of embodiments 121 - 123, wherein B2M is knocked out with the gRNAsequence as set forth in SEQ ID NOs: 34-42. 5125. The method of embodiments 122 or 123, wherein CIITA is knocked out with the gRNAsequence as set forth in SEQ ID NOs: 25-33.126. The method of any of embodiments 86-125, wherein the stem cells are further geneticallymodified to include a suicide gene.127. The method of embodiment 126, wherein the suicide gene includes CDK1 linked Herpes 10simplex virus-thymidine kinase/ganciclovir (CDK1/HSV-TK/GCV), TOP2A/HSV-TK/GCV,and/or inducible Casp9.128. The method of embodiments 126 or 127, wherein the suicide gene includes CDK1/HSV-TK/GCV.129. A method including differentiating a stem cell of any of embodiments 1-41 into a more 15differentiated cell type.130. The method of embodiment 129, wherein the more differentiated stem cells include CD34+hematopoietic stem cells, mesenchymal stem cells, or neural stem cells.131. The method of embodiments 129 or 130, wherein the more differentiated cell type includesmore differentiated stem cells, pancreatic cells, epithelial cells, cardiac cells, endothelial cells, 20liver cells, endocrine cells, connective tissue cells, muscle cells, brain cells, bone cells, kidneycells, cartilage cells, cancer cells, or immune cells.132. The method of embodiment 131, wherein the pancreatic cells include alpha cells, betacells, or delta cells.133. The method of embodiments 131 or 132, wherein the cardiac cells include cardiomyocytes 25134. The method of any of embodiments 131-133, wherein the liver cells include hepatocytes,hepatic stellate cells (HSCs), Kupffer cells (KCs), and liver sinusoidal endothelial cells(LSECs).135. The method of any of embodiments 131-134, wherein the connective tissue cells includefibroblasts. 30136. The method of any of embodiments 131-135, wherein the muscle cells include myoblasts.137. The method of any of embodiments 131-136, wherein the brain cells include neurons.138. The method of any of embodiments 131-137, wherein the bone cells include osteoblastsand osteoclasts. 139. The method of any of embodiments 131-138, wherein the cartilage cells includechondrocytes.140. The method of any of embodiments 131-139, wherein the immune cells include T-cells,NK cells, or macrophages.141. The method of any of embodiments 129-140, further including genetically modifying the 5more differentiated cell type to include an exogenous sequence that encodes an expressionproduct.142. The method of embodiment 141, wherein the expression product is a protein.143. The method of embodiment 142, wherein the protein includes a recombinant receptor, adetectable label, an antigen, an antibody, and/or an enzyme. 10144. The method of embodiments 142 or 143, wherein the protein includes CD70, αCD3, CD28,4-1BB, MICA, 4-1BBL, membrane-bound interleukin (IL)-15, and/or membrane-bound IL21.145. The method of any of embodiments 142-144, wherein the protein includes membrane-bound IL21.146. The method of any of embodiments 142-145, wherein the protein includes a cancer 15antigen.147. The method of embodiment 146, wherein the cancer includes multiple myeloma,lymphoma, acute lymphocytic leukemia (ALL), acute myelocytic leukemia (AML), chroniclymphocytic leukemia (CLL), breast cancer, colorectal cancer, ovarian cancer, renal cellcarcinoma (RCC), glioblastoma, prostate cancer, neuroblastoma, melanoma, Ewing sarcoma, 20and/or hepatocellular cancer (HCC).148. The method of embodiments 146 or 147, wherein the cancer antigen includes BCMA,CD4, CD5, CD7, CD19, CD20, CD22, CD33, CD73, CD123, CD133, CD138, CD244, CD276,CS1, EGFR, EGFRVIII, EpCAM, FLT3, GD2, GPA7, GPC3, HER2, Mesothelin, MUC1,NKG2D, PSMA, PSCA, and/or TF. 25149. The method of any of embodiments 146-148, wherein the cancer antigen includes BCMA,CD19, CD20, CD33, CD133, CD138, CS1, EGFR, EGFRVII, EpCAM, GD2, GPA7, HER2,NKG2D, MUC1, and/or PSCA.150. The method of any of embodiments 142-150, wherein the protein includes a viral,bacterial, fungal, and/or parasitic antigen. 30151. The method of any of embodiments 142-151, wherein the protein includes insulin, factorVIII, factor IX, factor XI, alpha-1 antitrypsin (A1AT), glucocerebrosidase (GC), acidsphingomyelinase, mucopolysaccharides, acid alpha-glucosidase, aspartylglucosaminidase,alpha-galactosidase A, palmitoyl protein thioesterase, tripeptidyl peptidase, lysosomal transmembrane protein, cysteine transporter, acid ceramidase, acid alpha-L-fucosidase,cathepsin A, acid beta-glucosidase, acid beta-galactosidase, iduronate-2-sulfatase, alpha-L-iduronidase, galactocerebrosidase, acid alpha-mannosidase, acid beta-mannosidase,arylsulfatase B, arylsulfatase A, N-acetylgalactosamine-6-sulfate, N-acetylglucosamine-1-phosphotransferase, acid sphingomyelinase, NPC-1, alpha-glucosidase, beta- 5hexosaminidase B, heparan N-sulfatase, alpha-N-acetylglucosaminidase, acetyl-CoA: alpha-glucosaminide, N-acetylglucosamine-6-sulfate, alpha-N-acetylgalactosaminidase, alpha-neuramidase, beta-glucuronidase, beta-hexosaminidase A, and/or acid lipase.152. The method of any of embodiments 143-151, wherein the recombinant receptor includesan extracellular component including a binding domain; an intracellular component including 10an effector domain; and a transmembrane domain linking the extracellular component to theintracellular component.153. The method of embodiment 143-152, wherein the recombinant receptor includes achimeric antigen receptor and/or an engineered T cell receptor.154. The method of embodiments 152 or 153, wherein the binding domain of the recombinant 15receptor binds a cancer antigen, a viral antigen, a bacterial antigen, and/or a fungal antigen.155. The method of embodiment 154, wherein the cancer antigen includes BCMA, CD4, CD5,CD7, CD19, CD20, CD22, CD33, CD73, CD123, CD133, CD138, CD244, CD276, CS1,EGFR, EGFRVIII, EpCAM, FLT3, GD2, GPA7, GPC3, HER2, Mesothelin, MUC1, NKG2D,PSMA, PSCA, and/or TF. 20156. The method of embodiments 154 or 155, wherein the cancer antigen includes BCMA,CD19, CD20, CD33, CD133, CD138, CS1, EGFR, EGFRVII, EpCAM, GD2, GPA7, HER2,NKG2D, MUC1, and/or PSCA.157. The method of any of embodiments 152-156, wherein the effector domain includes all ora portion of the signaling domain of CD3 ζ and/or 4-1BB. 25158. The method of any of embodiments 152-157, wherein the transmembrane domainincludes a CD28 transmembrane domain.159. The method of any of embodiments 143-158, wherein the recombinant receptor includesa CD19 binding domain.160. The method of any of embodiments 143-159, wherein the recombinant receptor includes 30a BCMA binding domain.161. The method of any of embodiments 143-160, wherein the detectable label includes afluorescent protein, a radioisotope, an enzyme label, and/or a fluorescent label.162. The method of embodiment 161, wherein the fluorescent protein includes luciferase. 163. The method of any of embodiments 129-162, further including genetically modifying themore differentiated cell type to knockout major histocompatibility complex (MHC).164. The method of embodiment 163, wherein the genetically modifying the more differentiatedcell type to knockout MHC includes knocking out B2M and/or CIITA.165. The method of embodiment 164, wherein the knocking out B2M and/or CIITA includes 5delivering the Cas9 nuclease, B2M gRNA, and CIITA gRNA to feeder cells.166. The method of embodiment 165, wherein the B2M gRNA includes SEQ ID NOs: 34-42.167. The method of embodiments 165 or 166, wherein the CIITA gRNA includes SEQ ID NOs:25-33.168. The method of any of embodiments 129-167, further including genetically modifying the 10more differentiated cell type to include a suicide gene.169. The method of embodiment 168, wherein the suicide gene includes CDK1 linked Herpessimplex virus-thymidine kinase/ganciclovir (CDK1/HSV-TK/GCV), TOP2A/HSV-TK/GCV,and/or inducible Casp9. [0235] (VIII) Experimental Examples. 15 [0236] Example 1. [0237] Materials and Methods. Culture of induced pluripotent stem cells (iPSCs) – thawing,passage, cryopreservation. Non-genetically modified and genetically modified, includingimmortalized, iPSC lines were thawed, cultured and cryopreserved according to protocolsdeveloped by Stemcell Technologies (Stemcell Technologies 100-0276). Frozen vials were 20thawed quickly at 37°C, gently resuspended in mTeSR-plus (Stemcell Technologies 100-0276),centrifuged, resuspended in pre-warmed mTeSR-Plus supplemented with Y-27632 (StemcellTechnologies 72302), and plated on cell-culture plastic dishes coated with Geltrex (Thermo FisherA1569601). Cultures were passages 1:5 – 1:10 every 4-6 days depending on cell growth, at anaverage of 75% confluency. Cultures were washed once with PBS and covered with ReLeSR 25(Stemcell Technologies 05872) for 1 minute. The ReLeSR was removed, the plates incubated for2-4 minutes at 37°C, gently resuspended in mTeSR-Plus and added to GelTrex treated culturedishes. For cryo-preservation, cells were dissociated with ReLeaSR as described above,centrifuged and resuspended in CryoStore CS10 (Stemcell Technologies 07959). Vials werefrozen at a rate on 1°C/min until reaching -80°C after which they were transferred to a -150°C 30freezer for long term storage. [0238] Differentiation of iPSC to CD34-positive hematopoietic progenitor cells andcharacterization of CD34-positive hematopoietic progenitor cells. Control unmodified iPSCs andTetON hTERT SV40 IPSCs were differentiated to CD34-positive hematopoietic progenitor cells using the StemDiff Hematopoietic Medium and Supplements (StemCell Technologies). For theTetON hTERT SV40 IPSC lines the iPSC culture and differentiation medium were supplementedwith 0.1 µM Doxycycline Hyclate (DOX, Sigma Aldrich). For the differentiation, adherent iPSCcultures were dissociated to single cells using Accutase (StemCell Technologies) and plated intoAggreWell 6-well plates (StemCell Technologies) at 3.5x10^6 cells/well to generate Embryoid 5Bodies (EBs). After 5 days of culture the EBs were transferred to non-tissue culture treated 6-wellplates and cultured for additional 7 days. On day 12 of differentiation, EBs were harvested anddissociated into single cells using Collagenase II (StemCell Technologies). CD34-positive cellswere isolated from the single cell suspension using the EasySep Human CD34 Positive SelectionKit (StemCell Technologies) per the manufacturer’s instructions. Freshly isolated CD34-positive 10cells were stained with anti-human CD34, CD45 and CD43 antibodies (Stem Cell Technologies)and analyzed via Flow Cytometry for surface marker expression using a CytoFlex flow cytometer(Beckman Coulter). [0239] Differentiation of iPSC to natural killer (NK) cells and characterization of NK cells. Controlunmodified iPSCs and TetON hTERT SV40 IPSCs were differentiated to NK cells using the 15StemDiff NK Cell Kit (StemCell Technologies) following a 3-step differentiation protocol per themanufacturer’s instructions: iPSC differentiation to CD34-positive hematopoietic progenitor cellsand selection of CD34-positive cells (12 days), differentiation of CD34-positive cells to CD5-positive and CD7-positive lymphoid progenitor cells (14 days), and differentiation of the lymphoidprogenitor cells to CD56-positive cells (14 days). For the TetON hTERT SV40 IPSC lines the 20iPSC culture and differentiation medium were supplemented with 0.1 µM Doxycycline Hyclate(DOX, Sigma Aldrich). In some instances, the Doxycylcine Hyclate differentiation mediumsupplementation was performed at either the lymphoid progenitor differentiation stage and/or NKdifferentiation stage. At each stage of the differentiation, cells were harvested and characterizedfor surface marker expression via antibody staining (anti-human CD34, CD45 and CD43 25antibodies (Stem Cell Technologies) for CD34-positive cells, anti-human CD7 antibody(BioLegend) and anti-human CD5 antibody (StemCell Technologies) for lymphoid progenitorcells, anti-human CD56, CD16 and CD3 antibodies (StemCell Technologies) for NK cells),followed by flow cytometry analysis (CytoFlex flow cytometer, Beckman Coulter). [0240] Transposon-based transgenes delivery in iPSC lines using Lipofectamine 3000. 30Mammalian Expression plasmid of PiggyBac or Sleeping beauty transposases and transposonvectors of transgenes were designed in-house and synthesized by VectorBuilder. For genedelivery, iPSCs were cultured in Geltrex (Thermo fisher) coated culture wares with iPSC growthmedia (mTeSR plus, StemCell Technologies) for at least 2 passages before transfection. On the day of the transfection, adherent iPSC cultures were dissociated to very small clamps usingReLeSR (StemCell Technologies) and plated into Geltrex-coated 6-well at 500,000 cells/ wells.The plasmids (transgene + transposase) and transfection reagents mix were prepared accordingto manufacture instructions (Lipofectamine 3000, Thermo Fisher). 2 hours after plating,plasmids/reagent mix was added to the corresponding well in the 6-well plate. For clones that 5were generated by the antibiotic selection, the antibiotics were introduced 24 hours (Neomycin)or 48 hours (Puromycin) after transfection. Antibiotic-resistant clones were collected 6-7 days(Puromycin), and 10 days (Neomycin) after the initial selection for downstream analysis. Antibioticresistant clones that were pooled from each experiment and pooled cells were cultured with iPSCgrowth media for 72 hours. Then transfected cells were collected, stained with antibodies (anti- 10FMC63 CAR, BioLegend), and single cells sorted into 96-well plates using a cell sorter (CytoFlexflow cytometer, Beckman Coulter) to generate clonal lines. [0241] Transgene copy number and expression level analysis. For genomic copy numberanalysis, genomic DNA from transgenic iPSC lines was extracted from cell pellets using DNeasyBlood & Tissue Kits (Qiagen). The transgene copy number in the transgenic iPSC line was 15measured using iCS-digital™ PSC kit. (Stemgenomics) Primer and probes used for ddPCR:Forward primer: 5’- GCTGCCGATTTCCAGAAGAAGA-3’ (SEQ ID NO: 55; Reverse primer: 5’-GTCTGCGCTCCTGCTGAA-3’ (SEQ ID NO: 56); probe: AAGGAGGATGTGAACTGA (SEQ IDNO: 57).For analysis of transgene transcript level by qPCR, the RNA from transgenic iPSC lines was 20extracted from cell pellets using RNeasy Mini Kit (Qiagen). cDNA was synthesized from extractedRNA using QuantiTect Reverse Transcription Kit (Qiagen). qPCR was set up using SensiFastSYBR No-Rox kit (FroggaBio BIO-98020) performed on CFX384 Touch Real-Time PCRDetection System. (BioRad). qPCR primer used:FMC63: Forward primer: 5’- TGGAGTGGCTGGGAGTAATA-3’ (SEQ ID NO: 58); Reverse primer: 255’- ACTTGGCTCTTGGAGTTGTC-3’ (SEQ ID NO: 59).TK007: Forward primer: 5’- CAACATCTACACCACACAGCAC-3’ (SEQ ID NO: 60); Reverseprimer: 5’- CGGCATTCCCATTGTGATCTGG-3’ (SEQ ID NO: 61).YWHAZ: Forward primer: 5’- ACTTTTGGTACATTGTGGCTTCAA-3’ (SEQ ID NO: 62); Reverseprimer: 5’- CCGCCAGGACAAACCAGTAT-3’ (SEQ ID NO: 63). 30hTERT: Forward primer: 5’-CTCCATCCTGAAAGCCAAGAA-3’ (SEQ ID NO: 64); Reverseprimer: 5’- AGTCAGCTTGAGCAGGAATG-3’ (SEQ ID NO: 65).SV40LT: Forward primer: 5’-CCAGAAGAAGCAGAGGAAACTA -3’ (SEQ ID NO: 66); Reverseprimer: 5’- CCAAGTACATCCCAAGCAATAAC -3’ (SEQ ID NO: 67). rtTA: Forward primer: 5’- GGCCTGGAGAAACAGCTAAA-3’ (SEQ ID NO: 68); Reverse primer:5’- TCAAGGTCAAAGTCGTCAAGG-3’ (SEQ ID NO: 69).CD19: Forward primer: 5’- AGCTGTGACTTTGGCTTATCT-3 (SEQ ID NO: 70)’; Reverse primer:5’- GGGTCAGTCATTCGCTTTCT-3’ (SEQ ID NO: 71).BCMA: Forward primer: 5’- GCGATTCTCTGGACCTGTTT-3’ (SEQ ID NO: 72); Reverse primer: 55’- AGGAGACCTGATCCTGTGTT-3’ (SEQ ID NO: 73).Luciferase: Forward primer: 5’- GTGGTGTGCAGCGAGAATAG-3’ (SEQ ID NO: 74); Reverseprimer: 5’- CGCTCGTTGTAGATGTCGTTAG-3’(SEQ ID NO: 75).For analysis of transgene protein level by FACS, 500,000 cells from transgenic iPSC lines werecollected and stained with anti-CD19, anti-BCMA antibodies (363039, 357517, BioLegend). The 10expression level of the transgenes was measured using a CytoFlex flow cytometer (BeckmanCoulter). [0242] Dox induction of Tet inducible expression of immortalization genes. Transgenic iPSC linesthat contain immortalization genes were cultured in iPSC growth media (mTeSR plus, StemcellTechnology) for at least two passages before Doxycycline induction. Doxycycline Hyclate (DOX, 15Sigma D9891) was diluted in iPSC growth media to a concentration of 0.3µM, 0.6µM, or 1µM.The transgenic iPSC lines were cultured in Doxycycline containing media for 72 hours. Cell pelletfrom each condition was collected for downstream analysis. [0243] Example 2. Growth and characteristics of immortalized induced pluripotent stem cell(iPSC) lines. 20 [0244] In this example, it is demonstrated that human iPSCs genetically modified to harbor theinducible immortalization construct (see Example 3) can be grown with the same techniques astheir unmodified parental line. It is further demonstrated that the immortalized iPSCs retain theirtypical morphology and pluripotency. Further, it is shown that immortalized iPSCs are capable todifferentiate into all three embryonic lineages. 25 [0245] To this aim, the SK005.3 iPSC line was cultured in parallel with its immortalized progenySK005.3-hTertSV40 under the same conditions outlined in the materials and methods section(See Example 1, Culture of iPSCs – thawing, passage, cryopreservation). Post thaw survival,growth rate and morphology was documented over several passages using cell counting andphase contrast photography (FIG. 4). It is also shown that the pluripotency of immortalized iPSC 30lines are maintained as evidence by alkaline phosphatase (AP) staining and expression of thepluripotency markers OCT4, NANOG, SOX2, SSEA3 and SSEA4. To this extent, bothfluorescence-activated cell sorting (FACS) analysis using antibodies for each marker as well asglobal gene expression analysis (Thermo Fisher Pluritest) was used. Finally, the immortalized iPSCs were demonstrated to be equally capable to differentiate into all three embryonic germlayers as their unmodified parental cell line. For this, in vitro differentiation was used followed byimaging and global gene expression analysis (Thermo Fisher Scorecard). [0246] Example 3. Editing a wildtype (WT) or previously edited induced pluripotent stem cell(iPSC) line genome to contain an inducible immortalization gene. I n this example, the ability of 5gene editing an human iPSC (hiPSC) line and further editing this previously edited iPSC line tocontain inducible immortalization genes is demonstrated. To this aim, an hiPSC line, SK005.3,was cultured and passaged twice in iPSC growth media (mTeSR plus, StemCell Technologies)before co-transfection with a plasmid containing PiggyBac transposase and PiggyBac-FMC63-IL15 CAR-TK plasmid using Lipofectamine 3000 (Thermo Fisher). 72 hours after the transfection, 10transfected cells were stained with anti-FMC63 antibody, and cells with correct insertion and goodFMC63 expression level were single-cell sorted vis fluorescence-activated cell sorting (FACS). 4clones were expanded, insertion copy number in each clone was analyzed by ddPCR, showed arange of 18 to 28 copies of inserted transgene. To further verify the expression of insertedtransgenes, cDNA samples from each clone were collected and the expression analysis of 15FMC63 and TK.007 was performed by qPCR. In comparison to unedited wild-type SK005.3 cells,FIG. 5 demonstrates an increase in FMC63 and TK.007 transcript levels in edited clones. Inaddition to the analysis of the FMC63 transcript level, 500,000 cells from each clone werecollected and stained with the anti-FMC63 antibody. The protein level in each clone was thenmeasured by FACS. As shown in FIG. 5, compared with the unedited wild-type SK005.3 line, a 20significant increase in FMC63 protein level was observed, further evidencing the successfulediting of the iPSC line. [0247] There are various ways to immortalize cells. FIG. 6B shows the design for the inducibleimmortalization vector. It contains an inducible immortalization cassette that includes humanTelomerase Transcriptase (hTERT) and SV40gp6 Large T Antigen (SV40 LT) under a 25Tetracycline (Tet)-inducible expression system. A Neomycin (Neo) resistant gene was placeddownstream of the inducible immortalization genes and flanked by two loxP sites. This designallows the enrichment and selection of cells that have the correct insertion by culturing with Neo,and removal of the Neo gene from the final edited iPSC line. The inducible immortalizationcassette was placed in a Sleeping Beauty (SB) transposon vector backbone. When co-transfected 30with SB transposase, the inducible immortalization can be effectively inserted by the SBtransposon system. [0248] Next, to demonstrate the ability to insert and activate the inducible immortalization genes,a pooled SK005.3 iPSC that express high-level FMC63 chimeric antigen receptor (CAR) and TK.007 were cultured and passaged twice in iPSC growth media (mTeSR plus, StemCellTechnologies) before co-transfected with the SB100x plasmid with the inducible immortalizationvector using Lipofectamine 3000 (Thermo Fisher). 24 hours after transfection, transfected cellswere cultured with iPSC growth media containing 150µg/mL Neo with daily media change. Neo-resistant colonies were collected and pooled 10 days after the selection. To test the activation of 5the Tet-inducible immortalization cassette, FMC63-Tet-hTERT-SV40 LT SK005.3 iPSCs werecultured without or with an increasing concentration of doxycycline (DOX) for 72 hours. cDNAfrom each condition and SK005.3 and TC1133 wildtype iPSC were collected and the transcriptlevels of hTERT, SV40 LT, rtTA, and FMC63 were measured by qPCR. As shown in FIG. 7, anincrease in transcripts levels of two genes under Tet-On promotor (hTERT and SV40 LT) can be 10observed after 72 hours of DOX treatment, in a dose-dependent manner. In comparison, thereare no significant changes in the transcript levels of rtTA and FMC63 in the presence or absenceof DOX. Overall, these data demonstrate the ability to insert inducible immortalization genes tohiPSC and successful activation of the expression of the immortalization gene with DOXtreatment. 15 [0249] Example 4. Differentiating an induced pluripotent stem cell (iPSC) line edited to contain aninducible immortalization gene into CD34+ cells. To demonstrate the ability of the gene-editediPSC line containing the inducible immortalization gene to differentiate into specific phenotypes,unmodified iPSCs and TetON hTERT SV40 IPSCs were differentiated to CD34-positivehematopoietic progenitor cells. To this aim, unmodified and hTERT SV40 iPSC were cultured for 20three passages in iPSC growth medium (TeSR E8TM Medium, StemCell technologies), with andwithout 0.1 µM Doxycycline Hyclate (DOX) before initiation of hematopoietic progenitor celldifferentiation. Both unmodified and TetON hTERT SV40 IPSCs displayed the typical iPSCmorphology, with tightly packed colonies and well-defined colony borders, and the cellmorphology was not affected by DOX treatment (FIG. 8A). To demonstrate the activation of the 25conditional immortalization construct, cell samples were collected from the iPSC cultures with andwithout DOX treatment and gene expression analysis was performed via RT-PCR. FIGs. 8A and8B shows increased expression of the hTERT and SV40 transcripts upon DOX treatment (DOX-inducible) and constitutive expression of the rtTA transcript (always ON) in the hTERT SV40 IPSCline only, while no significant expression for these transcripts was measured in the unmodified 30iPSC line. [0250] Next, unmodified iPSCs and TetON hTERT SV40 IPSCs were subjected to differentiationinto hematopoietic progenitor cells following the timeline depicted in FIG. 8C. Briefly, iPSCs wereseeded into AggreWell plates in StemDiff Hematopoietic Differentiation Medium (StemCell Technologies) for embryoid bodies (EB) formation and cultured for 5 days. EBs were thentransferred to 6-well plates and cultured for 7 days before harvest and selection of CD34-positivehematopoietic progenitor cells. FIG. 8D shows representative images of EBs generated from theTetON hTERT SV40 IPSC line cultured with and without DOX on day 2 of the differentiation inAggreWells and on day 12 of EB harvest. DOX treatment increased the size of EB compared to 5the no treatment control, suggesting the induction of immortalization genes promotes cellproliferation during EB formation. Upon EB harvest, dissociation and CD34-positive selection, theenriched CD34-positive cell fraction of the DOX-treated hTERT SV40 line showed higher viabilityand viable cell yield compared to the no treatment control (FIG. 8E). Further staining of theenriched fraction with CD34 antibody followed by flow cytometry analysis revealed a significantly 10higher percentage of CD34-positve cells upon DOX treatment compared to the unmodified line,with and without DOX, as shown in FIG. 8F (60.1% CD34-positive cells for the DOX-treatedhTERT SV40 line compared to 28.8% CD34-positive cells for the DOX-treated unmodified line). [0251] Overall, these data demonstrate that activation of the inducible immortalization constructincreases the viability and yield of CD34-positive cells during hematopoietic differentiation. 15 [0252] Example 5. Differentiating an induced pluripotent stem cell (iPSC) line edited to contain aninducible immortalization gene into mesenchymal stem cells (MSC) and natural killer (NK) cells. [0253] To demonstrate the ability of the gene-edited iPSC line containing the inducibleimmortalization gene to differentiate into specific phenotypes, unmodified iPSCs and TetONhTERT SV40 IPSCs were differentiated to NK cells using the StemDiff NK Cell Kit (StemCell 20Technologies) following a 3-step differentiation. FIG. 9 shows the diagram of the NK celldifferentiation process. iPSCs are thawed and expanded before they are passaged intoAggreWells to generate embryoid bodies (EBs). After 5 days of culture in AggreWells, the EBsare transferred to a 6-well plate. At day 12 of the differentiation, EBs are dissociated, positively-selected for CD34 expression, phenotypically characterized for hematopoietic progenitor cells 25surface marker expression, and seeded for Lymphoid Progenitor Cell differentiation. After 14 daysof culture, Lymphoid Progenitor Cells are harvested, phenotypically characterized for cell surfacemarker expression and seeded for NK Cell differentiation. [0254] In some instances, 0.1 µM Doxycycline Hyclate (DOX) was added to the iPSC culture anddifferentiation medium throughout the differentiation process. In other instances, 0.1 µM 30Doxycycline Hyclate was added to the differentiation medium at the initiation of either LymphoidProgenitor Cell differentiation stage, or NK Cell differentiation stage. The presence of DOX in thedifferentiation medium stimulates the generation of a higher percentage of CD5-positive, CD7-positve lymphoid progenitor cells as demonstrated by flow cytometry analysis of the differentiated population. Similarly, the presence of DOX increased the yield of CD56-positive NK cells at theend of the NK cell differentiation stage. [0255] Together, these data demonstrate that activation of the inducible immortalization constructincreases the yield of lymphoid progenitors and NK cells during the NK cell differentiation process. [0256] Example 6. CD19 expressing mesenchymal stem cells (MSC)-derived induced pluripotent 5stem cell (iPSC) line edited to contain an inducible immortalization gene are useful indemonstrating that chimeric antigen receptor (CAR) T-cells are properly prepared. [0257] CAR T cell therapy is a promising approach to cancer treatment that targets specificantigens expressed on the surface of cancer cells. One key step in CAR T cell preparation is totest whether the expanded CAR T cells are able to recognize and attack the cancer cells 10expressing the corresponding target antigen. [0258] In this section, the ability to generate transgenic iPSCs that express CD19 and B-cellmaturation antigen (BCMA) antigens and the potential of generating transgenic iPSCs thatexpress an array of commonly targeted antigens for CAR-T cell quality control is demonstrated. [0259] CD19 and BCMA are two commonly targeted antigens in treatments. CD19 is a cell 15surface antigen that is expressed on the surface of B cells. In cell therapy treatment of B-cellmalignancies such as acute lymphoblastic leukemia and non-Hodgkin lymphoma, CD19 is acommonly targeted antigen. CAR T cells targeting CD19 have shown promising results in clinicaltrials, with high rates of complete remission in patients with relapsed or refectory B cellmalignancies. BCMA, or B cell maturation antigen, is another cell surface antigen that is 20expressed on the surface of plasma cells, which produces antibodies. BCMA is a promising targetfor CAR T cell therapy in the treatment of multiple myeloma, a type of cancer that arises fromabnormal plasma cells. CAR T cells targeting BCMA have shown high response rates in clinicaltrials, with some patients achieving complete remission. [0260] First, a CD19 or BCMA expressing plasmid was designed in a PiggyBac plasmid 25backbone. As shown in FIG. 10, in addition to the antigen, a Puromycin (Puro) resistant gene wasplaced downstream of the antigen gene for the selection and enrichment of transfected cells.Furthermore, a Luciferase gene (Luc) was included in the design to facilitate cell tracking indownstream applications. [0261] To generate the transgenic iPSC lines that express CD19 and BCMA, PAN3 and SK005.3 30MHC class I/II KO hiPSC were cultured and passaged twice in iPSC growth media (mTeSR plus,StemCell Technologies) before co-transfected with a plasmid containing PiggyBac transposaseand PiggyBac-CD19-Luc or PiggyBac- BCMA -Luc plasmid using Lipofectamine 3000 (ThermoFisher). 48 hours after transfection, transfected cells were cultured with iPSC growth media containing 0.8µg/mL Puro with daily media change. Puro-resistant colonies were pooled andpassaged twice before frozen down. To verify the expression of inserted transgenes, cDNAsamples from each pooled transfected cell from each condition were collected and the expressionanalysis of CD19 or BCMA was performed by qPCR. In comparison to unedited wild-type hiPSClines, an increase in CD19 and BCMA transcript levels in edited pooled cells was demonstrated. 5In addition to the analysis of the transcript level, 500,000 cells from each clone were collectedand stained with the anti-CD19 or anti-BCMA antibody. The protein level in each condition wasthen measured by FACS. Compared with the unedited wild-type hiPSC line, a significant increasein CD19 or BCMA protein level was observed, further evidencing the successful editing of theiPSC line. In this case, the insertion and expression of CD19 and BCMA were done in both 10unedited (PAN3) as well as previously edited hiPSC (SK005.3 MHC class I/II KO), highlightingthe flexibility and adaptivity of the design and workflow. [0262] In addition to CD19 and BCMA, an array of commonly targeted antigens has beenidentified as the target antigens for CAR-T cells for various diseases. FIG. 11 listed a panel of 12common target antigens, including CD19 and BCMA. hiPSCs have the potential to differentiate 15into any cell type. Having hiPSCs that express these targeted antigens for CAR-T cells wouldgreatly reduce the cost and time to verify the efficacity and specificity of CAR-T cell products inthe QC phase. FIG. 12 shows two systems (PiggyBac- and lentivirus-based) which were designedfor insertion and expression of CAR-T targeted antigen or other cargos into hiPSC, highlightingthe capability of generating edited iPSC lines for QCing various clinical CAR T cells beyond just 20CD19 and BCMA. [0263] Example 7. The induced pluripotent stem cell (iPSC) line containing an inducibleimmortalization gene can be further manipulated to express luciferase or green fluorescent protein(GFP). [0264] Introduction. Fluorescence and bioluminescence have been widely used in biomedical 25research for decades. Both provide means of detecting cells expressing a protein as an emittedlight that can be captured and analyzed to provide detailed data on specific gene expression.Fluorescence is found in a large variety in nature, ranging from minerals and marine organismsto butterflies and arachnids and is based on the absorption of light of a specificwavelength (excitation light) and the subsequent emission of a lower frequency (emission light) 30(FIG. 13A). The resulting emission of the fluorochrome-specific wavelength can be captured withcameras equipped with the corresponding filters. Fluorochromes exist in a wide array of colors,the most widely used being GFP (Prasher 1992, Gene, 111(2):229-33, PMID: 1347277).Bioluminescense refers to the direct emission of visible light without the need for excitation.

Luciferase is an enzyme that catalyzes a light-producing biochemical reaction when it is in thepresence the substrate luciferin. Bioluminescence is found in nature (such as the firefly and theangler fish) (Shimomura 1995, Bio Bull. 189(1):1-5, PMID:7654844). Capturing this phenomenonfor biomedical research has allowed the detection of transgenes in living organisms usingBioluminescent Light Imaging (BLI) (FIG. 13B). Here the benefits of each system was combined, 5optimizing their utilities. While GFP was used as a tool to visualize transgene expression in vitroand ex-vivo, Luciferase allows us to rack immortalized iPSCs in in vivo pre-clinical studies usingappropriate mouse models. [0265] DNA transposons are designed to move from one genomic location to another by a cut-and-paste mechanism. They are powerful forces of genetic change and have played a significant 10role in the evolution of many genomes. As genetic tools, they can be used to introduce foreignDNA into a genome. To obtain high levels of transgene expression, the piggyBAC and SleepingBeauty transposon systems were used as briefly described in Example 3. Both piggyBAC andSleeping Beauty (Chen et al., Nature Biotechnology, 2020, 38:165-168) has been used for manyyears in biotechnology. 15 [0266] Human iPSCs that carry the inducible immortalization transgene hTertSV40 display thesame phenotype, pluripotency, ability to differentiate into all three embryonic lineages as are theirunmodified. These cells can be further modified both with transgene constructs using thetransposon technology as well as targeted genomic modifications (Knock-In and Knock-out)utilizing CRISPR/Cas9. In case of the former, the transposon system is used that was not utilized 20for the immortalization step (piggyBAC/Sleeping Beauty). [0267] The order of engineering can be done in reverse: immortalizing previously gene editediPSC lines. [0268] The current disclosure provides immortalized cell lines generated from immortal cells anduses thereof. 25 [0269] Particular embodiments utilize stem cells modified to include a drug-inducible growthsystem (e.g., Tert and SV40). These embodiments are particularly useful to differentiate intoimmortalized differentiated cell populations that can be maintained as immortal throughadministration of the growth controlling drug. [0270] Particular embodiments utilize stem cells modified to include a drug-inducible growth 30system and a suicide switch. These embodiments are particularly useful to differentiate intoimmortalized differentiated cell populations for a therapeutic purpose, the suicide switch providingan in vivo safety feature. Further, the suicide switch embodiment may be especially useful toprovide a safety feature allowing the removal of proliferating cells from cultured cells (in vitro) before use as a therapeutic, and after application as a therapeutic (in vivo). [0271] Particular embodiments utilize stem cells modified to include a drug-inducible growthsystem and factors that support use as feeder cells during cell culture. These embodiments areparticularly useful to differentiate into immortalized differentiated feeder cells. Examples includeadherent cells (e.g., mesenchymal stem cells) or suspension cells (e.g. CD34+ cells). 5Differentiated immortalized feeder cells can be genetically modified to support growth of particularcell types, such as expression of membrane-bound IL21 and MHC Class I and Class II knock-outto support growth of natural killer (NK) cells. These embodiments may also include a suicideswitch to reduce contamination of cell populations of interest with feeder cells. Further, theseembodiments may also utilize cells that express a viral antigen that can be used as a living vaccine 10allowing for extended antigenic presentation in a physiologically appropriate manner. Theseembodiments may also express a reporter, such as fluorescent proteins and/or luciferase. [0272] Particular embodiments utilize stem cells modified to include a drug-inducible growthsystem and factors that support use as tester cells during research and development. Theseembodiments are particularly useful to differentiate into immortalized differentiated tester cells. 15Examples include tester cells that express a cancer antigen or a viral antigen to test efficacy ofantibodies, chimeric antigen receptors, or similar recombinant molecules under development.When manufactured for in vivo use, these immortalized differentiated tester cells may alsoexpress a reporter, such as luciferase. These embodiments may also include a suicide switch. [0273] Example 8. Mesenchymal stem cell (MSC) line used in natural killer (NK) assays. 20 [0274] In this example, the method and results from functional tests on IL-21 expressing MSCs(also called feeder cells) are described. The functional test involves co-culture of NK cells withfeeder lines that express IL-21 on the surface membrane. If IL-21 is properly expressed on thecell surface, and it can be recognized properly by NK cells, that leads to activation and expansionof NK cells when co-cultured. 25 [0275] In the following, the methods to confirm the phenotype and genotype of these feeder celllines are described and results are presented. Then, the method of NK cell co-culture is described,and the results are presented. [0276] To demonstrate the function of IL-21 expression in activating and expanding NK cells, 3lines were used in NK activation assays as listed in Table 3 and shown in FIG. 15A. 30Table 3. List of MSC lines used in NK activation and expansion functional test. Cell Line Description Line A SK005 immortalized MSCLine B MSC, SK5 Class I/II null MB-IL21 iPSC-derivedLine C "Immortalized" MSC, SK5 Class I/II null MB-IL21 iPSC-derived id="p-277"

[0277] Line A served as the negative control for both IL-21 expression (i.e., had no expression ofIL-21) and for Class I/II null (i.e., expressed ClassI/II major histocompatibility class (MHC)). LineB served as control for immortalization (i.e., lacks hTert-SV40 expression). Line C is the targetproduct and is positive for expression of IL-21 and immortalization cassettes as well as negativefor expression of Class I/II MHC. In all tests and assays, Lines A and B were used as controls for 5Line C. [0278] The genotype of the cells were confirmed via qPCR (FIG. 15B). In brief, genomic DNAwas extracted via commercial kits (RNeasy® Plus Mini Kit, Qiagen, Germantown, MD) and qPCRprotocols were performed using the primer sets in Table 4.Table 4. Primers used for confirming genotype of the MSC lines for NK assay. 10 Primers Forward primer Reverse Primer MB-IL21 qPCR F1CGGCACCAGAAGATGTAGAAA(SEQ ID NO: 76)TCCTCTCGTTATTTCCCGTATTG(SEQ ID NO: 77)MB-IL21 qPCR F2TGTCCCTCTTGCGATTCTTAC(SEQ ID NO: 78)TCACTACCGTGTGTCCTACT(SEQ ID NO: 79)hTERT qPCR F1CTCCATCCTGAAAGCCAAGAA(SEQ ID NO: 80)AGTCAGCTTGAGCAGGAATG(SEQ ID NO: 81)SV40LT qPCR F1CCAGAAGAAGCAGAGGAAACTA(SEQ ID NO: 82)CCAAGTACATCCCAAGCAATAAC(SEQ ID NO: 83) [0279] The genotype of the cells were confirmed via qPCR (FIG. 15B) and Flow Cytometry (FIGs.and 17). A summary of results can be found in Table 5.Table 5: Summary of phenotyping and genotyping results for cells used in the NK assay. Phenotype Expected Genotype Expected Phenotype hTert SV40 MB- IL21 CD90 CD105 HLA ABC Line A Immortalized + + - + + + Line BMB-IL21,Class I/II null - - + + + - Line C Immortalized,MB-IL21,Class I/II null + + + + + - [0280] NK Activation and Expansion Assay. The intended function of membrane-bound IL-21(mbIL-21) expressing MSCs (feeder lines) was assessed via co-culture with primary human NK 15cells following the method described herein. [0281] Negatively-selected, cryopreserved, primary human NK cells were purchased from acommercial vendor (BloodWork NW, Seattle, US). On Day -1 of co-culture (i.e. one day prior tostart of co-culture), the MSC cells Lines A, B, and C were seeded at 1.75e5/well/line in 6 wellplates in StemXvivo media (R&D Systems, US). Also on Day -1, the NK cells were thawed and 20seeded at 0.7e5/mL in NK Xpander media (Thermo Fisher, US) supplemented with 500 IU IL-2 and 5% FBS. [0282] In a side arm, iNK cells generated from iPSC lines described in Example 5, were thawedand seeded with MSCs similarly to the primary NK cells as described above. [0283] On Day 0 of culture, MSC line cell counts were assessed via counting of cells from arepresentative plate, and the NK cells were seeded on MSCs at 5:1 MSC-to-NK ratio in NK cell 5media. A control plate of MSCs was cultured in NK media, without NK co-culture, to assess theimpact of NK media on MSC cells over the course of the experiment. Another control plate wasseeded with NK cells only in the NK media (no co-culture). All co-culture and control plates wereincubated at 37ºC and 5% CO 2. [0284] On Days 1 to 3 of co-culture, a respective plate containing the co-cultured cells was 10removed from the incubator, and the cells were imaged on a microscope to document the killingand activation/expansion on NK cells. Then, a sample of cell suspension was collected, and cellcount was performed to assess viability and count of cells in suspension. The plates were thenwashed with PBS and the MSC cells were lifted using a lifting reagent (Accutase, Thermo, US),and were enumerated on the cell counter. 15 [0285] For re-stimulation test, all NK cells from Day 3 co-culture were removed, resuspended infresh NK culture media, and were reseeded on a fresh plate of Line B and Line C MSC. Cellswere imaged and counted on Day 6 (Day 3 of re-stimulation). [0286] Results. FIGs. 18 and 19 are representative images of the state of the cells at thebeginning (Day 0) and the end (Day 3) of 3-day co-culture assay. Cell viability and counts over 20the course of co-culture experiment are presented in FIGs. 20, 21A, and 21B. Due to impact ofhandling on viability of NK, total nucleated cell count (TNC) is reported and used to calculatenormalized numbers instead of total viable count (TVC). It appears that the NK cells could be toofragile for sampling manipulation after activation, even when gently sampled. As such, thereported viabilities appear to be very low. This also seems consistent with iNK cells. Due to impact 25of handling on viability of NK, TNC is reported and used to calculate normalized numbers insteadof TVC. [0287] The results in FIG. 20 suggest that, in the absence of IL-21 expression, NK cells do notchange in number by Day 3 (120%) compared to those on Lines B (145%) and C (185%). Incomparison, NK cells in NK media without MSC co-culture maintain the viability and count through 30the 3-day assay period. This also suggests that when not activated, NK cells are more resilientagainst the sampling procedure, hence maintaining the viability, while those that were activatedshow fragility to pipetting as exhibited in low viability numbers for the co-cultured NK cells. [0288] iNK cells will be assessed on Day 3 of co-culture. The iNK cells are not expected to demonstrate any appreciable killing effect within the time window of this assay and the specificratio in which they will be seeded on MSCs. It is expected that iNK cells will also demonstrate thekilling effect if seeded at a higher ratio such as 1:1 or 5:1 NK to MSC. [0289] The MSCs on the other hand, significantly decrease in number for Lines B and C comparedto Line A which shows an increase instead of decrease. This suggests the killing action of NK 5cells on MSCs that expressed IL-21, while those which do not express IL-21 (Line A) continueexpanding in culture by Day 3. [0290] After restimulation, a significant increase in cell number was observed as documented byimaging and after performing cell count on the supernatant (FIGs. 21A, 21B). Cells activated onfeeder Line B and Line C expanded significantly by multiple folds (13 to 16 folds) after 3 days of 10re-stimulation on fresh Line B and Line C feeders. [0291] Example 9. Mesenchymal stem cell (MSC) lines lacking Class I or Class II HLA expression(individually, Class I null or Class II null) are tested in three different types of potency assays:natural killer (NK), natural killer T-cells (NKT), and T-cells assays. [0292] In this example, the method and results from functional potency tests on four different IL- 15expressing MSCs (also called feeder cells) are described. The functional test is described inExample 8 and involves co-culture of Lymphocytes (NK, NKT, or T) cells with feeder lines thatexpress IL-21 on the surface membrane. [0293] The methods to confirm the phenotype and genotype of these feeder cell lines are alsodescribed in Example 8 and expected results are presented below. The method of Lymphocytic 20cell co-culture is described in Example 8, and the expected results are presented below. [0294] To demonstrate the function of IL-21 expression in activating and expanding NK, NKT,and T-cells in the absence of MHC Class I or Class II expression, 5 lines are used in lymphocyticactivation assays as listed in Table 3.Table 6. List of MSC lines to be used in NK NKT, and T-cell activation and expansion functional 25tests. Cell Line Description Line A SK005 immortalized MSCLine D MSC, SK5 Class I null MB-IL21 iPSC-derivedLine E "Immortalized" MSC, SK5 Class I null MB-IL21 iPSC-derivedLine F MSC, SK5 Class II null MB-IL21 iPSC-derivedLine G "Immortalized" MSC, SK5 Class II null MB-IL21 iPSC-derived [0295] Line A serves as the negative control for both IL-21 expression (i.e., should have noexpression of IL-21) and HLA expression of both Class I and II major histocompatibility complexes(MHC)). Line D serves as control for immortalization (i.e., lacks hTert-SV40 expression) and is lacking proper Class I MHC expression (Class I null). Line E is the immortalized Class I null targetproduct and is positive for expression of IL-21 and immortalization cassettes as well as negativefor expression of Class I MHC. Line F serves as control for immortalization (i.e., lacks hTert-SV40expression) and is lacking proper Class II MHC expression (Class II null). Line G is theimmortalized Class II null target product and is positive for expression of IL-21 and immortalization 5cassettes as well as negative for expression of Class II MHC. .In all tests and assays, Lines Aand D are used as controls for Line E and Lines A and F are used as controls for line G. [0296] The genotype of the cells are confirmed via qPCR as described in Example 8. In brief,genomic DNA is extracted via commercial kits (RNeasy® Plus Mini Kit, Qiagen, Germantown,MD) and qPCR protocols are performed using the primer sets in Table 4. 10Table 7. Primers to be used for confirming genotype of the MSC lines for NK, NKT, and T-cellassays. Primers Forward primer Reverse Primer MB-IL21 qPCR F1CGGCACCAGAAGATGTAGAAA(SEQ ID NO: 76)TCCTCTCGTTATTTCCCGTATTG(SEQ ID NO: 77)MB-IL21 qPCR F2TGTCCCTCTTGCGATTCTTAC(SEQ ID NO: 78)TCACTACCGTGTGTCCTACT(SEQ ID NO: 79)hTERT qPCR F1CTCCATCCTGAAAGCCAAGAA(SEQ ID NO: 80)AGTCAGCTTGAGCAGGAATG(SEQ ID NO:81)SV40LT qPCR F1CCAGAAGAAGCAGAGGAAACTA(SEQ ID NO: 82)CCAAGTACATCCCAAGCAATAAC(SEQ ID NO: 83) [0297] The genotype of the cells are confirmed via qPCR and Flow Cytometry. A summary ofexpected results can be found in Table 8.Table 8: Summary of phenotyping and genotyping expected results for cells to be used in the NK, 15NKT, and T-cell potency assays. Phenotype Expected Genotype Expected Phenotype hTert SV40 MB-IL21 CD90 CD105 HLA- ABC (MHC class I) HLA-D (MCH class II) LineA Immortalized + + - + + ++ LineDMB-IL21,Class I null - - + + + - + LineE Immortalized,MB-IL21,Class I null + + + + + - +LineFMB-IL21,Class II null - - + + + + - LineG Immortalized,MB-IL21,Class II null + + + + + + - id="p-298"

[0298] NK, NKT, T-cell Activation and Expansion Assays. The intended function of membrane-bound IL-21 (mbIL-21) expressing MSCs (feeder lines) in the Class I only and Class II onlybackground is assessed via co-culture with primary human NK cells, primary human NKT, andprimary human T-cell following the method described herein. [0299] Negatively-selected, cryopreserved, primary human cytotoxic lymphocytes (NK, NKT, or 5T-cells) are purchased from a commercial vendor (BloodWork NW, Seattle, US). On Day -1 of co-culture (i.e. one day prior to start of co-culture), the MSC cell Lines A, B, and C are seeded at1.75e5/well/line in 6 well plates in StemXvivo media (R&D Systems, US). Also on Day -1, the NKcells are thawed and seeded at 0.7e5/mL in NK Xpander media (Thermo Fisher, US)supplemented with 500 IU IL-2 and 5% FBS. 10 [0300] In a side arm, cytotoxic lymphocyte cells (iNK, iNKT, or iT-cells) generated from iPSC linesdescribed in Example 5, are thawed and seeded with MSCs similarly to the primary cytotoxiclymphocytes as described above. [0301] On Day 0 of culture, MSC line cell counts are assessed via counting of cells from arepresentative plate, and the cytotoxic lymphocytes cells are seeded on MSCs at 5:1 MSC-to- 15cytotoxic lymphocytes ratio in cytotoxic lymphocyte cell media. A control plate of MSCs is culturedin cytotoxic lymphocyte media, without co-culture, to assess the impact of cytotoxic lymphocytemedia on MSC cells over the course of the experiment. Another control plate is seeded withcytotoxic lymphocyte cells only in cytotoxic lymphocyte media (no co-culture). All co-culture andcontrol plates are incubated at 37ºC and 5% CO 2. 20 [0302] On Days 1 to 3 of co-culture, a respective plate containing the co-cultured cells is removedfrom the incubator, and the cells are imaged on a microscope to document the killing andactivation/expansion on cytotoxic lymphocyte cells. Then, a sample of cell suspension iscollected, and cell count is performed to assess viability and count of cells in suspension. Theplates are then washed with PBS and the MSC cells are lifted using a lifting reagent (Accutase, 25Thermo, US), and are enumerated on the cell counter. [0303] For re-stimulation test, all cytotoxic lymphocyte cells from Day 3 co-culture are removed,resuspended in fresh cytotoxic lymphocyte culture media, and are reseeded on a fresh plate ofLine D, E, F, or G MSCs. Cells are imaged and counted on Day 6 (Day 3 of re-stimulation). [0304] Results. FIGs. 18 and 19 represent anticipated images of the state of the cells at the 30beginning (Day 0) and the end (Day 3) of 3-day co-culture assay. Cell viability and counts overthe course of co-culture experiment are conducted over the course of the 3-day potency assay.Due to impact of handling on viability of cytotoxic lymphocytes, total nucleated cell count (TNC)will be reported and used to calculate normalized numbers instead of total viable count (TVC).

Due to impact of handling on viability of cytotoxic lymphocytes, TNC is reported and used tocalculate normalized numbers instead of TVC. [0305] The results will show that, in the absence of IL stimulation, cytotoxic lymphocytes cells donot change in number by Day 3 compared to those cultured on Lines D E, F, and G, which isexpected to be between 150% to 200%. In comparison, cytotoxic lymphocyte cells in cytotoxic 5lymphocyte media without MSC co-culture maintain the viability and count through the 3-dayassay period. This will also suggest that when not activated, cytotoxic lymphocyte cells are moreresilient against the sampling procedure, hence maintaining viability, while those that areactivated will show fragility to pipetting as shown previously resulting in low viability numbers forthe co-cultured cytotoxic lymphocyte cells. 10 [0306] iPSC derived cytotoxic lymphocyte cells will be assessed on Day 3 of co-culture. The iPSCderived cytotoxic lymphocyte cells are not expected to demonstrate any appreciable killing effectwithin the time window of this assay and the specific ratio in which they will be seeded on MSCs.It is expected that iPSC derived cytotoxic lymphocyte cells will also demonstrate the killing effectif seeded at a higher ratio such as 1:1 or 5:1 NK to MSC. 15 [0307] The MSCs on the other hand, significantly decrease in number for Lines D, E, F, and Gcompared to Line A which shows an increase instead of decrease. This suggests the killing actionof iPSC derived cytotoxic lymphocyte cells on MSCs that expresses IL-21, while those which donot express IL-21 (Line A) continue expanding in culture by Day 3. [0308] After restimulation, a significant increase in cell number will be observed, documented by 20imaging and after performing cell count on the supernatant. Cells activated on feeder Lines D, E,F, and G will be shown to expand significantly by multiple folds (e.g., 13 to 16 folds) after 3 daysof re-stimulation on fresh feeders. [0309] (IX) Closing Paragraphs. The nucleic acid and amino acid sequences provided herein areshown using letter abbreviations for nucleotide bases and amino acid residues, as defined in 37 25C.F.R. §1.831-1.835 and set forth in WIPO Standard ST.26 (implemented on July 1, 2022). Onlyone strand of each nucleic acid sequence is shown, but the complementary strand is understoodas included in embodiments where it would be appropriate. [0310] Variants of the sequences disclosed and referenced herein are also included. Guidancein determining which amino acid residues can be substituted, inserted, or deleted without 30abolishing biological activity can be found using computer programs well known in the art, suchas DNASTAR™ (Madison, Wisconsin) software. Preferably, amino acid changes in the proteinvariants disclosed herein are conservative amino acid changes, i.e., substitutions of similarlycharged or uncharged amino acids. A conservative amino acid change involves substitution of one of a family of amino acids which are related in their side chains. [0311] In a peptide or protein, suitable conservative substitutions of amino acids are known tothose of skill in this art and generally can be made without altering a biological activity of aresulting molecule. Those of skill in this art recognize that, in general, single amino acidsubstitutions in non-essential regions of a polypeptide do not substantially alter biological activity 5(see, e.g., Watson et al. Molecular Biology of the Gene, 4th Edition, 1987, TheBenjamin/Cummings Pub. Co., p. 224). Naturally occurring amino acids are generally divided intoconservative substitution families as follows: Group 1: Alanine (Ala), Glycine (Gly), Serine (Ser),and Threonine (Thr); Group 2: (acidic): Aspartic acid (Asp), and Glutamic acid (Glu); Group 3:(acidic; also classified as polar, negatively charged residues and their amides): Asparagine (Asn), 10Glutamine (Gln), Asp, and Glu; Group 4: Gln and Asn; Group 5: (basic; also classified as polar,positively charged residues): Arginine (Arg), Lysine (Lys), and Histidine (His); Group 6 (largealiphatic, nonpolar residues): Isoleucine (Ile), Leucine (Leu), Methionine (Met), Valine (Val) andCysteine (Cys); Group 7 (uncharged polar): Tyrosine (Tyr), Gly, Asn, Gln, Cys, Ser, and Thr;Group 8 (large aromatic residues): Phenylalanine (Phe), Tryptophan (Trp), and Tyr; Group 9 (non- 15polar): Proline (Pro), Ala, Val, Leu, Ile, Phe, Met, and Trp; Group 11 (aliphatic): Gly, Ala, Val, Leu,and Ile; Group 10 (small aliphatic, nonpolar or slightly polar residues): Ala, Ser, Thr, Pro, and Gly;and Group 12 (sulfur-containing): Met and Cys. Additional information can be found in Creighton(1984) Proteins, W.H. Freeman and Company. [0312] In making such changes, the hydropathic index of amino acids may be considered. The 20importance of the hydropathic amino acid index in conferring interactive biologic function on aprotein is generally understood in the art (Kyte and Doolittle, 1982, J. Mol. Biol. 157(1), 105-32).Each amino acid has been assigned a hydropathic index on the basis of its hydrophobicity andcharge characteristics (Kyte and Doolittle, 1982). These values are: Ile (+4.5); Val (+4.2); Leu(+3.8); Phe (+2.8); Cys (+2.5); Met (+1.9); Ala (+1.8); Gly (−0.4); Thr ( −0.7); Ser ( −0.8); Trp ( −0.9); 25Tyr ( −1.3); Pro ( −1.6); His ( −3.2); Glutamate ( −3.5); Gln ( −3.5); aspartate ( −3.5); Asn ( −3.5); Lys ( −3.9); and Arg ( −4.5). [0313] It is known in the art that certain amino acids may be substituted by other amino acidshaving a similar hydropathic index or score and still result in a protein with similar biologicalactivity, i.e., still obtain a biological functionally equivalent protein. In making such changes, the 30substitution of amino acids whose hydropathic indices are within ±2 is preferred, those within ±1are particularly preferred, and those within ±0.5 are even more particularly preferred. It is alsounderstood in the art that the substitution of like amino acids can be made effectively on the basisof hydrophilicity. id="p-314"

[0314] As detailed in US 4,554,101, the following hydrophilicity values have been assigned toamino acid residues: Arg (+3.0); Lys (+3.0); aspartate (+3.0±1); glutamate (+3.0±1); Ser (+0.3);Asn (+0.2); Gln (+0.2); Gly (0); Thr ( −0.4); Pro ( −0.5±1); Ala ( −0.5); His ( −0.5); Cys ( −1.0); Met ( −1.3); Val ( −1.5); Leu ( −1.8); Ile ( −1.8); Tyr ( −2.3); Phe ( −2.5); Trp ( −3.4). It is understood that an amino acid can be substituted for another having a similar hydrophilicity value and still obtain 5a biologically equivalent, and in particular, an immunologically equivalent protein. In suchchanges, the substitution of amino acids whose hydrophilicity values are within ±2 is preferred,those within ±1 are particularly preferred, and those within ±0.5 are even more particularlypreferred. [0315] As outlined above, amino acid substitutions may be based on the relative similarity of the 10amino acid side-chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size,and the like. As indicated elsewhere, variants of gene sequences can include codon optimizedvariants, sequence polymorphisms, splice variants, and/or mutations that do not affect thefunction of an encoded product to a statistically-significant degree. [0316] Variants of the protein, nucleic acid, and gene sequences disclosed herein also include 15sequences with at least 70% sequence identity, 80% sequence identity, 85% sequence, 90%sequence identity, 95% sequence identity, 96% sequence identity, 97% sequence identity, 98%sequence identity, or 99% sequence identity to the protein, nucleic acid, or gene sequencesdisclosed herein. [0317] "% sequence identity" refers to a relationship between two or more sequences, as 20determined by comparing the sequences. In the art, "identity" also means the degree of sequencerelatedness between protein, nucleic acid, or gene sequences as determined by the matchbetween strings of such sequences. "Identity" (often referred to as "similarity") can be readilycalculated by known methods, including those described in: Computational Molecular Biology(Lesk, A. M., ed.) Oxford University Press, NY (1988); Biocomputing: Informatics and Genome 25Projects (Smith, D. W., ed.) Academic Press, NY (1994); Computer Analysis of Sequence Data,Part I (Griffin, A. M., and Griffin, H. G., eds.) Humana Press, NJ (1994); Sequence Analysis inMolecular Biology (Von Heijne, G., ed.) Academic Press (1987); and Sequence Analysis Primer(Gribskov, M. and Devereux, J., eds.) Oxford University Press, NY (1992). Methods to determineidentity are designed to give the best match between the sequences tested. Methods to determine 30identity and similarity are codified in publicly available computer programs. Sequence alignmentsand percent identity calculations may be performed using the Megalign program of theLASERGENE bioinformatics computing suite (DNASTAR, Inc., Madison, Wisconsin). Multiplealignment of the sequences can also be performed using the Clustal method of alignment (Higgins and Sharp CABIOS, 5, 151-153 (1989) with default parameters (GAP PENALTY=10, GAPLENGTH PENALTY=10). Relevant programs also include the GCG suite of programs (WisconsinPackage Version 9.0, Genetics Computer Group (GCG), Madison, Wisconsin); BLASTP,BLASTN, BLASTX (Altschul, et al., J. Mol. Biol. 215:403-410 (1990); DNASTAR (DNASTAR, Inc.,Madison, Wisconsin); and the FASTA program incorporating the Smith-Waterman algorithm 5(Pearson, Comput. Methods Genome Res., [Proc. Int. Symp.] (1994), Meeting Date 1992, 111-20. Editor(s): Suhai, Sandor. Publisher: Plenum, New York, N.Y.. Within the context of thisdisclosure it will be understood that where sequence analysis software is used for analysis, theresults of the analysis are based on the "default values" of the program referenced. As usedherein "default values" will mean any set of values or parameters, which originally load with the 10software when first initialized. [0318] Variants also include nucleic acid molecules that hybridize under stringent hybridizationconditions to a sequence disclosed herein and provide the same function as the referencesequence. Exemplary stringent hybridization conditions include an overnight incubation at 42 °Cin a solution including 50% formamide, 5XSSC (750 mM NaCl, 75 mM trisodium citrate), 50 mM 15sodium phosphate (pH 7.6), 5XDenhardt's solution, 10% dextran sulfate, and 20 µg/ml denatured,sheared salmon sperm DNA, followed by washing the filters in 0.1XSSC at 50 °C. Changes in thestringency of hybridization and signal detection are primarily accomplished through themanipulation of formamide concentration (lower percentages of formamide result in loweredstringency); salt conditions, or temperature. For example, moderately high stringency conditions 20include an overnight incubation at 37°C in a solution including 6XSSPE (20XSSPE=3M NaCl;0.2M NaH2PO4; 0.02M EDTA, pH 7.4), 0.5% SDS, 30% formamide, 100 µg/ml salmon spermblocking DNA; followed by washes at 50 °C with 1XSSPE, 0.1% SDS. In addition, to achieve evenlower stringency, washes performed following stringent hybridization can be done at higher saltconcentrations (e.g. 5XSSC). Variations in the above conditions may be accomplished through 25the inclusion and/or substitution of alternate blocking reagents used to suppress background inhybridization experiments. Typical blocking reagents include Denhardt's reagent, BLOTTO,heparin, denatured salmon sperm DNA, and commercially available proprietary formulations. Theinclusion of specific blocking reagents may require modification of the hybridization conditionsdescribed above, due to problems with compatibility. 30 [0319] "Specifically binds" refers to an association of a binding molecule to its cognate bindingmolecule with an affinity or Ka (i.e., an equilibrium association constant of a particular bindinginteraction with units of 1/M) equal to or greater than 10 M-1, while not significantly associatingwith any other molecules or components in a relevant environment sample. Binding molecules may be classified as "high affinity" or "low affinity". In particular embodiments, "high affinity"binding molecules refer to those binding molecules with a K a of at least 10 M-1, at least 10 M-1,at least 10 M-1, at least 10 M-1, at least 10 M-1, at least 10 M-1, or at least 10 M-1. In particularembodiments, "low affinity" binding molecules refer to those binding molecules with a Ka of up to7 M-1, up to 10 M-1, up to 10 M-1. Alternatively, affinity may be defined as an equilibrium 5dissociation constant (K d) of a particular binding interaction with units of M (e.g., 10-5 M to 10-13M). In certain embodiments, a binding molecule may have "enhanced affinity," which refers to aselected or engineered (i.e., genetically modified) binding molecules with stronger binding to acognate binding molecule than a wild type (or parent) binding molecule. For example, enhancedaffinity may be due to a K a (equilibrium association constant) for the cognate binding molecule 10that is higher than the reference binding molecule or due to a K d (dissociation constant) for thecognate binding molecule that is less than that of the reference binding molecule, or due to anoff-rate (Koff) for the cognate binding molecule that is less than that of the reference bindingmolecule. A variety of assays are known for detecting binding molecules that specifically bind aparticular cognate binding molecule as well as determining binding affinities, such as Western 15blot, ELISA, and BIACORE® analysis (see also, e.g., Scatchard, et al., 1949, Ann. N.Y. Acad. Sci.51:660; and U.S. Patent Nos. 5,283,173, 5,468,614, or the equivalent). [0320] Unless otherwise indicated, the practice of the present disclosure can employ conventionaltechniques of immunology, molecular biology, microbiology, cell biology and recombinant DNA.These methods are described in the following publications. See, e.g., Sambrook, et al. Molecular 20Cloning: A Laboratory Manual, 2nd Edition (1989); F. M. Ausubel, et al. eds., Current Protocolsin Molecular Biology, (1987); the series Methods IN Enzymology (Academic Press, Inc.); M.MacPherson, et al., PCR: A Practical Approach, IRL Press at Oxford University Press (1991);MacPherson et al., eds. PCR 2: Practical Approach, (1995); Harlow and Lane, eds. Antibodies, ALaboratory Manual, (1988); and R. I. Freshney, ed. Animal Cell Culture (1987). 25 [0321] As will be understood by one of ordinary skill in the art, each embodiment disclosed hereincan comprise, consist essentially of or consist of its particular stated element, step, ingredient orcomponent. Thus, the terms "include" or "including" should be interpreted to recite: "comprise,consist of, or consist essentially of." As used herein, the transition term "comprise" or "comprises"means has, but is not limited to, and allows for the inclusion of unspecified elements, steps, 30ingredients, or components, even in major amounts. The transitional phrase "consisting of"excludes any element, step, ingredient or component not specified. The transitional phrase"consisting essentially of" limits the scope of the embodiment to the specified elements, steps,ingredients or components and to those that do not materially affect the embodiment. id="p-322"

[0322] Unless otherwise indicated, all numbers expressing quantities of ingredients, propertiessuch as molecular weight, reaction conditions, and so forth used in the specification and claimsare to be understood as being modified in all instances by the term "about." Accordingly, unlessindicated to the contrary, the numerical parameters set forth in the specification and attachedclaims are approximations that may vary depending upon the desired properties sought to be 5obtained by the present invention. At the very least, and not as an attempt to limit the applicationof the doctrine of equivalents to the scope of the claims, each numerical parameter should at leastbe construed in light of the number of reported significant digits and by applying ordinary roundingtechniques. When further clarity is required, the term "about" has the meaning reasonablyascribed to it by a person skilled in the art when used in conjunction with a stated numerical value 10or range, i.e. denoting somewhat more or somewhat less than the stated value or range, to withina range of ±20% of the stated value; ±19% of the stated value; ±18% of the stated value; ±17%of the stated value; ±16% of the stated value; ±15% of the stated value; ±14% of the stated value;±13% of the stated value; ±12% of the stated value; ±11% of the stated value; ±10% of the statedvalue; ±9% of the stated value; ±8% of the stated value; ±7% of the stated value; ±6% of the 15stated value; ±5% of the stated value; ±4% of the stated value; ±3% of the stated value; ±2% ofthe stated value; or ±1% of the stated value. [0323] Notwithstanding that the numerical ranges and parameters setting forth the broad scopeof the invention are approximations, the numerical values set forth in the specific examples arereported as precisely as possible. Any numerical value, however, inherently contains certain 20errors necessarily resulting from the standard deviation found in their respective testingmeasurements. [0324] The terms "a," "an," "the" and similar referents used in the context of describing theinvention (especially in the context of the following claims) are to be construed to cover both thesingular and the plural, unless otherwise indicated herein or clearly contradicted by context. 25Recitation of ranges of values herein is merely intended to serve as a shorthand method ofreferring individually to each separate value falling within the range. Unless otherwise indicatedherein, each individual value is incorporated into the specification as if it were individually recitedherein. All methods described herein can be performed in any suitable order unless otherwiseindicated herein or otherwise clearly contradicted by context. The use of any and all examples, or 30exemplary language (e.g., "such as") provided herein is intended merely to better illuminate theinvention and does not pose a limitation on the scope of the invention otherwise claimed. Nolanguage in the specification should be construed as indicating any non-claimed elementessential to the practice of the invention. id="p-325"

[0325] Groupings of alternative elements or embodiments of the invention disclosed herein arenot to be construed as limitations. Each group member may be referred to and claimed individuallyor in any combination with other members of the group or other elements found herein. It isanticipated that one or more members of a group may be included in, or deleted from, a group forreasons of convenience and/or patentability. When any such inclusion or deletion occurs, the 5specification is deemed to contain the group as modified thus fulfilling the written description ofall Markush groups used in the appended claims. [0326] Certain embodiments of this invention are described herein, including the best modeknown to the inventors for carrying out the invention. Of course, variations on these describedembodiments will become apparent to those of ordinary skill in the art upon reading the foregoing 10description. The inventor expects skilled artisans to employ such variations as appropriate, andthe inventors intend for the invention to be practiced otherwise than specifically described herein.Accordingly, this invention includes all modifications and equivalents of the subject matter recitedin the claims appended hereto as permitted by applicable law. Moreover, any combination of theabove-described elements in all possible variations thereof is encompassed by the invention 15unless otherwise indicated herein or otherwise clearly contradicted by context. [0327] Furthermore, numerous references have been made to patents, printed publications,journal articles and other written text throughout this specification (referenced materials herein).Each of the referenced materials are individually incorporated herein by reference in their entiretyfor their referenced teaching. 20 [0328] In closing, it is to be understood that the embodiments of the invention disclosed hereinare illustrative of the principles of the present invention. Other modifications that may be employedare within the scope of the invention. Thus, by way of example, but not of limitation, alternativeconfigurations of the present invention may be utilized in accordance with the teachings herein.Accordingly, the present invention is not limited to that precisely as shown and described. 25 [0329] The particulars shown herein are by way of example and for purposes of illustrativediscussion of the preferred embodiments of the present invention only and are presented in thecause of providing what is believed to be the most useful and readily understood description ofthe principles and conceptual aspects of various embodiments of the invention. In this regard, noattempt is made to show structural details of the invention in more detail than is necessary for the 30fundamental understanding of the invention, the description taken with the drawings and/orexamples making apparent to those skilled in the art how the several forms of the invention maybe embodied in practice. [0330] Definitions and explanations used in the present disclosure are meant and intended to be controlling in any future construction unless clearly and unambiguously modified in the examplesor when application of the meaning renders any construction meaningless or essentiallymeaningless. In cases where the construction of the term would render it meaningless oressentially meaningless, the definition should be taken from Webster's Dictionary, 3rd Edition ora dictionary known to those of ordinary skill in the art, such as the Oxford Dictionary of 5Biochemistry and Molecular Biology (Eds. Attwood T et al., Oxford University Press, Oxford,2006).

Claims (170)

1. CLAIMSWhat is claimed is:1. A stem cell comprising a conditional immortalization gene.

2. The stem cell of claim 1, wherein the conditional immortalization gene encodes TERT.

3. The stem cell of claim 1, wherein the conditional immortalization gene encodes SV40 large Tantigen.

4. The stem cell of claim 1, wherein the conditional immortalization gene comprises TERT andSV40 large T antigen.

5. The stem cell of claim 1, wherein the conditional immortalization gene is induced by a drug.

6. The stem cell of claim 5, wherein the drug comprises tetracycline or doxycycline.

7. The stem cell of claim 1, wherein the stem cell is a totipotent stem cell, a pluripotent stem cell,a multipotent stem cell, or a unipotent stem cell.

8. The stem cell of claim 1, wherein the pluripotent stem cell is an embryonic stem cell, a cordblood stem cell, or an induced pluripotent stem cells (iPSC).

9. The stem cell of claim 1, wherein the multipotent stem cell is a hematopoietic stem cell, amesenchymal stem cell, or a neuronal stem cell.

10. The stem cell of claim 1, further comprising an exogenous sequence that encodes anexpression product.

11. The stem cell of claim 10, wherein the expression product is a protein.

12. The stem cell of claim 11, wherein the protein comprises a recombinant receptor, a detectablelabel, an antigen, an antibody, or an enzyme.

13. The stem cell of claim 11, wherein the protein comprises CD70, αCD3, CD28, 4-1BB, MICA,4-1BBL, membrane-bound interleukin (IL)-15, and/or membrane-bound IL2114.

14. The stem cell of claim 11, wherein the protein comprises membrane-bound IL21.

15. The stem cell of claim 11, wherein the protein comprises a cancer antigen.

16. The stem cell of claim 15, wherein the cancer comprises multiple myeloma, lymphoma, acutelymphocytic leukemia (ALL), acute myelocytic leukemia (AML), chronic lymphocytic leukemia(CLL), breast cancer, colorectal cancer, ovarian cancer, renal cell carcinoma (RCC),glioblastoma, prostate cancer, neuroblastoma, melanoma, Ewing sarcoma, or hepatocellularcancer (HCC).

17. The stem cell of claim 15, wherein the cancer antigen comprises BCMA, CD4, CD5, CD7,CD19, CD20, CD22, CD33, CD73, CD123, CD133, CD138, CD244, CD276, CS1, EGFR,EGFRVIII, EpCAM, FLT3, GD2, GPA7, GPC3, HER2, Mesothelin, MUC1, NKG2D, PSMA,PSCA, or TF.

18. The stem cell of claim 15, wherein the cancer antigen comprises BCMA, CD19, CD20, CD33,CD133, CD138, CS1, EGFR, EGFRVII, EpCAM, GD2, GPA7, HER2, NKG2D, MUC1, orPSCA.

19. The stem cell of claim 11, wherein the protein comprises a viral, bacterial, fungal, or parasiticantigen.

20. The stem cell of claim 11, wherein the protein comprises insulin, factor VIII, factor IX, factorXI, alpha-1 antitrypsin (A1AT), glucocerebrosidase (GC), acid sphingomyelinase,mucopolysaccharides, acid alpha-glucosidase, aspartylglucosaminidase, alpha-galactosidase A, palmitoyl protein thioesterase, tripeptidyl peptidase, lysosomaltransmembrane protein, cysteine transporter, acid ceramidase, acid alpha-L-fucosidase,cathepsin A, acid beta-glucosidase, acid beta-galactosidase, iduronate-2-sulfatase, alpha-L-iduronidase, galactocerebrosidase, acid alpha-mannosidase, acid beta-mannosidase,arylsulfatase B, arylsulfatase A, N-acetylgalactosamine-6-sulfate, N-acetylglucosamine-1-phosphotransferase, acid sphingomyelinase, NPC-1, alpha-glucosidase, beta-hexosaminidase B, heparan N-sulfatase, alpha-N-acetylglucosaminidase, acetyl-CoA: alpha-glucosaminide, N-acetylglucosamine-6-sulfate, alpha-N-acetylgalactosaminidase, alpha-neuramidase, beta-glucuronidase, beta-hexosaminidase A, or acid lipase.

21. The stem cell of claim 12, wherein the recombinant receptor comprises an extracellularcomponent comprising a binding domain; an intracellular component comprising an effectordomain; and a transmembrane domain linking the extracellular component to the intracellularcomponent.

22. The stem cell of claim 12, wherein the recombinant receptor comprises a chimeric antigenreceptor or an engineered T cell receptor.

23. The stem cell of claim 21, wherein the binding domain of the recombinant receptor binds acancer antigen, a viral antigen, a bacterial antigen, or a fungal antigen.

24. The stem cell of claim 23, wherein the cancer antigen comprises BCMA, CD4, CD5, CD7,CD19, CD20, CD22, CD33, CD73, CD123, CD133, CD138, CD244, CD276, CS1, EGFR,EGFRVIII, EpCAM, FLT3, GD2, GPA7, GPC3, HER2, Mesothelin, MUC1, NKG2D, PSMA,PSCA, or TF.

25. The stem cell of claim 23, wherein the cancer antigen comprises BCMA, CD19, CD20, CD33,CD133, CD138, CS1, EGFR, EGFRVII, EpCAM, GD2, GPA7, HER2, NKG2D, MUC1, orPSCA.

26. The stem cell of claim 21, wherein the effector domain comprises all or a portion of thesignaling domain of CD3 ζ and/or 4-1BB.

27. The stem cell of claim 21, wherein the transmembrane domain comprises a CD28transmembrane domain.

28. The stem cell of claim 12, wherein the recombinant receptor comprises a CD19 bindingdomain.

29. The stem cell of claim 12, wherein the recombinant receptor comprises a BCMA bindingdomain.

30. The stem cell of claim 12, wherein the detectable label comprises a fluorescent protein, aradioisotope, an enzyme label, or a fluorescent label.

31. The stem cell of claim 30, wherein the fluorescent protein comprises luciferase.

32. The stem cell of claim 1, wherein the stem cell is genetically modified to knockout a majorhistocompatibility complex (MHC).

33. The stem cell of claim 1, wherein the stem cell is genetically modified to knockout β2-microglobulin (B2M).

34. The stem cell of claim 1, wherein the stem cell is genetically modified to knockout Class IMajor Histocompatibility Complex Transactivator and/or Class II Major HistocompatibilityComplex Transactivator.

35. The stem cell of claim 1, wherein the stem cell is genetically modified to knockout B2M, CIITA,or B2M and CIITA.

36. The stem cell of claims 33 or 35, wherein B2M is knocked out with the gRNA sequence as setforth in SEQ ID NOs: 34-42.

37. The stem cell of claims 34 or 35, wherein CIITA is knocked out with the gRNA sequence asset forth in SEQ ID NOs: 25-33.

38. The stem cell of claim 1, wherein the stem cell further comprises a suicide gene.

39. The stem cell of claim 38, wherein the suicide gene comprises CDK1 linked Herpes simplexvirus-thymidine kinase/ganciclovir (CDK1/HSV-TK/GCV), TOP2A/HSV-TK/GCV, or inducibleCasp9.

40. The stem cell of claim 38, wherein the suicide gene comprises CDK1/HSV-TK/GCV.

41. The stem cell of claim 1, wherein the stem cell further comprises a sequence encoding a tagcassette, a transduction marker, selection cassette, or a skipping element.

42. A cell line differentiated from the stem cell of claim 1.

43. The cell line of claim 42, wherein the cell line comprises more differentiated stem cells thanthe stem cell of claim 1.

44. The cell line of claim 43, wherein the more differentiated stem cells are CD34+ hematopoieticstem cells or mesenchymal stem cells.

45. The cell line of claim 42, wherein the cell line comprises pancreatic cells, epithelial cells,cardiac cells, endothelial cells, liver cells, endocrine cells, connective tissue cells, musclecells, brain cells, bone cells, kidney cells, cartilage cells, or immune cells.

46. The cell line of claim 45, wherein the pancreatic cells comprise alpha cells, beta cells, or deltacells.

47. The cell line of claim 45, wherein the cardiac cells comprise cardiomyocytes.

48. The cell line of claim 45, wherein the liver cells comprise hepatocytes, hepatic stellate cells(HSCs), Kupffer cells (KCs), or liver sinusoidal endothelial cells (LSECs).

49. The cell line of claim 45, wherein the connective tissue cells comprise fibroblasts.

50. The cell line of claim 45, wherein the muscle cells comprise myoblasts.

51. The cell line of claim 45, wherein the brain cells comprise neurons.

52. The cell line of claim 45, wherein the bone cells comprise osteoblasts or osteoclasts.

53. The cell line of claim 45, wherein the cartilage cells comprise chondrocytes.

54. The cell line of claim 45, wherein the immune cells comprise T-cells, NK cells, ormacrophages.

55. The cell line of claim 42, wherein cells within the cell line are genetically modified to expressan expression product.

56. The cell line of claim 55, wherein the expression product is a protein.

57. The cell line of claim 56, wherein the protein comprises a recombinant receptor, a detectablelabel, an antigen, an antibody, or an enzyme.

58. The cell line of claim 56, wherein the protein comprises CD70, αCD3, CD28, 4-1BB, MICA, 4-1BBL, membrane-bound interleukin (IL)-15, and/or membrane-bound IL2159.

59. The cell line of claim 56, wherein the protein comprises membrane-bound IL21.

60. The cell line of claim 56, wherein the protein comprises a cancer antigen.

61. The cell line of claim 60, wherein the cancer comprises multiple myeloma, lymphoma, acutelymphocytic leukemia (ALL), acute myelocytic leukemia (AML), chronic lymphocytic leukemia(CLL), breast cancer, colorectal cancer, ovarian cancer, renal cell carcinoma (RCC),glioblastoma, prostate cancer, neuroblastoma, melanoma, Ewing sarcoma, or hepatocellularcancer (HCC).

62. The cell line of claim 60, wherein the cancer antigen comprises BCMA, CD4, CD5, CD7,CD19, CD20, CD22, CD33, CD73, CD123, CD133, CD138, CD244, CD276, CS1, EGFR,EGFRVIII, EpCAM, FLT3, GD2, GPA7, GPC3, HER2, Mesothelin, MUC1, NKG2D, PSMA,PSCA, or TF.

63. The cell line of claim 60, wherein the cancer antigen comprises BCMA, CD19, CD20, CD33, CD133, CD138, CS1, EGFR, EGFRVII, EpCAM, GD2, GPA7, HER2, NKG2D, MUC1, orPSCA.

64. The cell line of claim 56, wherein the protein comprises a viral, bacterial, fungal, or parasiticantigen.

65. The cell line of claim 56, wherein the protein comprises insulin, factor VIII, factor IX, factor XI,alpha-1 antitrypsin (A1AT), glucocerebrosidase (GC), acid sphingomyelinase,mucopolysaccharides, acid alpha-glucosidase, aspartylglucosaminidase, alpha-galactosidase A, palmitoyl protein thioesterase, tripeptidyl peptidase, lysosomaltransmembrane protein, cysteine transporter, acid ceramidase, acid alpha-L-fucosidase,cathepsin A, acid beta-glucosidase, acid beta-galactosidase, iduronate-2-sulfatase, alpha-L-iduronidase, galactocerebrosidase, acid alpha-mannosidase, acid beta-mannosidase,arylsulfatase B, arylsulfatase A, N-acetylgalactosamine-6-sulfate, N-acetylglucosamine-1-phosphotransferase, acid sphingomyelinase, NPC-1, alpha-glucosidase, beta-hexosaminidase B, heparan N-sulfatase, alpha-N-acetylglucosaminidase, acetyl-CoA: alpha-glucosaminide, N-acetylglucosamine-6-sulfate, alpha-N-acetylgalactosaminidase, alpha-neuramidase, beta-glucuronidase, beta-hexosaminidase A, or acid lipase.

66. The cell line of claim 57, wherein the recombinant receptor comprises an extracellularcomponent comprising a binding domain; an intracellular component comprising an effectordomain; and a transmembrane domain linking the extracellular component to the intracellularcomponent.

67. The cell line of claim 57, wherein the recombinant receptor comprises a chimeric antigenreceptor or an engineered T cell receptor.

68. The cell line of claim 66, wherein the binding domain of the recombinant receptor binds acancer antigen, a viral antigen, a bacterial antigen, or a fungal antigen.

69. The cell line of claim 68, wherein the cancer antigen comprises BCMA, CD4, CD5, CD7,CD19, CD20, CD22, CD33, CD73, CD123, CD133, CD138, CD244, CD276, CS1, EGFR,EGFRVIII, EpCAM, FLT3, GD2, GPA7, GPC3, HER2, Mesothelin, MUC1, NKG2D, PSMA,PSCA, or TF.

70. The cell line of claim 68, wherein the cancer antigen comprises BCMA, CD19, CD20, CD33,CD133, CD138, CS1, EGFR, EGFRVII, EpCAM, GD2, GPA7, HER2, NKG2D, MUC1, orPSCA.

71. The cell line of claim 66, wherein the effector domain comprises all or a portion of the signalingdomain of CD3 ζ and/or 4-1BB.

72. The cell line of claim 66, wherein the transmembrane domain comprises a CD28 transmembrane domain.

73. The cell line of claim 57, wherein the recombinant receptor comprises a CD19 binding domain.

74. The cell line of claim 57, wherein the recombinant receptor comprises a BCMA bindingdomain.

75. The cell line of claim 57, wherein the detectable label comprises a fluorescent protein, aradioisotope, an enzyme label, or a fluorescent label.

76. The cell line of claim 75, wherein the fluorescent protein comprises luciferase.

77. The cell line of claim 42, wherein cells within the cell line are genetically modified to knockouta major histocompatibility complex (MHC).

78. The cell line of claim 42, wherein cells within the cell line are genetically modified to knockoutβ2-microglobulin (B2M).

79. The cell line of claim 42, wherein cells within the cell line are genetically modified to knockoutClass I Major Histocompatibility Complex Transactivator and/or Class II MajorHistocompatibility Complex Transactivator.

80. The cell line of claim 42, wherein cells within the cell line are genetically modified to knockoutB2M, CIITA, or B2M and CIITA.

81. The cell line of claims 78 or 80, wherein B2M is knocked out with the gRNA sequence as setforth in SEQ ID NOs: 34-42.

82. The cell line of claims 79 or 80, wherein CIITA is knocked out with the gRNA sequence as setforth in SEQ ID NOs: 25-33.

83. The cell line of claim 42, wherein cells within the cell line further comprise a suicide gene.

84. The cell line of claim 83, wherein the suicide gene comprises CDK1 linked Herpes simplexvirus-thymidine kinase/ganciclovir (CDK1/HSV-TK/GCV), TOP2A/HSV-TK/GCV, or inducibleCasp9.

85. The cell line of claim 83, wherein the suicide gene comprises CDK1/HSV-TK/GCV.

86. A method comprising genetically modifying a stem cell to comprise a conditionalimmortalization gene.

87. The method of claim 86, wherein the genetically modifying comprises transfecting a stem cellwith the conditional immortalization gene using the Tet inducible system.

88. The method of claim 86, wherein the conditional immortalization gene encodes TERT.

89. The method of claim 86, wherein the conditional immortalization gene encodes SV40 large Tantigen.

90. The method of claim 86, wherein the conditional immortalization gene comprises TERT andSV40 large T antigen. 100

91. The method of claim 86, wherein the conditional immortalization gene is induced by a drug.

92. The method of claim 91, wherein the drug comprises tetracycline or doxycycline.

93. The method of claim 86, wherein the stem cell is a totipotent stem cell, a pluripotent stem cell,a multipotent stem cell, or a unipotent stem cell.

94. The method of claim 93, wherein the pluripotent stem cell is an embryonic stem cell, a cordblood stem cell, or an induced pluripotent stem cells (iPSC).

95. The method of claim 93, wherein the multipotent stem cell is a hematopoietic stem cell, amesenchymal stem cell, or a neuronal stem cell.

96. The method of claim 86, further genetically modifying the stem cell to comprise an exogenoussequence that encodes an expression product.

97. The method of claim 96, wherein the genetically modifying the stem cell to comprise anexogenous sequence comprises transfecting the stem cell with an expression construct usinga transposon-based system or a lentivirus system.

98. The method of claim 96, wherein the genetically modifying the stem cell to comprise anexogenous sequence comprises transfecting the stem cell with an expression construct usinga transposon-based system.

99. The method of claim 96, wherein the expression product is a protein.

100. The method of claim 99, wherein the protein comprises a recombinant receptor, adetectable label, an antigen, an antibody, or an enzyme.

101. The method of claim 99, wherein the protein comprises CD70, αCD3, CD28, 4-1BB, MICA,4-1BBL, membrane-bound interleukin (IL)-15, and/or membrane-bound IL21102.

102. The method of claim 99, wherein the protein comprises membrane-bound IL21.

103. The method of claim 99, wherein the protein comprises a cancer antigen.

104. The method of claim 103, wherein the cancer comprises multiple myeloma, lymphoma,acute lymphocytic leukemia (ALL), acute myelocytic leukemia (AML), chronic lymphocyticleukemia (CLL), breast cancer, colorectal cancer, ovarian cancer, renal cell carcinoma (RCC),glioblastoma, prostate cancer, neuroblastoma, melanoma, Ewing sarcoma, or hepatocellularcancer (HCC).

105. The method of claim 103, wherein the cancer antigen comprises BCMA, CD4, CD5, CD7,CD19, CD20, CD22, CD33, CD73, CD123, CD133, CD138, CD244, CD276, CS1, EGFR,EGFRVIII, EpCAM, FLT3, GD2, GPA7, GPC3, HER2, Mesothelin, MUC1, NKG2D, PSMA,PSCA, or TF.

106. The method of claim 103, wherein the cancer antigen comprises BCMA, CD19, CD20,CD33, CD133, CD138, CS1, EGFR, EGFRVII, EpCAM, GD2, GPA7, HER2, NKG2D, MUC1, 101 or PSCA.

107. The method of claim 99, wherein the protein comprises a viral, bacterial, fungal, orparasitic antigen.

108. The method of claim 99, wherein the protein comprises insulin, factor VIII, factor IX, factorXI, alpha-1 antitrypsin (A1AT), glucocerebrosidase (GC), acid sphingomyelinase,mucopolysaccharides, acid alpha-glucosidase, aspartylglucosaminidase, alpha-galactosidase A, palmitoyl protein thioesterase, tripeptidyl peptidase, lysosomaltransmembrane protein, cysteine transporter, acid ceramidase, acid alpha-L-fucosidase,cathepsin A, acid beta-glucosidase, acid beta-galactosidase, iduronate-2-sulfatase, alpha-L-iduronidase, galactocerebrosidase, acid alpha-mannosidase, acid beta-mannosidase,arylsulfatase B, arylsulfatase A, N-acetylgalactosamine-6-sulfate, N-acetylglucosamine-1-phosphotransferase, acid sphingomyelinase, NPC-1, alpha-glucosidase, beta-hexosaminidase B, heparan N-sulfatase, alpha-N-acetylglucosaminidase, acetyl-CoA: alpha-glucosaminide, N-acetylglucosamine-6-sulfate, alpha-N-acetylgalactosaminidase, alpha-neuramidase, beta-glucuronidase, beta-hexosaminidase A, or acid lipase.

109. The method of claim 100, wherein the recombinant receptor comprises an extracellularcomponent comprising a binding domain; an intracellular component comprising an effectordomain; and a transmembrane domain linking the extracellular component to the intracellularcomponent.

110. The method of claim 100, wherein the recombinant receptor comprises a chimeric antigenreceptor or an engineered T cell receptor.

111. The method of claim 109, wherein the binding domain of the recombinant receptor bindsa cancer antigen, a viral antigen, a bacterial antigen, or a fungal antigen.

112. The method of claim 111, wherein the cancer antigen comprises BCMA, CD4, CD5, CD7,CD19, CD20, CD22, CD33, CD73, CD123, CD133, CD138, CD244, CD276, CS1, EGFR,EGFRVIII, EpCAM, FLT3, GD2, GPA7, GPC3, HER2, Mesothelin, MUC1, NKG2D, PSMA,PSCA, or TF.

113. The method of claim 111, wherein the cancer antigen comprises BCMA, CD19, CD20,CD33, CD133, CD138, CS1, EGFR, EGFRVII, EpCAM, GD2, GPA7, HER2, NKG2D, MUC1,or PSCA.

114. The method of claim 109, wherein the effector domain comprises all or a portion of thesignaling domain of CD3 ζ and/or 4-1BB.

115. The method of claim 109, wherein the transmembrane domain comprises a CD28transmembrane domain. 102

116. The method of claim 100, wherein the recombinant receptor comprises a CD19 bindingdomain.

117. The method of claim 100, wherein the recombinant receptor comprises a BCMA bindingdomain.

118. The method of claim 100, wherein the detectable label comprises a fluorescent protein, aradioisotope, an enzyme label, or a fluorescent label.

119. The method of claim 118, wherein the fluorescent protein comprises luciferase.

120. The method of claim 86, wherein the stem cells are genetically modified to knockout amajor histocompatibility complex (MHC).

121. The method of claim 86, wherein the stem cells are genetically modified to knockout β2-microglobulin (B2M).

122. The method of claim 86, wherein the stem cells are genetically modified to knockout ClassI Major Histocompatibility Complex Transactivator and/or Class II Major HistocompatibilityComplex Transactivator.

123. The method of claim 86, wherein the stem cells are genetically modified to knockout B2M,CIITA, or B2M and CIITA.

124. The method of claims 121 or 123, wherein B2M is knocked out with the gRNA sequenceas set forth in SEQ ID NOs: 34-42.

125. The method of claims 122 or 123, wherein CIITA is knocked out with the gRNA sequenceas set forth in SEQ ID NOs: 25-33.

126. The method of claim 86, wherein the stem cells are further genetically modified tocomprise a suicide gene.

127. The method of claim 126, wherein the suicide gene comprises CDK1 linked Herpessimplex virus-thymidine kinase/ganciclovir (CDK1/HSV-TK/GCV), TOP2A/HSV-TK/GCV, orinducible Casp9.

128. The method of claim 126, wherein the suicide gene comprises CDK1/HSV-TK/GCV.

129. A method comprising differentiating a stem cell of claim 1 into a more differentiated celltype.

130. The method of claim 129, wherein the more differentiated stem cells comprise CD34+hematopoietic stem cells, mesenchymal stem cells, or neural stem cells.

131. The method of claim 129, wherein the more differentiated cell type comprises moredifferentiated stem cells, pancreatic cells, epithelial cells, cardiac cells, endothelial cells, livercells, endocrine cells, connective tissue cells, muscle cells, brain cells, bone cells, kidneycells, cartilage cells, cancer cells, or immune cells. 103

132. The method of claim 131, wherein the pancreatic cells comprise alpha cells, beta cells, ordelta cells.

133. The method of claim 131, wherein the cardiac cells comprise cardiomyocytes

134. The method of claim 131, wherein the liver cells comprise hepatocytes, hepatic stellatecells (HSCs), Kupffer cells (KCs), and liver sinusoidal endothelial cells (LSECs).

135. The method of claim 131, wherein the connective tissue cells comprise fibroblasts.

136. The method of claim 131, wherein the muscle cells comprise myoblasts.

137. The method of claim 131, wherein the brain cells comprise neurons.

138. The method of claim 131, wherein the bone cells comprise osteoblasts and osteoclasts.

139. The method of claim 131, wherein the cartilage cells comprise chondrocytes.

140. The method of claim 131, wherein the immune cells comprise T-cells, NK cells, ormacrophages.

141. The method of claim 129, further comprising genetically modifying the more differentiatedcell type to comprise an exogenous sequence that encodes an expression product.

142. The method of claim 141, wherein the expression product is a protein.

143. The method of claim 142, wherein the protein comprises a recombinant receptor, adetectable label, an antigen, an antibody, or an enzyme.

144. The method of claim 142, wherein the protein comprises CD70, αCD3, CD28, 4-1BB,MICA, 4-1BBL, membrane-bound interleukin (IL)-15, and/or membrane-bound IL21.

145. The method of claim 142, wherein the protein comprises membrane-bound IL21.

146. The method of claim 142, wherein the protein comprises a cancer antigen.

147. The method of claim 146, wherein the cancer comprises multiple myeloma, lymphoma,acute lymphocytic leukemia (ALL), acute myelocytic leukemia (AML), chronic lymphocyticleukemia (CLL), breast cancer, colorectal cancer, ovarian cancer, renal cell carcinoma (RCC),glioblastoma, prostate cancer, neuroblastoma, melanoma, Ewing sarcoma, or hepatocellularcancer (HCC).

148. The method of claim 146, wherein the cancer antigen comprises BCMA, CD4, CD5, CD7,CD19, CD20, CD22, CD33, CD73, CD123, CD133, CD138, CD244, CD276, CS1, EGFR,EGFRVIII, EpCAM, FLT3, GD2, GPA7, GPC3, HER2, Mesothelin, MUC1, NKG2D, PSMA,PSCA, or TF.

149. The method of claim 146, wherein the cancer antigen comprises BCMA, CD19, CD20,CD33, CD133, CD138, CS1, EGFR, EGFRVII, EpCAM, GD2, GPA7, HER2, NKG2D, MUC1,or PSCA.

150. The method of claim 142, wherein the protein comprises a viral, bacterial, fungal, or 104 parasitic antigen.

151. The method of claim 142, wherein the protein comprises insulin, factor VIII, factor IX, factorXI, alpha-1 antitrypsin (A1AT), glucocerebrosidase (GC), acid sphingomyelinase,mucopolysaccharides, acid alpha-glucosidase, aspartylglucosaminidase, alpha-galactosidase A, palmitoyl protein thioesterase, tripeptidyl peptidase, lysosomaltransmembrane protein, cysteine transporter, acid ceramidase, acid alpha-L-fucosidase,cathepsin A, acid beta-glucosidase, acid beta-galactosidase, iduronate-2-sulfatase, alpha-L-iduronidase, galactocerebrosidase, acid alpha-mannosidase, acid beta-mannosidase,arylsulfatase B, arylsulfatase A, N-acetylgalactosamine-6-sulfate, N-acetylglucosamine-1-phosphotransferase, acid sphingomyelinase, NPC-1, alpha-glucosidase, beta-hexosaminidase B, heparan N-sulfatase, alpha-N-acetylglucosaminidase, acetyl-CoA: alpha-glucosaminide, N-acetylglucosamine-6-sulfate, alpha-N-acetylgalactosaminidase, alpha-neuramidase, beta-glucuronidase, beta-hexosaminidase A, or acid lipase.

152. The method of claim 143, wherein the recombinant receptor comprises an extracellularcomponent comprising a binding domain; an intracellular component comprising an effectordomain; and a transmembrane domain linking the extracellular component to the intracellularcomponent.

153. The method of claim 143, wherein the recombinant receptor comprises a chimeric antigenreceptor or an engineered T cell receptor.

154. The method of claim 152, wherein the binding domain of the recombinant receptor bindsa cancer antigen, a viral antigen, a bacterial antigen, or a fungal antigen.

155. The method of claim 154, wherein the cancer antigen comprises BCMA, CD4, CD5, CD7,CD19, CD20, CD22, CD33, CD73, CD123, CD133, CD138, CD244, CD276, CS1, EGFR,EGFRVIII, EpCAM, FLT3, GD2, GPA7, GPC3, HER2, Mesothelin, MUC1, NKG2D, PSMA,PSCA, or TF.

156. The method of claim 154, wherein the cancer antigen comprises BCMA, CD19, CD20,CD33, CD133, CD138, CS1, EGFR, EGFRVII, EpCAM, GD2, GPA7, HER2, NKG2D, MUC1,or PSCA.

157. The method of claim 152, wherein the effector domain comprises all or a portion of thesignaling domain of CD3 ζ and/or 4-1BB.

158. The method of claim 152, wherein the transmembrane domain comprises a CD28transmembrane domain.

159. The method of claim 143, wherein the recombinant receptor comprises a CD19 bindingdomain. 105

160. The method of claim 143, wherein the recombinant receptor comprises a BCMA bindingdomain.

161. The method of claim 143, wherein the detectable label comprises a fluorescent protein, aradioisotope, an enzyme label, or a fluorescent label.

162. The method of claim 161, wherein the fluorescent protein comprises luciferase.

163. The method of claim 129, further comprising genetically modifying the more differentiatedcell type to knockout major histocompatibility complex (MHC).

164. The method of claim 163, wherein the stem cells are genetically modified to knockoutClass I Major Histocompatibility Complex Transactivator and/or Class II MajorHistocompatibility Complex Transactivator.

165. The method of claim 163, wherein the genetically modifying the more differentiated celltype to knockout MHC comprises knocking out B2M; B2M and CITA; B2M and CIITA; or B2M,CITA, and CIITA.

166. The method of claim 165, wherein the knocking out B2M and CIITA comprises deliveringthe Cas9 nuclease, B2M gRNA, and CIITA gRNA to feeder cells.

167. The method of claim 166, wherein the B2M gRNA comprises SEQ ID NOs: 34-42.

168. The method of claim 166, wherein the CIITA gRNA comprises SEQ ID NOs: 25-33.

169. The method of claim 129, further comprising genetically modifying the more differentiatedcell type to comprise a suicide gene.

170. The method of claim 169, wherein the suicide gene comprises CDK1 linked Herpessimplex virus-thymidine kinase/ganciclovir (CDK1/HSV-TK/GCV) or inducible Casp9. Dr. Hadassa Waterman Patent Attorney G.E. Ehrlich (1995) Ltd. 35 HaMasger Street Sky Tower, 13th Floor Tel Aviv 6721407