WO2013177133A2 - Generation of human ips cells by a synthetic self- replicative rna - Google Patents
Generation of human ips cells by a synthetic self- replicative rna Download PDFInfo
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- WO2013177133A2 WO2013177133A2 PCT/US2013/041980 US2013041980W WO2013177133A2 WO 2013177133 A2 WO2013177133 A2 WO 2013177133A2 US 2013041980 W US2013041980 W US 2013041980W WO 2013177133 A2 WO2013177133 A2 WO 2013177133A2
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Definitions
- iPS induced pluripotent stem cells
- Stems cells are a potential source from which organs may be regenerated, tissues may be repaired, biological factors prepared or delivered and disease or disorders treated.
- iPS induced Pluripotent Stem
- iPS cells require expression of several pluripotent transcription factors or Reprogramming Factors (RFs) , including Oct4, Sox2, Klf4, cMyc, Glisl (and potentially Nanog and Lin28) .
- RFs Reprogramming Factors
- the disclosure describes an approach to generate induced Pluripotent Stem (iPS) cells by ectopically expressing RFs using a synthetic self-replicating RNA from a modified alphavirus (e.g., Venezuelan Equine Encephalitis (VEE) virus) .
- VEE Venezuelan Equine Encephalitis
- the alphavirus was designed to express, in one embodiment, four RFs that resulted in the following advantages over mRNA transfection approaches: 1) utilized a single RNA species capable of self-replicating for a limited number cell divisions, thereby reducing the number of transfections ; 2) is capable of encoding at one, two, three, four, or more RF open reading frames (ORFs) ; and 3) consistently expressed all the RF genes at high threshold levels over multiple cellular divisions.
- ORFs RF open reading frames
- the generation of the alphavirus RF-RNA transcript utilizes a SP6 (or T7) in vitro transcription kit that does not require special conditions and thereby, further simplifies the approach for broad use.
- SP6 or T7 in vitro transcription kit
- the alphavirus-RF RNA approach solves both of the major inefficiency problems associated with attempting to generate iPS cells by daily repeated daily transfections for >14 days of four individual RF mRNAs .
- the alphavirus-RF RNA is an ectopic approach that does not utilize a DNA intermediate and therefore, there is no opportunity for integrative mutation that can occur with DNA vector-based iPS cell approaches.
- the disclosure provides an alphavirus replicon RNA comprising at least one non-structural replicase domain from an alphavirus and at least one non-alphavirus heterologous sequence encoding factors for inducing the generation of pluripotent stem cells when expressed in a somatic cell.
- the replicon comprises sequences obtained from an alphavirus selected from the group consisting of Eastern Equine Encephalitis virus
- the replicon comprises sequences obtained from an alphavirus selected from the group consisting of Sindbis virus, Semliki Forest virus, Middelburg virus, Chikungunya virus, 0 ' nyong-nyong virus, Ross River virus, Barmah Forest virus, Getah virus, Sagiyama virus, Bebaru virus, Mayaro virus, Una virus, Aura virus, Whataroa virus, Babanki virus, Kyzylagach virus, Highlands J virus, Fort Morgan virus, Ndumu virus and Buggy Creek virus.
- the at least one non-alphavirus heterologous sequence comprises at least 2, 3, 4 or 5 non-alphavirus heterologous sequences.
- non-alphavirus heterologous sequence is selected from a polynucleotide encoding a KLF
- polypeptide a SOX-2 polypeptide, a OCT-3/4 polypeptide, a c-MYC or n-MYC or L-MYC polypeptide, a GLIS1 polypeptide, a NANOG
- polypeptide and any combination thereof encodes a KLF polypeptide having at least 95% identity to a sequence of SEQ ID NO : 8.
- polynucleotide encoding the KLF polypeptide encodes a KLF polypeptide having a sequence of SEQ ID NO : 8.
- polynucleotide encoding the KLF polypeptide comprises a sequence as set forth in SEQ ID NO: 7, wherein "T" is "U” .
- the polynucleotide encoding the SOX-2 polypeptide encodes a SOX-2 polypeptide having at least 95% identity to a sequence of SEQ ID NO: 6. In another embodiment, the polynucleotide encoding the SOX-2 polypeptide encodes a SOX-2 polypeptide having a sequence of SEQ ID NO : 6. In yet another embodiment, the polynucleotide encoding the Sox-2 polypeptide comprises a sequence as set forth in SEQ ID NO: 5, wherein "T" is "U” . In another embodiment, the polynucleotide encoding the OCT-4 polypeptide encodes a OCT-4 polypeptide having at least 95% identity to a sequence of SEQ ID NO: 4.
- the polynucleotide encoding the OCT-4 polypeptide encodes a OCT-4 polypeptide having a sequence of SEQ ID NO : 4. In a further embodiment, the polynucleotide encoding the OCT-4
- polypeptide comprises a sequence as set forth in SEQ ID NO: 3, wherein "T” is "U” .
- the polynucleotide encoding the c-MYC polypeptide encodes a c-MYC polypeptide having at least 95% identity to a sequence of SEQ ID NO: 10.
- the polynucleotide encoding the c-MYC polypeptide encodes a c-MYC polypeptide having a sequence of SEQ ID NO: 10.
- the polynucleotide encoding the c-MYC polypeptide comprises a sequence as set forth in SEQ ID NO: 9, wherein "T" is "U” .
- the polynucleotide encoding the GLIS1 polypeptide encodes a GLIS1 polypeptide having at least 95% identity to a sequence of SEQ ID NO: 34. In a further embodiment, the polynucleotide encoding the GLIS1 polypeptide encodes a GLIS1 polypeptide having a sequence of SEQ ID NO: 34. In yet a further embodiment, the polynucleotide encoding the GLIS1 polypeptide comprises a sequence as set forth in SEQ ID NO: 33, wherein "T" is "U” .
- the polynucleotide encoding the NANOG polypeptide encodes a NANOG polypeptide having at least 95% identity to a sequence of SEQ ID NO: 2.
- the polynucleotide encoding the NANOG polypeptide encodes a NANOG polypeptide having a sequence of SEQ ID NO: 2.
- the polynucleotide encoding the NANOG polypeptide comprises a sequence as set forth in SEQ ID NO:l, wherein "T" is "U” .
- the replicon comprises from 5' to 3' : (VEE RNA replicases) - (promoter) - (RFi)-(self cleaving peptide) - (RF 2 ) - (self cleaving peptide )- (RF 3 ) - (IRES or core promoter )- (RF 4 ) -( IRES or optional promoter )- (optional selectable marker) - (VEE 3'UTR and polyA tail) - (optional selectable marker) -promoter ; wherein RFi_ 4 are factors that induce de- differentiation of a somatic cell to a pluripotent cells, wherein RF 2 -3 are optional, RF 3 _ 4 are optional, or RF 4 is optional; wherein RFi_ 4 are selected from the group consisting of Oct-4, Klf4, Sox-2, c-Myc, Nanog, and Glisl.
- the replicon comprise a sequence that is 90%, 95%, 98%, 99% or 100% identical to SEQ ID NO: 29, 30, 31, or 32 from about position 1 to about position 7561 wherein "T" of the sequence is substituted with "U", followed by one or more RFs, followed by a 3'UTR and polyA tail, wherein the one or more RFs are selected from the group consisting of Oct-3/4, Sox-2, Klf4, c-Myc, Nanog, and Glisl; wherein when more than one RF is present, the coding sequences may be separated by an internal ribosome entry site (IRES) or a small promoter.
- the replicon comprise a sequence that is at least 95%, 98%, 99% or 100% identical to a sequence selected from the group consisting of SEQ ID NO: 29, 30, 31, or 32, wherein "T" is "U” .
- composition comprising human cells transformed with a replicon as described in any of the foregoing embodiments and embodiments further described herein.
- the composition further comprises B18R conditioned media.
- the human cells are somatic cells.
- the human cells are fibroblasts.
- the disclosure also provides a method of making stem cells comprising culturing the composition described above and elsewhere herein, for at least 30 days under conditions to express the coding sequences of the replicon and isolating stem cells.
- the disclosure also provides a method of making stem cells comprising transforming somatic cells with a replicon of the disclosure, culturing the somatic cells under conditions to promote expression of the replicon and isolating stem cells.
- the culturing comprise culturing the cells in media conditioned with B18R.
- the B18R conditioned media is produced by transfection of B18R mRNA into primary human fibroblasts .
- the disclosure also provides isolated stem cells obtained from the methods described herein, wherein the stem cells are retroviral DNA- or RNA-free.
- the disclosure also provides a method comprising contacting a human somatic cell with an ectopic self-replicating RNA replicon comprising polynucleotides encoding at least four de- differentiation factors selected from the group consisting of a (i) KLF4, (ii) OCT4, (iii) SOX2, (iv) c-MYC or n-MYC or L-MYC, (v) GLIS1 and (vi) NANOG; culturing the somatic cell to express the de- differentiation factor; selecting cells that display a stem cell morphology and/or stem cell markers; and subculturing the cells to obtain a population of induced stem cells.
- the cells are selected by detecting expression of a Tumor Rejection Antigen 1-60 and/or 1-81.
- the disclosure also provides a vector system for producing human stem cells, comprising at least one self-replicating RNA replicon comprising one or more polynucleotides encoding de- differentiation factors selected from the group consisting of a KLF4, OCT4, SOX2, c-MYC or n-MYC or L-MYC, GLIS1, and NANOG .
- the replicon comprises (a)Oct4, Sox2, Klf4, and c- Myc, or (b)Oct4, Sox2, Klf4, and Glisl .
- the at least one self-replicating RNA vector is derived from an alphavirus.
- the alphavirus is VEE .
- the disclosure also provides an isolated human somatic cell comprising an ectopic RNA replicon comprising one or more de- differentiation polynucleotide sequences.
- the somatic cell upon culture conditions to express the de-differentiation polynucleotides in the ectopic RNA replicon, the somatic cell dedifferentiates .
- the disclosure also provides a cell population comprising the human somatic cell containing an ectopic RNA replicon
- the disclosure also provides a cell population obtained by contacting a human somatic cell with an ectopic self-replicating RNA replicon comprising polynucleotides encoding at least four de- differentiation factors selected from the group consisting of a (i) KLF4, (ii) OCT4, (iii) SOX2, (iv) c-MYC or n-MYC or L-MYC, (v) GLIS1 and (vi) NANOG; culturing the somatic cell to express the de- differentiation factor; selecting cells that display a stem cell morphology and/or stem cell markers; and subculturing the cells to obtain a population of induced stem cells.
- the cells are selected by detecting expression of a Tumor Rejection Antigen 1-60 and/or 1-81.
- the disclosure also provides a recombinant human fibroblast cells containing an ectopic RNA molecule encoding B18R.
- the RNA encoding B18R comprise SEQ ID NO: 39, wherein "T” is replaced with "U” .
- the RNA encodes a polypeptide comprising the sequence set forth in SEQ ID NO: 40.
- the disclosure also provides a method of making B18R conditioned media comprising culturing a human fibroblast cell transformed with RNA encoding B18R under conditions that allow expression of B18R and isolating media from the
- Figure 1A-E shows construction and Persistence of Synthetic VEE-RF RNA Replicons in Primary Human Fibroblasts.
- A Schematic of VEE-RF RNA replicon. 5' end nsPl-4: non-structural proteinsl-4 ; 3' end C, E2, El: Structural proteins. Locations of 26S internal promoter, ribosome shifting 2A peptide, IRES sequence, Puromycin
- Figure 2A-E shows generation of iPS cells by VEE-RF RNA.
- Human fibroblasts were plated on day 0 (dO) and co- transfected (Tfx) with VEE-RF RNA replicon plus B18R mRNA (3:1 ratio) on day 1 (confluent, ⁇ 4xl0 5 cells) and treated with puromycin until day 7 (or 10) as indicated.
- Cells were cultured in B18R-CM until iPS cell colonies were isolated on day 25-30.
- B iPS cell colonies stained with Alkaline Phosphatase were generated with VEE- OKS-iM RNA, but not VEE-OMKS RNA.
- FIG. 3A-E shows characterization of VEE-RF RNA iPS Cell Clones.
- A Expression of ES maker genes by qRT-RCR analysis of BJ and HFF VEE-OKS-iM iPS clones as indicated.
- B DNA methylation analysis of NANOG and OCT4 promoter regions. Solid circle, methylated; Open circle, demethylated . Top numbers indicate CpG number relative to the transcription start site.
- C Genome-wide mRNA sequence profile scatter plot analysis of BJ-OKS-iM #2 and BJ- OKS-iG #5 compared to parental human BJ fibroblasts and human HUES9 embryonic stem cells with pluripotency NANOG, OCT4, SOX2 indicated.
- cytokeratin cytokeratin
- NF-1 neurotrophic factor-1
- GFAP neurotrophic factor-1
- Desmin muscle cells
- AFP primary and definitive endoderm
- Figure 4 shows RT-PCR analysis for checking up the existence of RNA replicon. Measurement of PCR sensitivity with the plasmid of OKS-iM-RNA replicon. PCRs for nsP2, nsP4 and OCT4-T2A- KLF4 (OK) regions were performed with 100, 10 and 1 fg of plasmid
- Figure 5 shows Karyotype Analysis of iPS Cell Clones.
- G- Banded Karyotyping of HFF-OKS-iM-1 , BJ-OKS-iM-2, BJ-OKS-iM-21 and BJ-OKS-iG-5 clones was performed on twenty G-banded metaphase cells from each clone and judged as normal male human karyotype in all clones (Cell line GENETICS) .
- FIG. 6A-B shows iPS cell clones were cultured with STO feeder cells. Cells were collected, and then intramuscularly or subcutaneously injected into the hind limb muscles or dorsal flank of nude mice. After 5 to 8 weeks of injection, tumors were dissected and fixed with 4% paraformaldehyde.
- A Teratoma analysis of HFF-OKS-iM #1 clone in nude mice. AE1/AE3 (cytokeratin) and NF- 1 (neuronal cells) used for markers of ectoderm; Desmin (muscle cells) used for marker of mesoderm; and AFP (primitive and definitive endoderm) used for marker of endoderm. Bar, 100 ⁇ .
- B H&E staining of teratomas from BJ-OKS-iG clones 3 and 5. Bar, 100 ⁇ .
- Figure 7A-D shows (A) B18R Conditioned Media is useful for persistent existence of VEE RNA replicon. Top; % of GFP positive cells, Bottom: mean value of GFP fluorescence in GFP positive population. (B) Photographs of cells. Bar, 200 ⁇ . (C) Protein expression of RFs on day 10 as indicated. (D) B18R-CM is required for generation of iPS cells in feeder culture. HFFs were co- transfected with OKS-iM RNA and B18R mRNA as indicated, and then cells were cultured in the presence of B18R-CM and puromycin.
- Cells were passaged to STO feeder cells on day 10 (dl, 3, 8 transfections) or day 11 (dl, 4, 7, 10 transfections) , and cultured in the presence or absence of B18R-CM plus/minus puromycin.
- iPS cells induced pluripotent stem cells
- ES cells induced pluripotent stem cells
- Induced pluripotent stem cells are described by Shinya Yamanaka ' s team at Kyoto University, Japan. Yamanaka identified genes that are particularly active in embryonic stem cells, and used retroviruses to transfect mouse fibroblasts with a selection of those genes. Eventually, four key pluripotency genes essential for the production of pluripotent stem cells were isolated; Oct-3/4, SOX2, c-Myc, and Klf4. Cells were isolated by antibiotic selection for Fbxl5 + cells. The same group published a study along with two other independent research groups from
- RNA-based iPS cell approaches using Sendai virus or mRNA transfection avoid potential integration problems associated with DNA-based approaches and are inherently safer methods for clinical
- Sendai virus offers a reasonably efficient iPS approach
- problems associated with persistent Sendai virus replication in iPS cell clones requires a negative selection step followed by several recloning steps from the single cell level to isolate virus-free iPS cells, such processes result in excessive iPS cellular division and passage.
- One of the more promising non- DNA based approaches involves daily transfection of four individual RF mRNAs (plus GFP mRNA) over 16 days. Unfortunately, this approach remains problematic. For example, experiments to replace KLF4 and c-MYC retroviruses with corresponding transfected mRNAs were performed and the results validated; however OCT4 and SOX2 retroviruses could not be replaced with transfected mRNAs.
- the disclosure provides methods and compositions for generating iPS cells from somatic cells (e.g., fibroblast cells) .
- the compositions and method comprise the use of replicons derived from alphaviruses .
- the replicons comprise an RNA sequence encoding non-structural alphavirus proteins necessary for replication and 1, 2, 3, 4 or more coding sequences heterologous to the alphavirus and which induce dedifferentiation of somatic cells to stem cell phenotypes .
- alphavirus has its conventional meaning in the art, and includes the various species such as
- VEE Venezuelan Equine Encephalistis
- EEE Encephalistis virus
- EVE Everglades Virus
- MUC Manuvate Virus
- PIX Pixuna Virus
- Western Equine Encephalitis Virus all of which are members of the VEE/EEE Group of alphaviruses.
- alphaviruses include, e.g., Semliki Forest Virus (SFV) , Sindbis, Ross River Virus, Chikungunya Virus, S.A. AR86, Barmah Forest Virus, Middleburg Virus, 0 ' nyong-nyong Virus, Getah Virus, Sagiyama Virus, Bebaru Virus, Mayaro Virus, Una Virus, Aura Virus, Whataroa Virus, Banbanki Virus, Kyzylagach Virus, Highlands J Virus, Fort Morgan Virus, Ndumu Virus, and Buggy Creek Virus.
- Semliki Forest Virus SFV
- Sindbis Sindbis
- Ross River Virus Chikungunya Virus
- S.A. AR86 Barmah Forest Virus
- Middleburg Virus 0 ' nyong-nyong Virus
- Getah Virus Sagiyama Virus
- Bebaru Virus Mayaro Virus
- Una Virus Aura
- Alphaviruses particularly useful in the constructs and methods described herein are VEE/EEE group alphaviruses.
- alphavirus RNA replicon refers to an RNA molecule expressing nonstructural protein genes such that it can direct its own replication (amplification) and comprises, at a minimum, 5' and 3' alphavirus replication recognition sequences, coding sequences for alphavirus nonstructural proteins, and a polyadenylation tract. It may additionally contain one or more elements (e.g., IRES sequences, core or mini-promoters and the like) to direct the expression, meaning transcription and translation, of a
- heterologous RNA sequence The alphavirus replicon of the alphavirus replicon of the alphavirus replicon of the alphavirus replicon of the alphavirus replicon of the alphavirus replicon of the alphavirus replicon of the alphavirus replicon of the alphavirus replicon of the alphavirus replicon of the alphavirus replicon of the alphavirus replicon of the alphavirus replicon of the alphavirus replicon of the alphavirus replicon of the alphavirus replicon of the alphavirus replicon of the alphavirus replicon of the alphavirus replicon of the alphavirus replicon of the alphavirus replicon of the alphavirus replicon of the alphavirus replicon of the alphavirus replicon of the alphavirus replicon of the alphavirus replicon of the alphavirus replicon of the alphavirus
- disclosure can comprise, in one embodiment, 5' and 3' alphavirus replication recognition sequences, coding sequences for alphavirus nonstructural proteins, a polyadenylation tract and one or more of a coding sequence selected from the group consisting of SOX-2, c- Myc, OCT-3/4, Klf, Glisl and Nanog.
- nucleic acid refers to polynucleotides such as deoxyribonucleic acid (DNA) , and, where appropriate (particularly with reference to a replicon) , ribonucleic acid (RNA) .
- DNA deoxyribonucleic acid
- RNA ribonucleic acid
- polynucleotide refers to transcription of the gene or
- polynucleotide and, as appropriate, translation of an mRNA transcript to a protein or polypeptide.
- expression of a protein or polypeptide results from transcription and/or translation of the open reading frame.
- the disclosure provides polynucleotides in the form of recombinant DNA expression vectors, RNA replicons or plasmids, as described in more detail elsewhere herein, that encode one or more polypeptides .
- a polynucleotide of the disclosure can be amplified using cDNA, mRNA or alternatively, genomic DNA, as a template and appropriate oligonucleotide primers according to standard PCR amplification techniques and those procedures described in the Examples section below.
- the nucleic acid so amplified can be cloned into an appropriate vector and characterized by sequence analysis.
- nucleotide sequences can be prepared by standard synthetic techniques, e.g., using an automated DNA synthesizer.
- a replicon of the disclosure comprise a sequence that is 90%, 95%, 98%, 99% or 100% identical to SEQ ID NO:29, 30, 31, or 32 from about position 1 to about position 7561 (including wherein "T” of the sequence can be substituted with "U”) , followed by one or more RFs selected from the group
- the coding sequences may be separated by an internal ribosome entry site (IRES) or a small (e.g., a core) promoter such as SP1.
- IRS internal ribosome entry site
- SP1 small (e.g., a core) promoter
- the order of the RFs is not critical to the disclosure; thus the order may be Klf4, Oct-3/4, Sox-2, c-Myc or can be Sox-2, Klf4, Oct-3/4, c-Myc, or Oct4, Klf4, Sox2, c-Myc or any variation of the order of the RFs.
- the replicon may further comprise a selectable marker (e.g., an antibiotic resistance marker) .
- coding sequences of RFs may be separated by self-cleaving peptides such as T2A and/or E2A.
- the replicon comprises from 5' to 3' : (VEE RNA replicases ) - ( 26S promoter) - (RFi) - (self cleaving peptide) - (RF 2 ) - (self cleaving peptide )- (RF 3 ) -( IRES or core promoter )- (RF 4 ) -( IRES or optional promoter) - (optional selectable marker) - (VEE 3'UTR and polyA tail) ; wherein RFi_ 4 are factors that induce de- differentiation of a somatic cell to a pluripotent cells, wherein RF 2 -3 are optional, RF 3 _ 4 are optional, or RF 4 is optional; wherein RFi_ 4 are selected from the group consisting of Oct-4, Klf4, Sox-2, c-Myc, Nanog, and Glisl.
- the replicon of the foregoing is an RNA molecule.
- the replicon is derived from VEE and includes a mutation to reduce pathogenicity.
- the VEE is a TC-83 strain
- the RFs include variants and degenerate polynucleotide sequences.
- an RF can comprise homologs and variants of an OCT-4 polypeptide, KLF4 polypeptide, SOX-2 polypeptide, c-MYC polypeptide, NANOG
- an RF coding sequence for NANOG useful in any of the replicon embodiments described herein can comprise (i) a polynucleotide encoding a polypeptide of SEQ ID NO : 2 ; (ii) a polynucleotide comprising at least 95% identity to SEQ ID NO:l and which encodes a polypeptide having NANOG activity;
- an RF coding sequence for Oct-4 useful in any of the replicon embodiments described herein can comprise (i) a
- polynucleotide encoding a polypeptide of SEQ ID NO: 4; (ii) a polynucleotide comprising at least 95% identity to SEQ ID NO: 3 and which encodes a polypeptide having Oct-4 activity; (iii) a
- an RF coding sequence for Sox-2 useful in any of the replicon embodiments described herein can comprise (i) a
- polynucleotide encoding a polypeptide of SEQ ID NO: 6; (ii) a polynucleotide comprising at least 95% identity to SEQ ID NO: 5 and which encodes a polypeptide having SOX-2 activity; (iii) a polynucleotide having a sequence as set forth in SEQ ID NO: 5 or
- an RF coding sequence for KLF4 useful in any of the replicon embodiments described herein can comprise (i) a
- polynucleotide encoding a polypeptide of SEQ ID NO: 8; (ii) a polynucleotide comprising at least 95% identity to SEQ ID NO: 7 and which encodes a polypeptide having KLF4 activity; (iii) a polynucleotide having a sequence as set forth in SEQ ID NO: 7 or
- an RF coding sequence for c-MYC useful in any of the replicon embodiments described herein can comprise (i) a
- polynucleotide encoding a polypeptide of SEQ ID NO: 10; (ii) a polynucleotide comprising at least 95% identity to SEQ ID NO: 9 and which encodes a polypeptide having c-MYC activity; (iii) a polynucleotide having a sequence as set forth in SEQ ID NO : 9 or
- an RF coding sequence for GLIS1 useful in any of the replicon embodiments described herein can comprise (i) a
- polynucleotide encoding a polypeptide of SEQ ID NO: 34; (ii) a polynucleotide comprising at least 95% identity to SEQ ID NO: 33 and which encodes a polypeptide having GLIS1 activity; (iii) a polynucleotide having a sequence as set forth in SEQ ID NO: 33 or
- Nanog is a gene expressed in embryonic stem cells (ESCs) and plays a role in maintaining pluripotency . Nanog is thought to function with SOX2.
- a polynucleotide and polypeptide encoding a Nanog is set forth in SEQ ID NO : 1 and 2, respectively.
- SEQ ID NO : 1 comprises a DNA sequence it will be recognized that "T” can be replaced with “U” .
- NANOG is a 305 amino acid protein with a homeodomain motif that is localized to the nuclear component of cells. Similar to murine NANOG, N-terminal region of human NANOG is rich in Ser, Thr and Pro residues and the C-terminus comprises Trp repeats. The homeodomain in human NANOG ranges from about residue 95 to about residue 155. Homologs of human Nanog are known.
- Oct polypeptide refers to any of the naturally- occurring members of Octamer family of transcription factors, or variants thereof that maintain transcription factor activity, similar (within at least 50%, 80%, or 90% activity) compared to the closest related naturally occurring family member, or polypeptides comprising at least the DNA-binding domain of the naturally occurring family member, and can further comprise a transcriptional activation domain.
- Exemplary Oct polypeptides include, Oct-1, Oct- 2, Oct-3/4, Oct-6, Oct-7, Oct-8, Oct-9, and Oct-11. e.g. Oct3/4
- Opt4 contains the POU domain, a 150 amino acid sequence conserved among Pit-1, Oct-1, Oct-2, and uric-86. See, Ryan, A. K. & Rosenfeld, M. G. Genes Dev. 11, 1207-1225
- variants have at least 85%, 90%, or 95% amino acid sequence identity across their whole sequence compared to a naturally occurring Oct polypeptide family member such as to those listed above or such as listed in Genbank accession number NP002692.2 (human Oct4) or NP038661.1 (mouse Oct4) .
- Oct polypeptides e.g., Oct3/4 can be from human, mouse, rat, bovine, porcine, or other animals. Generally, the same species of protein will be used with the species of cells being
- Oct-4 (Octamer-4) is a homeodomain transcription factor of the POU family and regulates the expression of numerous genes (see, e.g., J. Biol. Chem., Vol. 282, Issue 29, 21551-21560, July 20, 2007, incorporated herein by reference) .
- a polynucleotide and polypeptide encoding an Oct4 is set forth in SEQ ID NO: 3 and 4, respectively.
- SEQ ID NO: 3 comprises a DNA sequence it will be recognized that "T” can be replaced with "U”.
- SRY (sex determining region Y) -box 2 also known as SOX2 is a transcription factor that plays a role in self-renewal of undifferentiated embryonic stem cells and transactivation of Fgf4 as well as modulating DNA bending (see, e.g., Scaffidi et al. J. Biol. Chem., Vol. 276, Issue 50, 47296-47302, December 14, 2001, incorporated herein by reference) .
- a "Sox polypeptide” refers to any of the naturally-occurring members of the SRY-related HMG-box
- Sox transcription factors characterized by the presence of the high-mobility group (HMG) domain, or variants thereof that maintain transcription factor activity similar (within at least 50%, 80%, or 90% activity) compared to the closest related naturally occurring family member, or polypeptides comprising at least the DNA-binding domain of the naturally occurring family member, and can further comprise a transcriptional activation domain.
- HMG high-mobility group
- Sox polypeptides include, e.g., Soxl, Sox-2, Sox3, Sox4, Sox5, Sox6, Sox7, Sox8, Sox9, SoxlO, Soxll, Soxl2, Soxl3, Soxl4, Soxl5, Soxl7, Soxl8, Sox-21, and Sox30.
- Soxl has been shown to yield iPS cells with a similar efficiency as Sox2, and genes Sox3, Soxl5, and Soxl8 have also been shown to generate iPS cells, although with somewhat less efficiency than Sox2. See, Nakagawa, et al . , Nature Biotechnology 26:101-106 (2007) .
- variants have at least 85%, 90%, or 95% amino acid sequence identity across their whole sequence compared to a naturally occurring Sox polypeptide family member such as to those listed above or such as listed in Genbank accession number CAA83435 (human Sox2) .
- Sox polypeptides e.g., Soxl, Sox2, Sox3, Soxl5, or Soxl8 can be from human, mouse, rat, bovine, porcine, or other animals. Generally, the same species of protein will be used with the species of cells being manipulated.
- SEQ ID NO: 5 comprises a DNA sequence it will be recognized that "T” can be replaced with "U”. Homologs of human Sox2 are known.
- Kruppel-like factor 4 also known as KLF4 plays a role in stem cell maintenance and growth.
- a "Klf polypeptide” refers to any of the naturally-occurring members of the family of Kruppel-like factors (Klfs) , zinc-finger proteins that contain amino acid sequences similar to those of the Drosophila embryonic pattern regulator Kruppel, or variants of the naturally-occurring members that maintain transcription factor activity similar (within at least 50%, 80%, or 90% activity) compared to the closest related naturally occurring family member, or polypeptides comprising at least the DNA-binding domain of the naturally occurring family member, and can further comprise a transcriptional activation domain. See, Dang, D. T., Pevsner, J.
- Klf family members include, Klfl, Klf2, Klf3, Klf-4, Klf5, Klf6, Klf7, Klf8, Klf9, KlflO, Klfll, Klfl2, Klfl3, Klfl4, Klfl5, Klfl6, and Klfl7.
- Klf2 and Klf-4 were found to be factors capable of generating iPS cells in mice, and related genes Klfl and Klf5 did as well, although with reduced efficiency. See, Nakagawa, et al . , Nature Biotechnology 26:101-106
- variants have at least 85%, 90%, or 95% amino acid sequence identity across their whole sequence compared to a naturally occurring Klf polypeptide family member such as to those listed above or such as listed in Genbank accession number CAX16088 (mouse Klf4) or CAX14962 (human Klf4) .
- Klf polypeptides e.g., Klfl, Klf4, and Klf5
- Klf polypeptides can be from human, mouse, rat, bovine, porcine, or other animals.
- the same species of protein will be used with the species of cells being manipulated.
- Essrb estrogen-related receptor beta
- a polynucleotide and polypeptide encoding an KLF4 is set forth in SEQ ID NO: 7 and 8, respectively.
- SEQ ID NO: 7 comprises a DNA sequence it will be recognized that "T” can be replaced with "U”.
- Homologs of human KLF4 are known and include NP_034767, NM_010637 (Mus musculus), which are incorporated herein by reference .
- the MYC family of cellular genes is comprised of c-myc, N- myc, and L-myc, three genes that function in regulation of cellular proliferation, differentiation, and apoptosis (Henriksson and Luscher 1996; Facchini and Penn 1998) .
- a "Myc polypeptide” refers any of the naturally-occurring members of the Myc family (see, e.g., Adhikary, S. & Eilers, M. Nat. Rev. Mol. Cell Biol. 6:635-645
- variants thereof that maintain transcription factor activity similar (within at least 50%, 80%, or 90% activity) compared to the closest related naturally occurring family member, or polypeptides comprising at least the DNA-binding domain of the naturally occurring family member, and can further comprise a transcriptional activation domain.
- Exemplary Myc polypeptides include, e.g., c-Myc, N-Myc and L-Myc.
- variants have at least 85%, 90%, or 95% amino acid sequence identity across their whole sequence compared to a naturally occurring Myc polypeptide family member, such as to those listed above or such as listed in Genbank accession number CAA25015 (human Myc) .
- Myc polypeptides can be from human, mouse, rat, bovine, porcine, or other animals. Generally, the same species of protein will be used with the species of cells being manipulated. Although myc family genes have common structural and biological activity. N-Myc is a member of the MYC family and encodes a protein with a basic helix-loop-helix (bHLH) domain.
- bHLH basic helix-loop-helix
- N- myc protein is found in the nucleus and dimerizes with another bHLH protein in order to bind DNA.
- a polynucleotide and polypeptide encoding an c-Myc is set forth in SEQ ID NO : 9 and 10, respectively.
- SEQ ID NO : 9 comprises a DNA sequence it will be recognized that "T” can be replaced with "U” .
- Homologs and variants of the Myc family of proteins are known in the art.
- Glisl (Glis Family Zinc Finger 1) is gene encoding a
- Kruppel-like protein of the same name whose locus is found on Chromosome lp32.3 The gene is enriched in unfertilised eggs and embryos at the one cell stage and it can be used to promote direct reprogramming of somatic cells to induced pluripotent stem cells. Glisl can be used as one of the four factors used in reprogramming somatic cells to induced pluripotent stem cells. The three other transcription factors used are Oct3/4, Sox2 and Klf4.
- a human Glisl (NM_147193) is set forth in SEQ ID NO: 33 and 34 (cDNA and polypeptide, respectively) .
- cDNA coding for the human oct4 (pour5fl), sox2, klf4, c-myc (n-myc or L-myc) , Glisl and nanog, variants and homologs thereof can be cloned and expressed using techniques known in the art.
- polynucleotides encoding one or more de-differentiation factors can be cloned into a suitable vector for expression in a cell type of interest.
- An RF "activity" refers the ability to de-differentiate a somatic cell when expressed in combination with other RFs as known in the art.
- an Oct-4 variant can be measured for Oct-4 activity by co-expressing the Oct-4 variant in a somatic cell with klf4, Sox-2 and c-myc and determining if a somatic cell de-differentiates. If the cell dedifferentiates than the Oct-4 variant can be said to have Oct-4 activity .
- the replicon comprises a sequence as set forth in SEQ ID NO:29, 30, 31, or 32.
- the replicon comprises a sequence that is about 90%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% identical to SEQ ID NO: 29, 30, 31, or 32, and wherein when the replicon is transfected into a somatic cells, the somatic cell is "induced” to become a stem cell.
- SEQ ID NO: 29 provides a replicon of the disclosure.
- sequence of SEQ ID NO:29 has "T” replaced with "U”.
- the replicon comprises VEE RNA replicases from nucleotide 1 to about nucleotide 7561, a human Oct-4 sequence from nucleotide 7592 to 8671, a coding sequence for a T2A self- cleaving peptide from nucleotide 8678-8731, a human Klf4 sequence from 8738-10147, a coding sequence for a self-cleaving E2A peptide from nucleotide 10154-10213, a human Sox-2 sequence from 10223- 11176, an internal ribosome entry site from 11195-11805, a human c- Myc sequence from 11818-13140, an internal ribosome entry site from 13165-13776, a puromycin resistance gene from 13777-14376, the VEE 3'UT
- SEQ ID NO: 30 provides a replicon of the disclosure.
- sequence of SEQ ID NO: 30 has "T” replaced with "U”.
- the replicon comprises VEE RNA replicases from nucleotide 1 to about nucleotide 7561, a human Oct-4 sequence from nucleotide 7592 to 8671, a coding sequence for a T2A self- cleaving peptide from nucleotide 8678-8731, a human Klf4 sequence from 8738-10147, a coding sequence for a self-cleaving E2A peptide from nucleotide 10154-10213, a human Sox-2 sequence from 10223- 11176, an internal ribosome entry site from 11195-11805, a human c- Myc sequence from 11818-13140, an internal ribosome entry site from 13165-13776, a puromycin resistance gene from 13777-14376, the VEE 3'UT
- SEQ ID NO: 31 provides a replicon of the disclosure.
- sequence of SEQ ID NO: 31 has "T” replaced with "U”.
- the replicon comprises VEE RNA replicases from nucleotide 1 to about nucleotide 7561, a human Oct-4 sequence from nucleotide 7592 to 8671, a coding sequence for a T2A self- cleaving peptide from nucleotide 8678-8731, a human Klf4 sequence from 8738-10147, a coding sequence for a self-cleaving E2A peptide from nucleotide 10154-10213, a human Sox-2 sequence from 10223- 11176, an internal ribosome entry site from 11195-11805, a human Glisl sequence from 11818-13680, an internal ribosome entry site from 13689-14300, a puromycin resistance gene from 14301-14900, the VEE 3'UTR
- SEQ ID NO: 32 provides a replicon of the disclosure.
- sequence of SEQ ID NO: 32 has "T” replaced with "U”.
- the replicon comprises VEE RNA replicases from nucleotide 1 to about nucleotide 7561, a human Oct-4 sequence from nucleotide 7592 to 8671, a coding sequence for a T2A self- cleaving peptide from nucleotide 8678-8731, a human Klf4 sequence from 8738-10147, a coding sequence for a self-cleaving E2A peptide from nucleotide 10154-10213, a human Sox-2 sequence from 10223- 11176, an internal ribosome entry site from 11195-11805, a human Glisl sequence from 11818-13680, an internal ribosome entry site from 13689-14300, a puromycin resistance gene from 14301-14900, the VEE 3'UTR
- each replicon comprising one or more coding sequences for factors that induce a somatic cell to become a stem cell, wherein the combination of the more than one alphavirus replicons include all the coding sequence for all RFs necessary for inducing de-differentiation into a stem cell.
- an alphavirus replicon comprises coding sequences for expression of OCT-3/4, SOX-2, KLF, c-MYC, GLIS1 and/or NANOG .
- the coding sequences for expression of OCT-3/4, SOX-2, KLF, c-MYC, GLIS1 and/or NANOG comprises coding sequences for expression of OCT-3/4, SOX-2, KLF, c-MYC, GLIS1 and/or NANOG .
- alphavirus replicon comprises coding sequences for OCT-4, KLF4, SOX-2, GLIS1 and c-MYC.
- the replicon may also be engineered to express alphavirus structural proteins.
- U.S. Pat. Nos . 7,045,335, 7,078,218, 7,425,337 and 7,442,381 describe numerous constructs for such alphavirus RNA replicons consisting of the 5' and 3' alphavirus replication recognition sequences, coding sequences for alphavirus
- nonstructural proteins and a polyadenylation tract, and such constructs are incorporated herein by reference. Specific
- embodiments of the alphavirus RNA replicons may contain one or more attenuating mutations, an attenuating mutation being a nucleotide deletion, addition, or substitution of one or more nucleotide (s) , or a mutation that comprises rearrangement or chimeric construction which results in a loss of virulence in a live virus containing the mutation as compared to the appropriate wild-type alphavirus.
- alphavirus structural protein/protein refers to one or a combination of the structural proteins encoded by alphaviruses . These are produced by the virus as a polyprotein and are represented generally in the literature as C-E3-E2-6k-El . E3 and 6k serve as membrane translocation/transport signals for the two glycoproteins, E2 and El.
- E3 and 6k serve as membrane translocation/transport signals for the two glycoproteins, E2 and El.
- E2 herein can refer to El, E3-E1, 6k-El, or E3-6k-El
- E2 herein can refer to E2, E3-E2, 6k-E2, or E3-6k-E2.
- Attenuating mutations can be introduced into any one or more of the alphavirus structural proteins.
- homologs used with respect to an original enzyme or gene of a first family or species refers to distinct enzymes or genes of a second family or species which are determined by functional, structural or genomic analyses to be an enzyme or gene of the second family or species which corresponds to the original enzyme or gene of the first family or species. Most often, homologs will have
- homologs of an enzyme or gene can readily be cloned using genetic probes and PCR. Identity of cloned sequences as homolog can be confirmed using functional assays and/or by genomic mapping of the genes.
- a protein has "homology” or is "homologous” to a second protein if the nucleic acid sequence that encodes the protein has a similar sequence to the nucleic acid sequence that encodes the second protein.
- a protein has homology to a second protein if the two proteins have "similar" amino acid sequences.
- homologous proteins are defined to mean that the two proteins have similar amino acid sequences.
- two proteins are substantially homologous when the amino acid sequences have at least about 30%, 40%, 50% 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity.
- the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment and non-homologous sequences can be disregarded for comparison purposes) .
- the length of a reference sequence aligned for comparison purposes is at least 30%, typically at least 40%, more typically at least 50%, even more typically at least 60%, and even more typically at least 70%, 80%, 90%, 100% of the length of the reference sequence.
- the amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position (as used herein amino acid or nucleic acid "identity" is equivalent to amino acid or nucleic acid "homology”) .
- the percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences.
- the percent sequence identity or degree of homology may be adjusted upwards to correct for the conservative nature of the substitution. Means for making this adjustment are well known to those of skill in the art (see, e.g., Pearson et al . , 1994, hereby incorporated herein by reference) .
- a "conservative amino acid substitution” is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain.
- Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine) , acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine) , nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan) , beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryp
- the following six groups each contain amino acids that are conservative substitutions for one another: 1) Serine (S) , Threonine (T) ; 2) Aspartic Acid (D) , Glutamic Acid (E) ; 3) Asparagine (N) , Glutamine
- Sequence homology for polypeptides is typically measured using sequence analysis software. See, e.g., the Sequence Analysis Software Package of the Genetics Computer Group (GCG) , University of Wisconsin Biotechnology Center, 910 University Avenue, Madison, Wis. 53705. Protein analysis software matches similar sequences using measure of homology assigned to various substitutions, deletions and other modifications, including conservative amino acid substitutions. For instance, GCG contains programs such as "Gap” and "Bestfit” which can be used with default parameters to determine sequence homology or sequence identity between closely related polypeptides, such as homologous polypeptides from different species of organisms or between a wild type protein and a mutein thereof. See, e.g., GCG Version 6.1.
- BLAST Altschul, 1990; Gish, 1993; Madden, 1996; Altschul, 1997; Zhang, 1997), especially blastp or tblastn (Altschul, 1997) .
- Typical parameters for BLASTp are: Expectation value: 10 (default); Filter: seg (default); Cost to open a gap: 11 (default); Cost to extend a gap: 1 (default); Max. alignments: 100 (default); Word size: 11 (default); No. of descriptions: 100 (default); Penalty Matrix: BLOWSUM62.
- polypeptide sequences can be compared using FASTA, a program in GCG Version 6.1.
- FASTA provides alignments and percent sequence identity of the regions of the best overlap between the query and search sequences (Pearson, 1990, hereby incorporated herein by reference) .
- percent sequence identity between amino acid sequences can be determined using FASTA with its default parameters (a word size of 2 and the PAM250 scoring matrix), as provided in GCG Version 6.1, hereby incorporated herein by reference.
- compositions and methods of the disclosure provide the ability to de-differentiate somatic cells to form stem cells (e.g., induce the formation of stem cells) .
- stem cells are cells capable of differentiation into other cell types, including those having a particular, specialized function (e.g., tissue specific cells, parenchymal cells and progenitors thereof) .
- tissue specific cells e.g., tissue specific cells, parenchymal cells and progenitors thereof
- progenitor cells can be either multipotent or pluripotent.
- Progenitor cells are cells that can give rise to different terminally differentiated cell types, and cells that are capable of giving rise to various progenitor cells. The term
- pluripotent refers to cells with the ability to give rise to progeny cells that can undergo differentiation, under the appropriate conditions, into cell types that collectively demonstrate characteristics associated with cell lineages from all of the three germinal layers (endoderm, mesoderm, and ectoderm) .
- Pluripotent stem cells can contribute to all embryonic derived tissues of a prenatal, postnatal or adult animal.
- a standard art- accepted test such as the ability to form a teratoma in 8-12 week old SCID mice, can be used to establish the pluripotency of a cell population; however identification of various pluripotent stem cell characteristics can also be used to detect pluripotent cells.
- Pluripotent stem cell characteristics refer to characteristics of a cell that distinguish pluripotent stem cells from other cells. The ability to give rise to progeny that can undergo
- pluripotent stem cell characteristic expression or non-expression of certain combinations of molecular markers are also pluripotent stem cell characteristics.
- human pluripotent stem cells express at least some, and in some
- pluripotent stem cells are also pluripotent stem cell characteristics.
- a multipotent stem cell is capable of differentiating into a subset of cells compared to a pluripotent stem cell.
- a multipotent stem cell may be able to undergo differentiation into one or two of the three germinal layers.
- non-pluripotent cells refer to mammalian cells that are not pluripotent cells.
- Examples of such cells include differentiated cells as well as multipotent cells.
- Examples of differentiated cells include, but are not limited to, cells from a tissue selected from bone marrow, skin, skeletal muscle, fat tissue and peripheral blood.
- Exemplary cell types include, but are not limited to, fibroblasts, hepatocytes, myoblasts, neurons, osteoblasts, osteoclasts, and T-cells.
- pluripotent stem cells uncommitted to a particular differentiation fate
- pluripotent stem cells are the so-called "totipotent" stem cells (e.g., fertilized oocytes, cells of embryos at the two and four cell stages of development) , which have the ability to differentiate into any type of cell of the particular species.
- totipotent stem cell could give rise to a complete animal, as well as to any of the myriad of cell types found in the
- Pluripotent stem cells are a type of cells that undergo self-renewal while maintaining an ability to give rise to all three germ layer-derived tissues and germ cell lineages.
- pluripotent human embryonic stem (hES) cells derived from human blastocysts are promising sources for cell-based therapies to treat diseases and disorders such as Parkinson's disease, cardiac infarction, spinal cord injury, and diabetes mellitus, their clinical potentials has been hampered by their immunogenicity and ethical concerns .
- progenitor cell and “stem cell” are used interchangeably in the art and herein and refer either to a pluripotent, or lineage-uncommitted, progenitor cell, which is potentially capable of an unlimited number of mitotic divisions to either renew its line or to produce progeny cells which will differentiate into fibroblasts or a lineage-committed progenitor cell and its progeny, which is capable of self-renewal and is capable of differentiating into a parenchymal cell type.
- lineage-committed progenitor cells are generally considered to be incapable of giving rise to numerous cell types that phenotypically differ from each other. Instead, they give rise to one or possibly two lineage-committed cell types.
- terminally differentiated human cells e.g., human dermal fibroblasts
- an ectopic mRNA expression system e.g., a replicon system
- the disclosure contemplates the use of a variety of de-differentiation (also referred to as Reprogramming Factors
- RFs RFs ) coding sequence
- a polynucleotide that encodes KLF4, OCT4, SOX2, c-MYC or n-MYC (L-Myc) , GLIS1, NANOG or any combination thereof e.g., KLF4, OCT4, SOX2, c-MYC or n-MYC
- De-differentiation may be achieved by contacting a cell, in vivo or in vitro, with one or more self- replicating RNA vectors that remain ectopic to the host cell genome and encode factors that induce de-differentiation.
- the ectopic self-replicating RNA vector of the disclosure can be controlled by culturing a host cell transformed with the self-replicating RNA vector in the presence of B18R.
- Methods for promoting de-differentiation provide methods of promoting regeneration of mammalian cells and tissues damaged by injury or disease.
- the disclosure also provides methods for enriching for induced stem cells and populations comprising such enriched stem cells.
- the generation of patient-specific pluripotent stem cells has the potential to dramatically speed the implementation of stem cells into clinical use to treat degenerative diseases.
- the disclosure provides methods to employ easily donated stromal cells, such as dermal fibroblasts, from a patient and generate Human Induced Pluripotent Stem (hiPS or iPS) cells by ectopic expression of a set of de-differentiation factors comprising RNA encoding (i) KLF4, OCT4, SOX2, c-MYC or n-MYC (L-Myc) , NANOG or any combination thereof; (ii) KLF4, OCT4, SOX2, and GLIS1; and (iii) KLF4, OCT4, SOX2, and NANOG.
- the cell lines generated are physiologically and morphologically indistinguishable from Human Embryonic Stem Cells
- HESC human embryo
- de-differentiation is familiar to the person skilled in the relevant art. In general de-differentiation signifies the regression of lineage committed cell to the status of a stem cell, for example, by "inducing" a de-differentiated phenotype .
- KLF4, OCT4, SOX2, c-MYC or n-MYC or L-MYC, GLIS1 and/or Nanog can induce de- differentiation and induction of mitosis in lineage committed mitotically inhibited cells.
- the disclosure provides a cell culture comprising human somatic cells that have been transformed with a replicon of the disclosure.
- the somatic cells are fibroblasts.
- the somatic cells are keratinocytes .
- the replicon comprises a sequence that is 90%, 95%, 98%, 99% or 100% identical to SEQ ID NO:29, 30, 31, or 32 from about position 1 to about position 7561
- the coding sequences may be separated by an internal ribosome entry site (IRES) or a small (e.g., a core) promoter such as SP1.
- IRS internal ribosome entry site
- SP1 small (e.g., a core) promoter
- the order of the RFs is not critical to the disclosure; thus the order may be Klf4, Oct-3/4, Sox-2, c-Myc or can be Sox-2, Klf4, Oct-3/4, c-Myc, or Oct4, Klf4, Sox2, c-Myc or any variation of the order of the RFs.
- the replicon comprises a sequence that is at least about 95%, 98%, 99% or 100% identical to a sequence as set forth in SEQ ID NO:29, 30, 31, or 32.
- the cells are cultured in conditioned media comprising B18R and/or are co-trasformed with a polynucleotide encoding B18R.
- the disclosure also provide methods of making a stem cell from a somatic cell comprising transforming the somatic cell with an RNA replicon as described in the disclosure and culturing the somatic cell under conditions to promote expression of coding sequences in the replicon and culturing the cells for a sufficient period of time to de-differentiate the cells to stem cells.
- the cells are passaged at least 5, 10, 15, 20 or more times.
- the cells are cultured for at least 10, 20, 30 or more days.
- the cells are cultured in conditioned media comprising B18R or are co-transformed with a polynucleotide encoding B18R.
- the disclosure also provides induced stem cell cultures obtained by the methods described herein.
- the stem cells do not contain any heterologous RF factors in the genomic DNA of the cell.
- the stem cells do not contain any retroviral DNA or RNA (e.g., stem cells that are retroviral DNA- or RNA-free) .
- the disclosure provides isolated induced stem cells, individually or in populations.
- isolated or “purified” when referring to stem cells of the disclosure means cells that are substantially free of cells carrying markers associated with lineage dedication.
- the human induced pluripotent stem cells are at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99% free of such contaminating cell types.
- the isolated stem cells also are substantially free of soluble, naturally occurring molecules.
- a substantially purified stem cell of the disclosure can be obtained, for example, by extraction (e.g., via density gradient
- a stem cell of the disclosure can be 99%-100% purified by, for example, flow cytometry (e.g., FACS analysis), as discussed herein. Such purified iPS cells will lack any retroviral DNA or RNA.
- the disclosure provides an enriched population of induced stem cells.
- An "enriched population of induced stem cells” is one wherein induced stem cells of the disclosure have been partially separated from other cell types, such that the resulting population of cells has a greater
- the enriched population of induced stem cells can have greater than about a 10-fold, 100-fold, 500-fold, 1,000-fold, 2,000-fold, 3,000-fold, 4,000-fold, 5,000-fold, 6,000-fold, 7,000- fold, 8,000-fold, 9,000-fold, 10,000-fold or greater concentration of induced stem cells than the original population had prior to separation.
- Induced stem cells of the disclosure can, for example, make up at least 5%, 10%, 15%, 20%, 35%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or more of the enriched population of stem cells.
- the enriched population of induced stem cells may be obtained by, for example, selecting against cells displaying markers associated with differentiated cells, or other undesired cell types, and/or selecting for cells displaying markers (e.g., TRA-1-81 and/or TRA-1-60) associated with the human induced pluripotent stem cells of the disclosure, and/or by regenerating isolated stem cells in defined culture systems.
- markers e.g., TRA-1-81 and/or TRA-1-60
- the loss of expression of a marker may also be used for enrichment.
- Such enriched iPS cells will lack any retroviral RNA or DNA typically used to transform cells with RFs .
- the disclosure provides cell lines of induced stem cells.
- a "cell line” means a culture of stem cells of the disclosure, or progeny cells thereof, that can be reproduced for an extended period of time, preferably
- a "culture” means a population of induced stem cells grown in a medium and optionally passaged accordingly.
- a stem cell culture may be a primary culture (e.g., a culture that has not been passaged) or may be a secondary or subsequent culture (e.g., a population of cells which have been subcultured or passaged one or more times) .
- the disclosure provides cells that are de-differentiated to stem cells (i.e., induced stem cells) comprising characteristics including the ability of self-renewal and differentiation into mesoderme, endoderm and epiderm, wherein the de-differentiated cells can be produced by expression of one or more RFs ectopic to the host cell genome using a replicating RNA vector.
- the replicon vector is derived from an alphavirus (e.g., Venezuelan Equine Encehalitis virus) .
- Therapeutic uses of the human induced pluripotent stem cells of the disclosure include transplanting the human induced pluripotent stem cells, stem cell populations, or progeny thereof into individuals to treat a variety of pathological states including diseases and disorders resulting from cancers, neoplasms, injury, viral infections, diabetes and the like.
- Stem cells or stem cell populations are introduced into a subject in need of such stem cells or progeny or in need of a KLF4, OCT4, SOX2, c-MYC or n-MYC or L-MYC, NANOG, GLIS1 or any combination thereof protein or molecule encoded or produced by the genetically altered cell.
- the human induced pluripotent stem cells can be administered to cancer patients who have undergone chemotherapy that have killed, reduced, or damaged stem cells or other cells of a subject, wherein the induced stems cells replace the damaged or dead cells.
- the human induced pluripotent stem cells can be transfected or transformed (in addition to the de-differentiation factors) with at least one additional
- the stem cells may be transformed with a polynucleotide encoding a therapeutic polypeptide .
- Such a method and compositions can provide stem cell bioreactors for the
- the iPS cells may be isolated, transformed with a polynucleotide encoding a therapeutic polypeptide and may then be implanted or administered to a subject, or may be differentiated to a desired cell type and implanted and delivered to the subject. Under such conditions the polynucleotide is expressed within the subject for delivery of the polypeptide product .
- the human cells are derived from a heterologous (non- autologous/allogenic) source compared to the recipient subject, concomitant immunosuppression therapy is typically administered, e.g., administration of the immunosuppressive agent cyclosporine or FK506.
- concomitant immunosuppression therapy is typically administered, e.g., administration of the immunosuppressive agent cyclosporine or FK506.
- the human induced pluripotent stem cells of the disclosure can be administered to a recipient in the absence of immunomodulatory
- the cells can be encapsulated in a membrane, which permits exchange of fluids but prevents cell/cell contact. Transplantation of microencapsulated cells is known in the art, e.g., Balladur et al . , 1995, Surgery 117:189-94, 1995; and Dixit et al . , 1992, Cell Transplantation 1 : 275-79.
- the cells may be introduced directly into the peripheral blood or deposited within other locations throughout the body, e.g., a desired tissue, or on microcarrier beads in the peritoneum.
- 10 2 to 10 9 cells can be transplanted in a single procedure, and additional transplants can be performed as required.
- Differentiation of the human induced pluripotent stem cells or de-differentiation of lineage committed (mitotically inhibited) cells can be induced ex vivo, or alterantively may be induced by contact with tissue in vivo, (e.g., by contact with fibroblasts or cell matrix components) .
- a differentiating agent or de- differentiation agent e.g., KLF4, OCT4, SOX2, c-MYC or n-MYC or L- MYC, NANOG, GLIS1, or any combination thereof or an agonist thereof
- KLF4, OCT4, SOX2, c-MYC or n-MYC or L- MYC, NANOG, GLIS1, or any combination thereof or an agonist thereof may be co-administered or subsequently administered to the subj ect .
- the human induced pluripotent stem cells of the disclosure provide an alternative source of islet cells to prevent or treat diabetes.
- induced pluripotent stem cells of the disclosure can be generated, isolated and
- the induced pluripotent stem cells can be delivered to the pancreas of the subject and differentiated to islet cells in vivo. Accordingly, the cells are useful for transplantation in order to prevent or treat the occurrence of diabetes .
- the disclosure contemplates that the in vitro methods described herein can be used for autologous transplantation of dedifferentiated or redifferentiated cells (e.g., the cells are harvested from and returned to the same individual) .
- the disclosure further contemplates that the in vitro methods described herein can be used for non-autologous transplantations.
- the transplantation occurs between a genetically related donor and recipient.
- the transplantation occurs between a genetically un-related donor and recipient.
- the disclosure contemplates that dedifferentiated cells can be expanded in culture and stored for later retrieval and use.
- redifferentiated cells can be can be expanded in culture and stored for later retrieval and use.
- compositions and methods of the disclosure may be applied to a procedure wherein differentiated (lineage committed) cells are removed from the a subject, de-differentiated in culture, and then either reintroduced into that individual or, while still in culture, manipulated to redifferentiate along specific differentiation pathways (e.g., pancreatic cells, neuronal cells, liver cells, skin cells, cardiovascular cells, gastrointestinal cells and the like) .
- specific differentiation pathways e.g., pancreatic cells, neuronal cells, liver cells, skin cells, cardiovascular cells, gastrointestinal cells and the like.
- Such redifferentiated cells can then be introduced to the individual.
- differentiated lineage committed
- fibroblasts can be removed, de-differentiated (e.g., with ectopic expression of of a replicon of the disclosure comprising KLF4, OCT4, SOX2, c-MYC or n-MYC or L-MYC, GLIS1, NANOG or any
- the method comprises removing differentiated cells from an injured or diseased subject.
- Cells de-differentiated from cells harvested from an injured subject can later be returned to the injured or diseased subject to treat an injury or degenerative disease.
- the dedifferentiated cells can be reintroduced at the site or injury, or the cells can be reintroduced at a site distant from the injury.
- cells can be harvested from an injured subject, dedifferentiated in vitro, redifferentiated in vitro, and
- the human induced pluripotent stem cells of the disclosure can be isolated from a sample obtained from a mammalian subject.
- the subject can be any mammal (e.g., bovine, ovine, porcine, canine, feline, equine, primate), including a human.
- the sample of cells may be obtained from any of a number of different sources including, for example, bone marrow, fetal tissue (e.g., fetal liver tissue) , peripheral blood, umbilical cord blood, pancreas and the like.
- the disclosure provides methods of establishing and/or maintaining populations of stem cells, or the progeny thereof, as well as mixed populations comprising both stem cells and progeny cells, and the populations of cells so produced.
- the human induced pluripotent stem cells of the disclosure once a culture of cells or a mixed culture of stem cells is established, the population of cells is mitotically expanded in vitro by passage to fresh medium as cell density dictates under conditions conducive to cell proliferation, with or without tissue formation.
- Such culturing methods can include, for example, passaging the cells in culture medium lacking particular growth factors that induce differentiation (e.g., IGF, EGF, FGF, VEGF, and/or other growth factor) , in the presence of an agent that stimulates (e.g., an agonist) of KLF4, OCT4, SOX2, c-MYC or n-MYC or L-MYC, NANOG, GLIS1 or any combination thereof, in the presence of KLF4, OCT4, SOX2, c-MYC or n-MYC or L-MYC, NANOG, Glisl or any combination thereof, or any combination of the foregoing.
- growth factors that induce differentiation e.g., IGF, EGF, FGF, VEGF, and/or other growth factor
- an agent that stimulates e.g., an agonist
- Cultures comprising fibroblast or fibroblast-like cells and mixed cultures comprising stem cells and fibroblast cells can be transferred to fresh medium when sufficient cell density is reached.
- Some stem cell types do not demonstrate typical contact inhibition-apoptosis or they become quiescent when density is maximum. Accordingly, appropriate passaging techniques can be used to reduce contact inhibition and quiescence.
- transferring a portion of the cells to a new culture vessel with fresh medium can be done in any culture vessel .
- human induced pluripotent stem cells of the disclosure may be maintained or stored in cell "banks" comprising either continuous in vitro cultures of cells requiring regular transfer or cells which have been cryopreserved .
- Cryopreservation of stem cells, or other cell of the disclosure may be carried out according to known methods, such as those described in Doyle et al . , (eds.), 1995, Cell & Tissue
- cells may be suspended in a "freeze medium" such as, for example, culture medium further comprising 15-20% fetal bovine serum (FBS) and 10%
- a "freeze medium” such as, for example, culture medium further comprising 15-20% fetal bovine serum (FBS) and 10%
- DMSO dimethylsulfoxide
- glycerol dimethylsulfoxide
- the cells are dispensed into glass or plastic vials which are then sealed and transferred to a freezing chamber of a programmable or passive freezer.
- the optimal rate of freezing may be determined empirically. For example, a freezing program that gives a change in temperature of -1 ° C/min through the heat of fusion may be used.
- vials containing the cells Once vials containing the cells have reached -80 °C, they are transferred to a liquid nitrogen storage area. Cryopreserved cells can be stored for a period of years, though they should be checked at least every 5 years for maintenance of viability.
- the cryopreserved cells of the disclosure constitute a bank of cells, portions of which can be withdrawn by thawing and then used to produce a stem cell culture comprising stem cells, as needed.
- Thawing should generally be carried out rapidly, for example, by transferring a vial from liquid nitrogen to a 37 °C water bath.
- the thawed contents of the vial should be immediately transferred under sterile conditions to a culture vessel containing an appropriate medium. It is advisable that the cells in the culture medium be adjusted to an initial density of about l-3xl0 5 cells/ml.
- the cells may be examined daily, for example, with an inverted microscope to detect cell proliferation, and subcultured as soon as they reach an appropriate density.
- the human induced pluripotent stem cells of the disclosure may be withdrawn from a cell bank as needed, and used for the production of new stem cells, either in vitro, for example, as a three dimensional tissue culture, as described below, or in vivo, for example, by direct administration of cells to the site where new fibroblasts or tissue is needed.
- the human induced pluripotent stem cells of the disclosure may be used to produce new tissue for use in a subject where the cells were originally isolated from that subject's own blood or other tissue (i.e., autologous cells).
- disclosure may be used as ubiquitous donor cells to produce new tissue for use in any subject (i.e., heterologous cells).
- a culture of stem cells may be used to produce progeny cells and/or fibroblasts capable of producing new tissue. Differentiation of stem cells to fibroblasts or other cell types, followed by the production of tissue therefrom, can be triggered by specific exogenous growth factors or by changing the culture conditions (e.g., the density) of a stem cell culture.
- the cells are pluripotent, they can be used to reconstitute an irradiated subject 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.
- 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
- LIF Steel Factor
- Stl Steel Factor
- the human induced pluripotent stem cells are genetically engineered to express genes for specific types of growth factors for successful and/or improved
- the cells of the disclosure may be used to treat subjects requiring the repair or replacement of tissue resulting from disease or trauma. Treatment may entail the use of the cells of the disclosure to produce new tissue, and the use of the tissue thus produced, according to any method presently known in the art or to be developed in the future.
- the induced cells e.g., cells comprising an ectopic expression vector expressing KLF4, OCT4, SOX2, c-MYC or n-MYC or L-MYC, NANOG, Glisl or any combination thereof
- administration includes the administration of genetically modified stem cells.
- a formulation comprising the cells of the disclosure is prepared for injection directly to the site where the production of new tissue is desired.
- the cells of the disclosure may be suspended in a hydrogel solution for injection.
- the hydrogel solution containing the cells may be allowed to harden, for instance in a mold to form a matrix having cells dispersed therein prior to implantation. Once the matrix has hardened, the cell 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 convalent, ionic, or hydrogen bonds to create a three-dimensional open-lattice structure, which entraps water molecules to form a gel. Examples of materials which can be used to form a hydrogel include
- polysaccharides such as alginate and salts thereof
- polyphosphazines, and polyacrylates which are cross-linked ionically, polyethylene oxide-polypropylene glycol block copolymers which are cross-linked by temperature or pH, respectively.
- Methods of synthesis of the hydrogel materials, as well as methods for preparing such hydrogels, are known in the art.
- Such cell formulations may further comprise one or more other components, including selected extracellular matrix
- growth factors which may be usefully incorporated into the cell formulation include one or more tissue growth factors known in the art such as, but not limited to, any member of the TGF- ⁇ family, IGF-I and -II, growth hormone, BMPs such as BMP-13, and the like.
- tissue growth factors known in the art such as, but not limited to, any member of the TGF- ⁇ family, IGF-I and -II, growth hormone, BMPs such as BMP-13, and the like.
- the cells of the disclosure may be genetically engineered to express and produce growth factors such as BMP-13 or TGF- ⁇ .
- Other components may also be included in the formulation include, for example, buffers to provide appropriate pH and isotonicity, lubricants, viscous materials to retain the cells at or near the site of
- administration e.g., alginates, agars and plant gums
- other cell types that may produce a desired effect at the site of administration (e.g., enhancement or modification of the formation of tissue or its physicochemical characteristics, support for the viability of the cells, or inhibition of inflammation or
- the cells can be covered by an appropriate wound covering to prevent cells from leaving the site.
- wound coverings are known to those of skill in the art.
- the human induced pluripotent stem cells of the disclosure may be seeded onto a three-dimensional framework or scaffold and cultured to allow the cells to differentiate, grow and fill the matrix or immediately 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.
- 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 or improve the practice of the disclosure.
- the human induced pluripotent stem cells of the disclosure can be used in conjunction with a three-dimensional culture system in a "bioreactor" to produce tissue constructs which possess critical biochemical, physical and structural properties of native human tissue by culturing the cells and resulting tissue under environmental conditions which are typically experienced by native tissue.
- the bioreactor may include a number of designs.
- the culture conditions will include placing a physiological stress on the construct containing cells similar to what will be encountered in vivo.
- the human induced pluripotent stem cells, their progeny, and tissue of the disclosure can be used in a variety of diseases and conditions.
- differentiated form an undifferentiated form, a de-differentiated form.
- Such cells and tissues serve to repair, replace or augment tissue that has been damaged due to disease or trauma, or that failed to develop normally.
- the human induced pluripotent stem cells and tissue produced according to the disclosure can be used to repair or replace damaged or destroyed tissue or to augment existing tissue.
- the cells or tissue of the disclosure can be used, for example, to screen in vitro for the efficacy and/or cytotoxicity of compounds, allergens, growth/regulatory factors, pharmaceutical compounds, and the like on stem cells, to elucidate the mechanism of certain diseases by determining changes in the biological activity of the stem cells (e.g., changes in KLF4, OCT4, SOX2, c-MYC or n-MYC or L-MYC, NANOG, Glisl or any combination thereof expression or activity, proliferative capacity, adhesion) , to study the mechanism by which drugs and/or growth factors operate to modulate stem cell biological activity (e.g., KLF4, OCT4, SOX2, c-MYC or n-MYC or L-MYC, NANOG, Glisl or any combination thereof expression or activity) , to diagnose and monitor cancer in a patient, for gene therapy, gene delivery or protein delivery; and to produce biologically active products .
- stem cells e.g., changes
- the human induced pluripotent stem cells also can be used in the isolation and evaluation of factors associated with the differentiation and maturation of stem cells.
- the human induced pluripotent stem cells may be used in assays to determine the activity of media, such as conditioned media, evaluate fluids for cell growth activity, involvement with dedication of particular lineages, or the like.
- media such as conditioned media
- fluids for cell growth activity involvement with dedication of particular lineages, or the like.
- Various systems are applicable and can be designed to induced differentiation of the human induced
- pluripotent stem cells based upon various physiological stresses.
- the human induced pluripotent stem cells, progeny thereof, and tissues derived therefrom of the disclosure may be used in vitro to screen a wide variety of agents for effectiveness and cytotoxicity of pharmaceutical agents, growth/regulatory factors, anti-inflammatory agents, and the like.
- the cells or tissue cultures of the disclosure can be maintained in vitro and exposed to the agent to be tested.
- the activity of a cytotoxic agent can be measured by its ability to damage or kill stem cells or their progeny in culture. This can be assessed readily by staining techniques.
- the effect of growth/regulatory factors can be assessed by analyzing the number of living cells in vitro, e.g., by total cell counts, and differential cell counts.
- the effect of various drugs on the cells of the disclosure can be assessed either in a suspension culture or in a three-dimensional system.
- the effect of a test agent on the human induced pluripotent stem cells of the disclosure can be analyzed.
- Stem cells which express a gene product of interest, or tissue produced in vitro therefrom, can be implanted into a subject who is otherwise deficient in that gene product.
- genes that express products capable of preventing or ameliorating symptoms of various types of vascular diseases or disorders, or that prevent or promote inflammatory disorders are of particular interest.
- the cells of the disclosure are genetically engineered 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.
- a stem cell of the disclosure can be genetically engineered to express one or more anti-inflammatory gene products including, for example, peptides or polypeptides corresponding to the idiotype of antibodies that neutralize granulocyte-macrophage colony stimulating factor (GM-CSF) , TNF, IL-1, IL-2, or other inflammatory cytokines.
- GM-CSF granulocyte-macrophage colony stimulating factor
- TNF granulocyte-macrophage colony stimulating factor
- IL-1 has been shown to decrease the synthesis of proteoglycans and collagens type II, IX, and XI (Tyler et al . , 1985, Biochem. J. 227:69-878; Tyler et al . , 1988, Coll. Relat. Res. 82:393-405; Goldring et al . , 1988, J.
- TNF also inhibits synthesis of proteoglycans and type II collagen, although it is much less potent than IL-1 (Yaron, I., et al . , 1989, Arthritis Rheum. 32:173-80; Ikebe, T., et al . , 1988, J. Immunol. 140:827-31; and Saklatvala, J., 1986, Nature 322:547-49).
- the cells of the disclosure may be engineered to express the gene encoding the human complement regulatory protein that prevents rejection of a graft by the host. See, for example, McCurry et al . , 1995, Nature Medicine 1:423-27.
- the human induced pluripotent stem cells may be engineered to include a gene or polynucleotides sequence that expresses or causes to be expressed an angiogenic factor.
- the induced stem cells of the disclosure express one or more markers associated with a human pluripotent stem cell phenotype and/or lack one or more markers associated with a differentiated cell (e.g., a cell having a reduced capacity for self-renewal, regeneration, or differentiation) and/or a cell of neuronal origin.
- a molecule is a "marker" of a desired cell type if it is found on a sufficiently high percentage of cells of the desired cell type, and found on a sufficiently low percentage of cells of an undesired cell type.
- a marker can be displayed on, for example, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or more of the desired cell type, and can be displayed on fewer than 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, 1% or fewer of an undesired cell type.
- the disclosure or induced stem cells that have been differentiated are characterized by the presence and/or the absence of certain markers that are specifically recognized by a molecule. Accordingly, in one aspect, the disclosure provides methods of labeling induced stem cells of the disclosure.
- the human induced pluripotent stem cells are labeled with a molecule (e.g., an antibody) that specifically recognizes a marker that is associated with an induced stem cell of the disclosure.
- a molecule e.g., an antibody
- a population of cells is contacted with a molecule that specifically binds to a marker (e.g., TRA-1-81) under conditions that allow the molecule to bind to the marker, wherein the population of cells comprises at least one stem cell having said marker.
- a population of cells is contacted with a molecule that specifically binds to a marker under
- the molecule used can be, for example, an antibody, an antibody derivative, or a ligand.
- the molecule optionally can comprise an additional moiety, for example, one that is detectable (e.g., a fluorescent or colorimetric label) or one that aids in the isolation of the labeled cells (e.g., a moiety that is bound by another molecule or a magnetic particle) .
- the population of transformed somatic cells undergoes live staining for a Tumor Rejection Antigen 1-61 and 1-81 (TRA-1-60, TRA-1-81) .
- TRA-1-60 and TRA-1-81 may be obtained commercially, for example from Chemicon International, Inc
- a population of somatic cells that have been transformed with at least one ectopic RNA vector comprising a KLF4, OCT4, SOX2, c-MYC or n-MYC or L-MYC, and optionally or alternatively NANOG or Glisl are enriched for cells comprising TRA- 1-81 or TRA-1-60 expression.
- the cells may also be enriched for the loss of a detectable marker associated with a retroviral vector.
- the disclosure provides methods of isolating induced stem cells of the disclosure.
- the human induced pluripotent stem cells of the disclosure can be isolated by, for example, utilizing molecules (e.g., antibodies, antibody
- a marker e.g., a TRA-1-81, a TRA-1-60 or a combination of markers
- positive selection methods include methods of preferentially promoting the growth of a desired cell type in a mixed population of desired and undesired cell types.
- positive selection methods include methods of preferentially promoting the growth of a desired cell type in a mixed population of desired and undesired cell types.
- the undesired cells containing such markers can be removed from the desired cells (i.e., a negative selection) .
- negative selection methods include preferentially killing or inhibiting the growth of an undesired cell type in a mixed
- an enriched population of stem cell can be made.
- Procedures for separation may include magnetic separation, using antibody-coated magnetic beads, affinity chromatography, cytotoxic agents joined to a monoclonal antibody, or such agents used in conjunction with a monoclonal antibody, e.g., complement and cytotoxins, and "panning" with antibody attached to a solid matrix (e.g., plate), or other convenient technique.
- Techniques providing accurate separation include fluorescence activated cell sorters, which can have varying degrees of sophistication, e.g., a plurality of color channels, low angle and obtuse light scattering detecting channels, and impedance channels. Conveniently,
- antibodies may be conjugated with markers, such as magnetic beads, which allow for direct separation, biotin, which can be removed with avidin or streptavidin bound to a support, fluorochromes , which can be used with a fluorescence activated cell sorter, or the like, to allow for ease of separation of the particular cell type. Any technique may be employed which is not unduly detrimental to the viability of the human induced pluripotent stem cells.
- the cells are incubated with an antibody against a marker (e.g., a TRA-1-81 antibody) and the cells that stain positive for the marker are manually selected and subcultured.
- a marker e.g., a TRA-1-81 antibody
- Combinations of enrichment methods may be used to improve the time or efficiency of purification or enrichment.
- the cells may be further separated or enriched by a fluorescence activated cell sorter (FACS) or other methodology having high specificity.
- FACS fluorescence activated cell sorter
- Multicolor analyses may be employed with a FACS.
- the cells may be separated on the basis of the level of staining for a particular antigen or lack thereof.
- Fluorochromes may be used to label antibodies specific for a particular antigen. Such fluorochromes include phycobiliproteins , e.g., phycoerythrin and
- Induced stem cell markers useful for enrichment comprise expressed markers such as TRA-1-81 and loss of markers (e.g., GFP) associated with a retroviral vector or other exogenous vector.
- stem cells Once stem cells have been isolated, they optionally can be propagated in appropriate medium in the presence of absence of a feeder layer.
- the human induced pluripotent stem cells of the invention may be cultured in a bioreactor system.
- the human induced pluripotent stem cells of the disclosure may be maintained or stored in cell "banks" comprising either continuous in vitro cultures of cells requiring regular transfer or cells which have been cryopreserved .
- the banked cells are used for autologous treatment of a subject.
- Fibroblasts may be readily isolated by disaggregating an appropriate organ or tissue which is to serve as the source of the fibroblasts. This may be readily accomplished using techniques known to those skilled in the art.
- the tissue or organ can be disaggregated mechanically and/or treated with digestive enzymes and/or chelating agents that weaken the connections between neighboring cells making it possible to disperse the tissue into a suspension of individual cells without appreciable cell breakage.
- Enzymatic dissociation can be accomplished by mincing the tissue and treating the minced tissue with any of a number of digestive enzymes either alone or in combination.
- the suspension can be fractionated into
- cell separation accomplished using standard techniques for cell separation including, but not limited to, cloning and selection of specific cell types, selective destruction of unwanted cells (negative selection) , separation based upon differential cell agglutinability in the mixed population, freeze-thaw procedures, differential adherence properties of the cells in the mixed population, filtration, conventional and zonal centrifugation, centrifugal elutriation (counterstreaming centrifugation) , unit gravity separation, countercurrent distribution, electrophoresis and fluorescence-activated cell sorting.
- clonal selection and cell separation techniques see Freshney, Culture of Animal Cells. A Manual of Basic Techniques, 2d Ed., A.R. Liss, Inc., New York, 1987, Ch . 11 and 12, pp. 137-168.
- fibroblasts may, for example, be carried out as follows: fresh tissue samples are thoroughly washed and minced in Hanks balanced salt solution (HBSS) in order to remove serum. The minced tissue is incubated from 1-12 hours in a freshly prepared solution of a dissociating enzyme such as trypsin. After such incubation, the dissociated cells are suspended, pelleted by centrifugation and plated onto culture dishes. All fibroblasts will attach before other cells, therefore, appropriate stromal cells can be selectively isolated and grown.
- HBSS Hanks balanced salt solution
- the de-differentiated cells are to be used for transplantation or implantation in vivo it is useful to obtain the stromal cells from the patient's own tissues.
- Oligonucleotide probes and primers can be used to identify expression of various factors described herein as well as in cloning and amplification procedures.
- An oligonucleotide probe or a primer refers to a nucleic acid molecule of between 8 and 2000 nucleotides in length. More particularly, the length of these oligonucleotides can range from about 8, 10, 15, 20, or 30 to 100 nucleotides, but will typically be about 10 to 50 (e.g., 15 to 30 nucleotides) .
- the appropriate length for oligonucleotides in assays of the disclosure under a particular set of conditions may be empirically determined by one of skill in the art.
- Oligonucleotide primers and probes can be prepared by any suitable method, including, for example, cloning and restriction of appropriate sequences and direct chemical synthesis based upon the known KLF4, OCT4, SOX2, c-MYC or n-MYC or L-MYC, NANOG or any combination thereof polynucleotide and polypeptide sequence.
- Oligonucleotide probes and primers can comprise nucleic acid analogs such as, for example, peptide nucleic acids, locked nucleic acid (LNA) analogs, and morpholino analogs.
- the 3' end of the probe can be functionalized with a capture or detectable label to assist in detection of a KLF4, OCT4, SOX2, c-MYC or n-MYC or L- MYC, NANOG, Glisl or any combination thereof nucleic acid.
- Any of the oligonucleotides or nucleic acid of the disclosure can be labeled by incorporating a detectable label measurable by spectroscopic, photochemical, biochemical,
- such labels can comprise radioactive substances ( 32 P, 35 S, 3 H, 125 I) , fluorescent dyes (5-bromodesoxyuridin, fluorescein, acetylaminofluorene ,
- oligonucleotides are typically labeled at their 3' and 5' ends.
- the oligonucleotide primers and probes can be immobilized on a solid support.
- Solid supports are known to those skilled in the art and include the walls of wells of a reaction tray, test tubes, polystyrene beads, magnetic beads, nitrocellulose strips, membranes, microparticles such as latex particles, glass and the like.
- the solid support is not critical and can be selected by one skilled in the art.
- microtiter wells glass or silicon chips and the like are all suitable examples.
- Suitable methods for immobilizing are:
- oligonucleotides on a solid phase include ionic, hydrophobic, covalent interactions and the like.
- the solid support can be chosen for its intrinsic ability to attract and immobilize the capture reagent.
- the oligonucleotide probes or primers can be attached to or immobilized on a solid support individually or in groups of about 2-10,000 distinct oligonucleotides of the disclosure to a single solid support.
- a substrate comprising a plurality of oligonucleotide primers or probes of the disclosure may be used either for detecting or amplifying KLF4, OCT4, SOX2, c-MYC or n-MYC or L-MYC, NANOG, Glisl or any combination thereof.
- the oligonucleotide probes can be used in an oligonucleotide chip such as those marketed by Affymetrix and described in U.S. Pat. No.
- arrays can be produced using mechanical synthesis methods or light directed synthesis methods which incorporate a combination of photolithographic methods and solid phase oligonucleotide
- the disclosure further contemplates antibodies capable of specifically binding to a KLF4, OCT4, SOX2, c-MYC or n-MYC or L- MYC, NANOG, or Glisl polypeptide.
- a reference or control population refers to a group of subjects or individuals who are predicted to be representative of the general population.
- a test sample is measured for the amount of KLF4, OCT4, SOX2, c-MYC or n-MYC or L-MYC, NANOG, Glisl or any combination thereof in the sample, wherein the amount is compared to a control sample .
- the disclosure provides methods of differentiating stem cells along a committed lineage comprising inhibiting the expression or activity of KLF4, OCT4, SOX2, c-MYC or n-MYC or L-MYC, NANOG, Glisl or any combination thereof.
- Differentiation agents useful in this regard include, for example, antibodies, antisense oligonucleotides, RNAi constructs, or ribozymes .
- BJ foreskin fibroblasts and STO cell line were obtained from ATCC .
- Primary human foreskin fibroblasts (HFF) and HUES-9 human ES cell line were obtained from existing sources.
- BJ, HFFs and STO were cultured in DMEM containing 10% FBS, MEM Non- Essential Amino Acids (NEAA) , Pyruvate, penicillin, and
- HUES-9 and iPS cells were cultured with ES culture medium in Knockout D-MEM containing 20% Knockout SR, GlutaMAX, NEAA, 2-Mercaptoethanol (all from Invitrogen) , penicillin, streptomycin, and bFGF (10 ng/ml) .
- STO feeder cells were prepared by mitomycin C treatment (10 g/ml, Sigma) .
- iPS cell clones and HUES-9 cells were passaged on MatrigelTM (BD Bioscience) coated wells and cultured in the conditioned medium prepared from STO feeder cells with ES culture medium .
- the cDNAs were used as templates for PCR amplification to add restriction enzyme sites and/or Kozak sequence, and cloned into pBluescript SK+ vector for checking of cDNA sequences. Then cDNAs were cloned into pTNT vector (Promega) for mRNA synthesis and pCX4bsrl for the retrovirus production.
- pTNT vector Promega
- F2A oligos, T2A oligos and E2A oligos (Table 1) were annealed and cloned into EcoRI/Spel, Spel/Xbal and Xbal/Notl sites of
- pBluescript SK+ vector pBluescript SK+ vector, respectively.
- cDNAs of reprogramming factors were linked with 2A peptide sequences in frame, and then cloned into pVEE-S-IRES-Puro .
- pVEE-S-IRES-Puro were constructed from p5' VEE/S/GFP/Pac3 to clone reprogramming factors.
- GFP/Pac genes and partial 3'UTR in p5' VEE/S/GFP/Pac were deleted with Xbal/Mfel digestion, and then introduced the multiple cloning sites (MCS; Ndel, Ascl, BbvCI, Clal, Mfel, Fsel and Notl) (Table 1), IRES and Puromycin resistance gene from pCX4puro.
- MCS multiple cloning sites
- IRES Puromycin resistance gene from pCX4puro.
- This vector was renamed as pVEE-IRES-Puro for convenience.
- the SP6 promoter ATTTAGGTGACACTATAG (see, e.g., SEQ ID NO:31 from 16844-16861) was replaced to T7 promoter
- E2A-R 5'-GGCCGCGGGCGCGCCAGGACCGGGGTTACTTTCAACATCGCCAGC E2A-oligo
- VEE-MCS-F1 5'-CTAGCATATGGGCGCCCTCAGCATCGATGGCCGGCCTCTAGAGC MCS-oligo
- VEE-MCS-R1 5'-GGCCGCGGCCGCTCTAGAGGCCGGCCATCGATGCTGAGGGCGCGC MCS-oligo
- nsP2a-F1 5'-CAGGACGATCTCATTCTCAC (SEQ ID NO:19) PCR, nsP2 nsP2a-R1 5'-GCTTGCCACTCCTCTATCGTG (SEQ ID NO:20) PCR, nsP2 nsP4a-F1 5'-CCACAATACGATCGGCAGTG (SEQ ID NO:21 ) PCR, nsP4 nsP4a-R1 5'-ATGTCCTGCAACATATTCAAA (SEQ ID NO:22) PCR, nsP4 hOct4RTa-F1 5'-CGGCGCCAGAAGGGCAAGCG (SEQ ID NO:23) PCR, OK hKlf4RTb-R1 5'-CACCTGCTTGACGCAGTGTC (SEQ ID NO:24) PCR, OK hKlf4GC2For 5 -GCAGGAGGCGGTCTCTTCGTGCACC (SEQ ID NO
- pTNT-Bl 8R plasmid was used for the synthesis of B18R mRNA.
- the pTNT vector contains a 5' ⁇ -globin leader sequence and a synthetic poly (A) tail (30 bases) to enhance the expression of genes. 30 bases of poly (A) were not enough to stabilize mRNA, so additional poly (A) tail was added by poly (A) tail polymerase.
- B18R-mRNA synthesis was performed with modified nucleotides using the RiboMAX Large Scale RNA Production System-SP6 (Promega) kit. Modification was performed with replacement of 100% of UTP with psuedouridine (Psi) (TriLink
- the mRNA was purified by extraction with
- PCI Phenol/Chloroform/Isoamyl alcohol
- CI Chloroform/Isoamyl alcohol
- RNA precipitation (2.5 M) , which is selectively precipitates RNA, while leaving most of the protein, DNA and unincorporated NTPs in the supernatant according to the manufacture's protocol (Epicentre) .
- 10 g of linearized plasmid for 100 ⁇ reaction scale was used and received about 400 g mRNA.
- ScriptCap m7G Capping SystemTM was used and ScriptCap 2'-0- Methyltransferase (Epicentre, currently available from CELLSCRIPT) to produce cap 1-capped RNA, which proceeds to quantitative completion of capping.
- RNA was briefly purified by ammonium acetate precipitation, and then additional poly (A) tail was added by Poly (A) Polymerase (Epicentre, currently available from CELLSCRIPT) .
- the mRNA bearing 5' -Capping and poly (A) tail was purified by extraction with PCI and CI, followed by ammonium precipitation.
- template plasmid was linearized by digestion with Mlul, and then used for RNA synthesis in the same way with mRNA synthesis. The synthesis of RNA replicon was performed without RNA modification.
- RNA was purified by ammonium acetate precipitation without organic purification because most of large RNA was trapped into intermediate phase after organic extraction.
- the replicon RNA was added 5' -Capping and poly (A) tail as described above, and then purified by ammonium acetate precipitation without organic purification. All RNAs were resuspended in the RNA Storage Solution (Ambion) at l g/ l concentration and stored at -80 °C until use.
- B18 conditioned medium B18R-CM
- 25% double modified B18R mRNA (1 g for 1 well of 6-well plate) was transfected into HFFs with Lipofectamine 2000 (Invitrogen) . After 3 hr, cells were cultured in Advanced DMEM (Invitrogen) containing 15% FCS (ES cell qualified, Millipore) , penicillin, and
- B18R-CM 20% B18R-CM
- the activity of B18R-CM was briefly measured by the efficiency of repeated transfection of mRNAs .
- Lipofectamine 2000 25% double modified B18-mRNA or 100% Psi modified mRNA were used for co-transfection . After 3 hr,
- transfection medium was changed to the Advanced DMEM (Invitrogen) containing 15% FCS (ES cell qualified, Millipore) , penicillin, and streptomycin.
- Cells were cultured in medium containing B18R-CM and puromycin (0.8 g/ml) from day-2. Medium was changed every day and transfections were performed every 3 days (day-1, 4, 7, 10 or 14) .
- ES medium was used from day-7. Puromycin was removed at day-7 or day-11.
- ES medium was changed every day and cultured until iPS cell colonies were generated.
- Colonies were mechanically picked for isolation of clones or stained with Alkaline Phosphase Detection kit (Millipore) or manually prepared AP-staining solution containing 1 mg/ml of FastRed TR (Sigma) and 0.4 mg/ml of 1-Naphthyl phosphate (Sigma) in AP buffer (100 mM Tris, 100 mM NaCl and 50 mM MgCl 2 , pH 9.5)
- RNA replicon was detected with RNeasy mini kit (Qiagen) or TRIzol (Invitrogen) .
- RNAs were then purified with ammonium acetate precipitation.
- Synthesis of cDNAs was performed with QuantiTect Rev. Transcription Kit (Qiagen) or iScript cDNA synsethis kit (Bio-Rad) from 1 g of total RNA. 1-2 ⁇ of 20 ⁇ RT reaction was used for PCR amplification. PCR was performed with Taq DNA plolymerase (NEB) supplemented with PCRx enhancer (Invitrogen) : 3 min at 94 °C for initial denature; 36 cyles of 94 °C for 25 sec, 56 °C for 25 sec, 68 °C for 30 sec; followed by 72 °C for 5 min.
- NEB Taq DNA plolymerase
- PCRx enhancer Invitrogen
- RNAs from feeder free culture of iPSCs clones, HUES-9, BJ and HFFs were isolated with RNeasy mini kit.
- TaqMan RT-PCR reactions were carried out using RNA-to-Ct one- step reaction (Applied Biosystem) according to manufacturer's protocol. 10 ng of total RNA were used per reaction.
- Nanog Hs02387400_gl ; TDGF1 Hs02339499_gl Quantitative PCR reactions were carried out in triplicate, and conditions were as followed: 20 min 55 °C, 10 min 95 °C, 40 cycles of 95 °C for 0.15 min, 65 °C for 1 min. Data were analyzed on the 7300 real-time PCR system (Applied Biosystems) using the delta-delta Ct method.
- iPSC clones were cultured with STO feeder cells. Cells were collected by accutase treatment, and then intramuscularly or subcutaneously injected into the hind limb muscles or dorsal flank of nude mice (approximately 10 cm dish cultured cells for 1 shot of injection) . After 5 to 8 weeks of injection, tumors were dissected and fixed with 4%
- cytokeratin cytokeratin
- NF-1 neurotrophic factor-1
- GFAP neurotrophic factor
- Primary antibodies such as rabbit anti-Oct4, goat anti- Nanog and anti-Sox2, mouse anti-SSEA4, anti-Tra-1-60 and anti-Tra- 1-81 antibodies were used at 1:100 to 1:500 dilutions. Alexa Fluor 488 (BD Biosciences) secondary antibodies were used at 1:800 dilutions .
- Antibodies used in this research are as follows; anti-OCT4 (sc-9081), anti-KLF4 (sc-20691), anti-GLISl (sc- 67584), anti-c-MYC (sc-42), anti-LIN28 (sc-54030), TRA-1-60 (sc- 21705), SSEA1 (sc-21702) and SSEA4 (sc-21704) from Santa Cruz;
- TRA-1- 81 (09-0011) from Stemgent
- AE1/AE3 (RB-9010P0)
- Desmin MS-376- SO
- AFP RB-365
- GFAP RB-087
- Labvision NF-1 (NB-300- 155) from Novus Biological.
- RNA Sequence Total RNAs were isolated with RNeasy mini kit (Qiagen) , and cDNA library of each cells were synthesized and analyzed as known in the art.
- VEE Venezuelan Equine Encephalitis
- VEE encodes four non-structural replication complex proteins (nsP) as a single ORF in the 5' end of the RNA that is separated from the viral structural protein ORF in the 3' end (Fig. la) .
- nsP non-structural replication complex proteins
- Petrakova et al . showed the ability to express exogenous proteins by replacing the 3' structural proteins ORF with GFP.
- exposure of cells to single stranded VEE RNA induces a strong IFN-alpha/beta innate immune response that has severely limited this approach.
- VEE-GFP RNA was produced using a standard SP6 polymerase in vitro transcription kit followed by 5' -capping, and poly (A) tail addition resulting in a high yield, full length 11,500 nt RNA transcript.
- Western Vaccinia virus was used, which binds to and neutralizes type I IFNs.
- a comparison of transfection of primary human foreskin fibroblasts (HFFs) with VEE-GFP RNA alone was performed, in the presence of recombinant B18R protein or with co-transfection of B18R mRNA. Consistent with induction of a strong innate immune response to cells exposed to single stranded RNA, in the absence of B18R, little to no GFP expression was observed (Fig. lb) . Although addition of recombinant B18R protein increased GFP expression, the GFP fluorescence level was very low.
- VEE-GFP RNA/B18R mRNA transfected cells showed a high level of GFP expression on day 1, the expression level was rapidly reduced over the next several days to baseline values by day 7 (Fig. le) .
- VEE-GFP RNA transfected cells stopped growing and/or were killed by the innate immune response (Fig. Id) .
- B18R- CM/puro treated VEE-GFP RNA/B18R mRNA transfected cells maintained persistent high levels of GFP expression in >90% of cells with healthy growth characteristics (Fig. ld,e) .
- VEE RNA replicon 3' ORF was engineered to encode a single combined ORF of three reprogramming factors, OCT4, KLF4, SOX2, separated by internal ribosomal skipping 2A peptides.
- the ORFs were followed by an IRES then either c-MYC (OKS-iM) or GLIS18
- VEE-RF RNAs were produced by SP6 in vitro transcription, 5' -capping, and poly (A) tail addition resulting in a high yield, full length
- RNA-based generation iPS cell protocol To develop an RNA-based generation iPS cell protocol, several parameters were evaluated, including number and timing of VEE-RF RNA transfections , selection for VEE-RF RNA replicon retention by puromycin, and the genetic organization of the VEE-RF RNA replicon (Fig. la, 2a) . Although even a single or double transfection of RF-RNA resulted in iPS cell generation, three or four transfections in the presence of B18R consistently resulted in the highest generation of Alkaline Phosphatase positive (AP+) colonies (Fig. 2b-d) .
- AP+ Alkaline Phosphatase positive
- >100 iPS cell colonies were mechanically isolated from the VEE-OKS-iM and VEE-OKS-iG RNA protocols and had a >95% success rate for the ability of isolated iPS-like clones to continuously divide and retain a human embryonic stem cell (hESC) morphology.
- hESC human embryonic stem cell
- VEE RF-RNA replicons To confirm the complete loss of VEE RF-RNA replicons, a highly sensitive and specific qRT-PCR protocol was developed capable of detecting ⁇ 10 femtogram of the VEE RF-RNA replicon (Fig. 4) . As expected, qRT-PCR analysis showed that all iPS cell clones had lost the VEE RF-RNA replicon (Table 2) . Moreover, karyotype analysis of 4 independent iPS cell clones
- iPS clones generated from both parental BJ and HFF fibroblasts with either the OKS-iM or OKS-iG VEE-RF RNA protocol expressed robust levels of endogenous OCT4, SOX2, NANOG, LIN28, TDGF1, DNMT3B and TERT, in contrast to low or no expression levels in starting parental BJ and HFF fibroblasts (Fig. 3a) .
- a hallmark of induced pluripotency is reduced DNA methylation of CpG dinucleotides in the OCT4 and NANOG promoter regions. Bisulfite genomic sequencing of both the OCT4 and NANOG promoter regions showed extensive
- RNA-seq whole genome RNA sequencing
- VEE-RF RNA generated iPS cells acquired full pluripotency by rigorous in vivo biological and molecular criterion that paralleled human ES cells.
- the generation of the VEE RF-RNA transcript utilizes a standard SP6 in vitro transcription kit that does not require special conditions and thereby, further simplifies the approach for broad use.
- the VEE-RF RNA approach solves both of the major inefficiency problems associated with attempting to generate iPS cells by daily repeated daily transfections for >14 days of four individual RF mRNAs .
- the VEE-RF RNA is an ectopic hit-and-run approach that does not utilize a DNA intermediate and therefore, there is no opportunity for integrative mutation that can occur with DNA vector-based iPS cell approaches.
- the timing of VEE-RF RNA replicon loss by degradation can be regulated by B18R withdrawal from the media. Using the VEE-RF RNA approach, >100 independent iPS cell clones were generated from both
- VEE-RF RNA protocols from two independent parental human fibroblast populations .
- the VEE-RF RNA approach can be engineered to express alternative RF combinations and/or insertion of additional RF ORFs into the RF-RNA backbone for refining iPS cell generation from specific cell types or for use in driving transdifferentiation .
- the VEE-RF RNA replicon approach has broad applicability for the efficient generation of human iPS cells for ultimate use in human stem cell therapies and regenerative medicine.
- RNA-Seq data have been submitted and can be accessed by the Gene Expression Omnibus (GEO) accession number GSE38265.
- GEO Gene Expression Omnibus
Abstract
Description
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DK13794196.9T DK2852671T3 (en) | 2012-05-21 | 2013-05-21 | CREATION OF HUMAN IPS CELLS WITH A SYNTHETIC SELF REPLICATING RNA |
CN201380038648.1A CN104508131B (en) | 2012-05-21 | 2013-05-21 | People iPS cell is formed by the RNA of the self-replacation of synthesis |
SG11201407595UA SG11201407595UA (en) | 2012-05-21 | 2013-05-21 | Generation of human ips cells by a synthetic self- replicative rna |
AU2013266496A AU2013266496B2 (en) | 2012-05-21 | 2013-05-21 | Generation of human iPS cells by a synthetic self- replicative RNA |
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CA2873964A CA2873964A1 (en) | 2012-05-21 | 2013-05-21 | Generation of human ips cells by a synthetic self-replicative rna |
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JP2015514104A JP6396893B2 (en) | 2012-05-21 | 2013-05-21 | Production of human iPS cells with synthetic self-replicating RNA |
US14/402,924 US9862930B2 (en) | 2012-05-21 | 2013-05-21 | Generation of human iPS cells by a synthetic self-replicative RNA |
EP13794196.9A EP2852671B1 (en) | 2012-05-21 | 2013-05-21 | Generation of human ips cells by a synthetic self- replicative rna |
IL235628A IL235628B (en) | 2012-05-21 | 2014-11-11 | Generation of human ips cells by a synthetic self-replicative rna |
US15/860,356 US10370646B2 (en) | 2012-05-21 | 2018-01-02 | Generation of human iPS cells by a synthetic self-replicative RNA |
US16/515,767 US10793833B2 (en) | 2012-05-21 | 2019-07-18 | Generation of human iPS cells by a synthetic self-replicative RNA |
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US17/025,836 US20210108179A1 (en) | 2012-05-21 | 2020-09-18 | GENERATION OF HUMAN iPS CELLS BY A SYNTHETIC SELF-REPLICATIVE RNA |
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US10370646B2 (en) | 2019-08-06 |
US20150159143A1 (en) | 2015-06-11 |
IL235628B (en) | 2020-03-31 |
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PL2852671T3 (en) | 2019-06-28 |
US20190338252A1 (en) | 2019-11-07 |
ES2698527T3 (en) | 2019-02-05 |
EP2852671A2 (en) | 2015-04-01 |
SG11201407595UA (en) | 2014-12-30 |
PT2852671T (en) | 2018-11-29 |
EP2852671B1 (en) | 2018-08-22 |
HUE041853T2 (en) | 2019-05-28 |
AU2013266496A1 (en) | 2014-11-27 |
EP2852671A4 (en) | 2015-12-23 |
CN104508131A (en) | 2015-04-08 |
CN104508131B (en) | 2018-11-23 |
US20180216079A1 (en) | 2018-08-02 |
JP6396893B2 (en) | 2018-09-26 |
DK2852671T3 (en) | 2018-12-10 |
IL272917A (en) | 2020-04-30 |
AU2013266496B2 (en) | 2018-08-09 |
IL235628A0 (en) | 2015-01-29 |
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JP2015519898A (en) | 2015-07-16 |
WO2013177133A3 (en) | 2014-03-20 |
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