WO2017134628A1 - Méthodes et compositions de culture de cellules souches - Google Patents

Méthodes et compositions de culture de cellules souches Download PDF

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WO2017134628A1
WO2017134628A1 PCT/IB2017/050613 IB2017050613W WO2017134628A1 WO 2017134628 A1 WO2017134628 A1 WO 2017134628A1 IB 2017050613 W IB2017050613 W IB 2017050613W WO 2017134628 A1 WO2017134628 A1 WO 2017134628A1
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cells
naive
cell
agent
ssea4
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Amander CLARK
Di Chen
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The Regents Of The University Of California
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C12Q1/24—Methods of sampling, or inoculating or spreading a sample; Methods of physically isolating an intact microorganisms
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0603—Embryonic cells ; Embryoid bodies
    • C12N5/0606—Pluripotent embryonic cells, e.g. embryonic stem cells [ES]
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56966—Animal cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • C12N2501/115—Basic fibroblast growth factor (bFGF, FGF-2)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • C12N2501/16—Activin; Inhibin; Mullerian inhibiting substance
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/20—Cytokines; Chemokines
    • C12N2501/23—Interleukins [IL]
    • C12N2501/235—Leukemia inhibitory factor [LIF]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/70—Enzymes
    • C12N2501/72—Transferases (EC 2.)
    • C12N2501/727—Kinases (EC 2.7.)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/999—Small molecules not provided for elsewhere

Definitions

  • the present invention relates generally to the field of cell culture and development.
  • hESCs Human embryonic stem cells
  • hESCs are derived from pre-implantation human blastocysts, they are morphologically and transcriptionally similar to murine epiblast stem cells (EpiSCs), which are derived from post-implantation mouse embryos. As such, hESCs and EpiSCs are said to exhibit a "primed pluripotent state" while, mouse ESCs derived from the pre-implantation blastocyst exhibit a "naive pluripotent state" corresponding to an earlier stage of development
  • EpiSCs murine epiblast stem cells
  • hESCs have many applications in basic science research and in therapeutic methods. However, it is not clear how to recapitulate or identify a naive hESC in culture, given the predominance of the primed state in standard culturing protocols.
  • the naive state may not only represent a more undifferentiated cell, but may also represent a cell that has the potential to differentiate in a more appropriate and biologically relevant way than a primed cell. Therefore, there is a need in the art for methods for identifying and isolating naive human ESCs.
  • SSEA-4 is known as a marker of human embryonic stem cells and as a marker of induced pluripotent stem cells. Surprisingly, it was found that a more undifferentiated and perhaps more appropriately programmed cell can be detected on the basis of SSEA-4 antigen expression. SSEA-4-negative cells represent a stem cell population that has many research and therapeutic applications, and methods of the disclosure relate to novel methods for isolating and identifying such cells as well as therapeutic methods using such cells.
  • aspects of the disclosure relate to a method for separating naive human embryonic stem cells (hESCs) or naive human induced pluroipotent cells (hiPSCs) from non-naive hESCs or non-naive hiPSCs in vitro, the method comprising: contacting the cells in vitro with an agent that binds to Stage Specific Embryonic Antigen 4 (SSEA4) antigen on the cells; and separating SSEA4-negative unbound cells from the SSEA4-positive agent-bound cells; wherein the fraction of unbound cells represents the naive hESCs or naive hiPSCs and the fraction of bound cells represents the non-naive hESCs or non-naive hiPSCs.
  • SSEA4 Stage Specific Embryonic Antigen 4
  • a further method aspect relates to a method for selectively labeling non-naive hESCs or hiPSCs in a composition comprising naive and non-naive hESCs or iPSCs, the method comprising: contacting the cells in vitro with a labeled agent that binds to SSEA4 antigen on the cells; wherein the binding of the labeled agent to the non-naive hESCs or iPSCs selectively labels the non-naive hESCs or hiPSCs.
  • there is a method comprising: selectively labeling non-naive hESCs or hiPSCs by contactacting the cells in vitro with a labeled agent that binds to SSEA-4; separating labeled cells from unlabeled cells, wherein the unlabeled cells are naive hESCs or hiPSCs; culturing the unlabeled naive hESCs or hiPSCs.
  • the naive hESCs or hiPSCs are undifferentiated or have not been contacted with a differentiation medium.
  • Further method aspects relate to a method for culturing or for maintaining a population of naive hESCs or naive hiPSCs comprising contacting the cells with an agent that binds to Stage Specific Embryonic Antigen 4 (SSEA4) antigen on the cells; and separating SSEA4-negative unbound cells from the SSEA4-positive agent-bound cells; and culturing the unbound cells in cell media.
  • SSEA4 Stage Specific Embryonic Antigen 4
  • the methods of the disclosure further comprises culturing the fraction of unbound cells.
  • the cells may be culutured for at least, at most, or exactly 1, 2, 3, 4, 5, 6, 7, 10, 20, 30, 40, 50 or more passages or any range derivable therein.
  • the cells are cultured in media comprising one or more inhibitors.
  • the inhibitors are inhibitors of MAPK/ERK kinase, ROCK, Src, LCK, B-raf kinase, and/or GSK-3p. Such inhibitors are commercially available.
  • StemoleculeTM PD0325901 is an inhibitor of MAPK/ERK kinase or MEK
  • StemoleculeTM Y27632 is an inhibitor of Rho-associated kinase (ROCK)
  • StemoleculeTM WH-4-023 is an inhibitor of Src and LCK
  • StemoleculeTM SB590885 is an inhibitor of B-RAF kinase
  • StemoleculeTM IM-12 and StemoleculeTM CHIR99021 are inhibitors of glycogen synthase kinase 3 ⁇ (GSK-3P).
  • the media further comprises one or more of fibroblast growth factor (FGF), Activin A, and/or leukemia inhibitor factor (LIF).
  • the media comprises a compound or composition described herein.
  • the agent is labeled with a detectable label. Detectable labels are known in the art and described herein.
  • the agent is an antibody.
  • the method further comprises contacting the cells with a second agent that binds to the SSEA4-binding agent.
  • the SSEA-4 binding agent and/or second agent are linked to a solid support and/or detectable label.
  • the agent-bound cells and unbound cells are separated by fluorescent-activated cell sorting (FACS), chromatography, solid-support assays, magnetic activated cell sorting (MACS), and panning.
  • FACS fluorescent-activated cell sorting
  • chromatography solid-support assays
  • MCS magnetic activated cell sorting
  • panning Other suitable methods for separating cell populations are also described herein.
  • the non-naive cell of the disclosure is a primed cell.
  • the method further comprises culturing or passaging the naive hESCs or hiPSCs (i.e. SSEA-4 negative cells). In some embodiments, the method further comprises expanding the naive hESCs or naive hiPSCs. In some embodiments, the the method further comprises freezing the naive hESCs or naive hiPSCs. In some embodiments, the method further comprises thawing the frozen cells.
  • compositions comprising cells isolated, identified, or cultured according to methods of the disclosure.
  • a further aspect relates to a therapeutic method comprising administering the cell isolated, identified, or cultured cell (i.e. a naive human pluripotent cell) according to methods of the disclosure, or a progeny of such cell.
  • a cell isolated, identified, or cultured cell i.e. a naive human pluripotent cell
  • the SSEA-4-negative cell is a naive pluripotent cell.
  • the cell is a progeny of the naive pluripotent cell and may be SSEA-4 negative or SSEA-4 positive.
  • the progeny is SSEA-4 positive.
  • the SSEA-4 negative cell is a naive human pluripotent cell.
  • the cell may be a mouse cell. Therefore, in some embodiments, the cell is a naive mouse ESC, iPC, pluripotent cell.
  • a further method aspect relates for a method for evaluating a cell culture medium for cultunng hESCs or hiPSCs, the method comprising: culturing the hESCs or hiPSCs in the medium; contacting the hESCs or hiPSCs with a detectable agent that binds to SSEA4 antigen on cells; detecting the agent binding to the antigen; and evaluating the medium on the basis of the detected agent.
  • a medium with a percentage of SSEA-4 negative cells of less than 25% is determined to be a naive hESC or hiPSC maintenance medium. In some embodiments, a medium with a percentage of SSEA-4 negative cells of less than 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1% (or any derivable range therein) is determined to be a naive hESC or hiPSC maintenance medium.
  • aspects of the disclosure relate to a cell medium evaluated according to any methods of the disclosure.
  • the method further comprises quantifying the SSEA4-negative unbound cells, SSEA4-positive bound cells, or both. Quantification methods are known in the art, and quantification may be done based on the detected label, such as a quantification of fluorescence, chemiluminescence, or enzyme activity.
  • a further aspect of the disclosure relates to a method for identifying culturing compositions that revert a primed hESC or hiPSC to a naive hESC or hiPSC comprising: contacting the primed hESC or hiPSC cultured in vitro with a candidate culturing composition; contacting the cell with a detectable agent that binds to SSEA4 antigen on cells; detecting the presence or absence of the agent binding to the antigen; and identifying the culturing composition as a composition that reverts a primed hESC or hiPSC to a naive hESC or hiPSC when the absence or reduction of agent-antigen binding is detected.
  • the method further comprises determining SSEA-4 status prior to contact with the candidate composition.
  • the reduction in SSEA-4-expressing cells is at least, at most, or exactly 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, or 99 % (or any derivable range therein).
  • the candidate culturing composition comprises one or more of the inhibitors of MAPK/ERK kinase, ROCK, Src, LCK, B-raf kinase, and GSK-3p.
  • the culturing composition comprises or further comprises one or more of FGF, Activin A, and LIF.
  • Further aspects relate to a culturing composition that revers a primed hESC or hiPSC to a naive hESC or hiPSC, wherein the composition is identified according to a method of the disclosure.
  • Further method aspects relate to a method for distinguishing a naive hESC or a primed hESC, the method comprising: contacting the cell with a detectable agent that binds to Stage Specific Embryonic Antigen 4 (SSEA4)-positive cells; and detecting a naive hESC as a cell not bound with the detectable agent; and/or detecting a primed hESC as a cell bound with the detectable agent.
  • SSEA4 Stage Specific Embryonic Antigen 4
  • a therapeutic method comprising contacting a naive human pluripotent cell in vitro with a detectable agent that binds to SSEA4 antigen on cells; administering the naive human pluripotent cell to the patient; wherein the binding of the agent to the SSEA4 antigen is not detectable.
  • the method comprises contacting a naive human pluripotent cell and wherein the naive human pluripotent cell is in a blastocyst.
  • the therapeutic method is in vitro fertilization (IVF).
  • compositions comprising an isolated naive hESC or hiPSC and cell culture media, wherein the cell is SSEA4-negative.
  • the cell is a naive hiPSC.
  • the cell is cryopreserved.
  • the cell is in cell culture media comprising one or more components described herein.
  • Further method aspects relate to methods for making a naive iPSC cell comprising: isolating an adult stem cell; inducing pluripotence in the cell by methods known in the art or described herein; and screening the cell for SSEA-4 negativity, wherein a negative SSEA-4 cell represents a naive iPSC.
  • the culturing temperature can be about 20 to 40°C, for example, at least, at most, or about 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40°C (or any range derivable therein), but particularly not limited to them.
  • the C0 2 concentration can be about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10% (or any range derivable therein), such as about 2% to 10%, for example, about 2 to 5%), or any range derivable therein.
  • the oxygen tension can be at least or about 1, 5, 8, 10, 20%), or any range derivable therein.
  • the methods further comprise differentiating the cell into a hematopoietic stem or progenitor cell or an immune cell such as a T cell.
  • any of the cell population may comprise at least, about, or at most, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 1 x 10 3 , 2 x 10 3 , 3 x 10 3 , 4 x 10 3 , 5 x 10 3 , 6 x 10 3 , 7 x 10 3 , 8 x 10 3 , 9 x 10 3 , 1 x 10 4 , 2 x 10 4 , 3 x 10 4 , 4 x 10 3 ,5 x 10 4 , 6 x 10 4 , 7 x 10 4 , 8 x 10 4 , 9 x 10 4 , 1 x 10 5 , 2
  • the subject or patient of the therapeutic methods may be any animal, in particular a mouse, non-human primate, or human.
  • the subject is a woman that has experienced infertility or has been diagnosed with infertility.
  • the subject is a woman that has not been successful with prior infertility treatments.
  • FIG. 1A-F 5iLAF SSEA4 negative subpopulation recapitulates naive expression pattern.
  • n 2.
  • FIG. 2A-H Naive hESCs fail to recapitulate naive-specific methylation pattern.
  • FIG. 3A-K Properties of 5iLAF SSEA4 negative and SSEA4 positive cells generated by reversion of primed hESCs.
  • A-C Immunofluorescence for SSEA4 and OCT4. Note that all colonies are OCT4 positive.
  • A) A colony of SSEA4 positive primed UCLAl hESCs.
  • B) A colony of 5iLAF SSEA4 positive UCLAl hESCs.
  • C) A colony of 5iLAF SSEA4 negative UCLAl hESCs.
  • D-F Immunofluorescence for TRA-1-81 and NANOG. Note that all populations are NANOG positive.
  • D A colony of TRA-1-81 positive UCLAl hESCs.
  • FIG. 4A-H A) DNA methylation over transiently imprinted CG islands. The average methylation level of each imprint in a given sample is represented as one point in the box and whisker point.
  • Gafni 2013 data was generated by RRBS, only CGs that had coverage in the Gafni 2013 dataset are included in this analysis to make the data comparable. Only sixteen stable imprints had sufficient coverage for robust analysis.
  • G) Karyotyping results from reverted UCLAl lines and new lines derived from blastocyst in 5iLAF.
  • FIG. 5 depicts the SSEA-4 antigen.
  • the terms "separating,” “isolating,” and “purifying” refer to a segregation of one component from one or more other components, such as a separation of one cell type from another cell type.
  • the separation is from one component defined in the method from another component defined in the method (eg. separation of naive and primed cells).
  • the separation, isolation, or purification may be substantial in that less than 10% of the other component remains.
  • the separation may also be such that less than 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 % of the isolated, separated, or purified composition comprises the other component defined in the claims.
  • antigen refers to a molecule that binds to Ag-specific receptors, such as an antibody.
  • Antigens are usually peptides, polysaccharides or lipids.
  • xeno-free (XF) or "animal component-free (ACF)" or “animal free,” when used in relation to a medium, an extracellular matrix, or a culture condition, refers to a medium, an extracellular matrix, or a culture condition which is essentially free from heterogeneous animal-derived components. For culturing human cells, any proteins of a non- human animal, such as mouse, would be xeno components.
  • a “chemically defined medium” refers to a medium in which the chemical nature of approximately all the ingredients and their amounts are known. These media are also called synthetic media.
  • a "conditioned medium” is prepared by culturing a first population of cells in a medium, and then harvesting the medium.
  • the conditioned medium (along with anything secreted into the medium by the cells) may then be used to support the growth of a second population of cells.
  • a particular ingredient or factor is described as having been added to the medium, what is meant is that the factor (or a cell or particle engineered to secrete the factor) has been mixed into the medium by deliberate manipulation.
  • fresh medium is a medium that has not been purposely conditioned by culturing with a different cell type before being used with the cell type it is ultimately designed to support. Otherwise, no limitations are intended as to its manner of preparation, storage, or use. It is added fresh (by exchange or infusion) into the ultimate culture, where it may be consumed or otherwise processed by the cell types that are present.
  • cell culture is meant cells or tissues that are maintained, cultured or grown in an artificial, in vitro or ex vivo environment.
  • stem cell attachment means binding of the stem cell to the support surface such that the stem cell is not eluted by conventional washing or handling procedures.
  • stem cell “survival,” particularly in regards to an undifferentiated stem cell, is meant sustained viability.
  • proliferation and “propagation” are used interchangeably herein to denote an increase in the number of cells.
  • expansion is also intended to mean that the resultant cell population is derived from ex vivo culture of stem cells, where the outgoing (cultured) number of cells exceeds the ingoing (non-cultured) number of cells.
  • expanded is not to be construed or limited by any mechanism or theory of cellular origin and may comprise cells that originate de novo in culture.
  • Cells are "substantially free” of certain reagents or elements, such as serum, signaling inhibitors, animal components or feeder cells, exogenous genetic elements or vector elements, as used herein, when they have less than 10% of the element(s), and are "essentially free” of certain reagents or elements when they have less than 1% of the element(s).
  • certain reagents or elements such as serum, signaling inhibitors, animal components or feeder cells, exogenous genetic elements or vector elements, as used herein, when they have less than 10% of the element(s), and are “essentially free” of certain reagents or elements when they have less than 1% of the element(s).
  • cell populations wherein less than 0.5% or less than 0.1%) of the total cell population comprise exogenous genetic elements or vector elements.
  • a culture, matrix or medium are "essentially free" of certain reagents or elements, such as serum, signaling inhibitors, animal components or feeder cells, when the culture, matrix or medium respectively have a level of these reagents lower than a detectable level using conventional detection methods known to a person of ordinary skill in the art or these agents have not been extrinsically added to the culture, matrix or medium.
  • the serum-free medium may be essentially free of serum.
  • a "gene,” “polynucleotide,” “coding region,” “sequence,” “segment,” “fragment,” or “transgene” which "encodes” a particular protein is a nucleic acid molecule which is transcribed and optionally also translated into a gene product, e.g., a polypeptide, in vitro or in vivo when placed under the control of appropriate regulatory sequences.
  • the coding region may be present in either a cDNA, genomic DNA, or RNA form. When present in a DNA form, the nucleic acid molecule may be single-stranded (i.e., the sense strand) or double- stranded.
  • a gene can include, but is not limited to, cDNA from prokaryotic or eukaryotic mRNA, genomic DNA sequences from prokaryotic or eukaryotic DNA, and synthetic DNA sequences.
  • a transcription termination sequence will usually be located 3' to the gene sequence.
  • cell is herein used in its broadest sense in the art and refers to a living body which is a structural unit of tissue of a multicellular organism, is surrounded by a membrane structure which isolates it from the outside, has the capability of self-replicating, and has genetic information and a mechanism for expressing it.
  • Cells used herein may be naturally-occurring cells or artificially modified cells (e.g., fusion cells, genetically modified cells, etc.).
  • stem cell refers to a cell capable of self-replication and pluripotency or multipotency. Typically, stem cells can regenerate an injured tissue.
  • Stem cells herein may be, but are not limited to, embryonic stem (ES) cells, induced pluripotent stem cells or tissue stem cells (also called tissue-specific stem cell, or somatic stem cell).
  • Embryonic stem (ES) cells are pluripotent stem cells derived from early embryos. An ES cell was first established in 1981, which has also been applied to production of knockout mice since 1989. In 1998, a human ES cell was established, which is currently becoming available for regenerative medicine.
  • naive hESC or “naive iPC” refers to cells that are phenotypically, morphologically, and/or transcriptionally most similar to endogenous human pre- implantation state.
  • the naive cells are distinct from primed cells based on their comparative hypomethylated state, for example.
  • naive cells exhibit a DNA methylation pattern of the pre-implantation blastocyst.
  • primed hESC or “primed iPC” refers to cells that are phenotypically, morphologically, and/or transcriptionally most similar to endogenous human post- implantation state.
  • the primed cells are distinct from naive cells based on their comparative methylation state, for example.
  • primed cells exhibit a DNA methylation pattern of the post-implantation blastocyst.
  • tissue stem cells have a limited differentiation potential. Tissue stem cells are present at particular locations in tissues and have an undifferentiated intracellular structure. Therefore, the pluripotency of tissue stem cells is typically low. Tissue stem cells have a higher nucleus/cytoplasm ratio and have few intracellular organelles. Most tissue stem cells have low pluripotency, a long cell cycle, and proliferative ability beyond the life of the individual. Tissue stem cells are separated into categories, based on the sites from which the cells are derived, such as the dermal system, the digestive system, the bone marrow system, the nervous system, and the like. Tissue stem cells in the dermal system include epidermal stem cells, hair follicle stem cells, and the like.
  • Tissue stem cells in the digestive system include pancreatic (common) stem cells, liver stem cells, and the like.
  • Tissue stem cells in the bone marrow system include hematopoietic stem cells, mesenchymal stem cells, and the like.
  • Tissue stem cells in the nervous system include neural stem cells, retinal stem cells, and the like.
  • iPS cells Induced pluripotent stem cells
  • iPS cells refer to a type of pluripotent stem cell artificially prepared from a non-pluripotent cell, typically an adult somatic cell, or terminally differentiated cell, such as fibroblast, a hematopoietic cell, a myocyte, a neuron, an epidermal cell, or the like, by introducing certain factors, referred to as reprogramming factors.
  • hiPSCs or hiPS cells refers to human induced pluripotent stem cells. In this case, the stem cell is derived from a human cell.
  • Pluripotency refers to a stem cell that has the potential to differentiate into all cells constituting one or more tissues or organs, or particularly, any of the three germ layers: endoderm (interior stomach lining, gastrointestinal tract, the lungs), mesoderm (muscle, bone, blood, urogenital), or ectoderm (epidermal tissues and nervous system).
  • endoderm internal stomach lining, gastrointestinal tract, the lungs
  • mesoderm muscle, bone, blood, urogenital
  • ectoderm epidermal tissues and nervous system.
  • Pluripotent stem cells used herein refer to cells that can differentiate into cells derived from any of the three germ layers, for example, direct descendants of totipotent cells or induced pluripotent cells.
  • operably linked with reference to nucleic acid molecules is meant that two or more nucleic acid molecules (e.g., a nucleic acid molecule to be transcribed, a promoter, and an enhancer element) are connected in such a way as to permit transcription of the nucleic acid molecule.
  • "Operably linked” with reference to peptide and/or polypeptide molecules is meant that two or more peptide and/or polypeptide molecules are connected in such a way as to yield a single polypeptide chain, i.e., a fusion polypeptide, having at least one property of each peptide and/or polypeptide component of the fusion.
  • the fusion polypeptide is particularly chimeric, i.e., composed of heterologous molecules.
  • binding refers to an action that is discriminating.
  • selective labeling non-naive hESCs refers to a labeling action that discriminates between non-naive and naive hESCs and lables non-naive hESCs (eg. primed cells).
  • the phrase "maintaining a population" with respect to a specific cell type refers to the culturing of cells for a period of time, wherein the cells are maintained in a specific state.
  • the maintenance may be done by periodically removing any cells not of the specified cell type or by culturing the specified cell type in conditions that do not allow the cells to differentiate or drift into another cell type.
  • Embryonic stem (ES) cells are pluripotent cells derived from the inner cell mass of a blastocyst.
  • ES cells can be isolated by removing the outer trophectoderm layer of a developing embryo, then culturing the inner mass cells on a feeder layer of non-growing cells. Under appropriate conditions, colonies of proliferating, undifferentiated ES cells are produced. The colonies can be removed, dissociated into individual cells, then replated on a fresh feeder layer. The replated cells can continue to proliferate, producing new colonies of undifferentiated ES cells. The new colonies can then be removed, dissociated, replated again and allowed to grow.
  • a “primary cell culture” is a culture of cells directly obtained from a tissue such as the inner cell mass of a blastocyst.
  • a “subculture” is any culture derived from the primary cell culture.
  • Human ES cells can be obtained from blastocysts using previously described methods (Thomson et al. Proc. Natl. Acad. Scie. USA, 92:7844-7848, 1995; Thomson and Marshall, Curr. Top. Dev. Biol, 38: 133- 165, 1998). In one method, day-5 human blastocysts are exposed to rabbit anti -human spleen cell antiserum, then exposed to a 1 :5 dilution of Guinea pig complement to lyse trophectoderm cells.
  • the inner cell mass After removing the lysed trophectoderm cells from the intact inner cell mass, the inner cell mass is cultured on a feeder layer of gamma-inactivated mouse embryonic fibroblasts and in the presence of fetal bovine serum. After 9 to 15 days, clumps of cells derived from the inner cell mass can be chemically (i.e. exposed to trypsin) or mechanically dissociated and replated in fresh medium containing fetal bovine serum and a feeder layer of mouse embryonic fibroblasts. Upon further proliferation, colonies having undifferentiated morphology are selected by micropipette, mechanically dissociated into clumps, and replated (see U.S. Patent No. 6,833,269).
  • ES-like morphology is characterized as compact colonies with apparently high nucleus to cytoplasm ratio and prominent nucleoli. Resulting ES cells can be routinely passaged by brief trypsinization or by selection of individual colonies by micropipette. In some methods, human ES cells can be grown without serum by culturing the ES cells on a feeder layer of fibroblasts in the presence of basic fibroblast growth factor (Amit et al, Dev. Bio., 227:271-278, 2000).
  • human ES cells can be grown without a feeder cell layer by culturing the cells on a protein matrix such as MatrigelTM or laminin in the presence of "conditioned" medium containing basic fibroblast growth factor (Xu et al., Nat. Biotechnol, 19:971-974, 2001). The medium is previously conditioned by coculturing with fibroblasts.
  • a protein matrix such as MatrigelTM or laminin
  • the medium is previously conditioned by coculturing with fibroblasts.
  • ES cell lines Another source of ES cells are established ES cell lines. Various human ES cell lines are known and conditions for their growth and propagation have been defined.
  • the source of ES cells can be a blastocyst, cells derived from culturing the inner cell mass of a blastocyst, or cells obtained from cultures of established cell lines.
  • ES cells can refer to inner cell mass cells of a blastocyst, ES cells obtained from cultures of inner mass cells, and ES cells obtained from cultures of ES cell lines.
  • Induced pluripotent stem (iPS) cells are cells which have the characteristics of ES cells but are obtained by the reprogramming of differentiated somatic cells. Induced pluripotent stem cells have been obtained by various methods. In one method, adult human dermal fibroblasts are transfected with transcription factors Oct4, Sox2, and Klf4 using retroviral transduction (Takahashi et al., Cell, 126(4):663-76, 2007; Takahashi et al, Cell, 131 :861-872, 2007.). In some embodiments, the cells are also transfected with c-myc.
  • the transfected cells are plated on SNL feeder cells (a mouse cell fibroblast cell line that produces LIF) in medium supplemented with basic fibroblast growth factor (bFGF). After approximately 25 days, colonies resembling human ES cell colonies appear in culture. The ES cell-like colonies are picked and expanded on feeder cells in the presence of bFGF.
  • SNL feeder cells a mouse cell fibroblast cell line that produces LIF
  • bFGF basic fibroblast growth factor
  • cells of the ES cell-like colonies are induced pluripotent stem cells.
  • the induced pluripotent stem cells are morphologically similar to human ES cells, and express various human ES cell markers. Also, when growing under conditions that are known to result in differentiation of human ES cells, the induced pluripotent stem cells differentiate accordingly. For example, the induced pluripotent stem cells can differentiate into cells having neuronal structures and neuronal markers.
  • human fetal or newborn fibroblasts are transfected with four genes, Oct4, Sox2, Nanog and Lin28 using lentivirus transduction (Yu et al, Science, 318: 1917-1920, 2007).
  • colonies with human ES cell morphology become visible.
  • the colonies are picked and expanded.
  • the induced pluripotent stem cells making up the colonies are morphologically similar to human ES cells, express various human ES cell markers, and form teratomas having neural tissue, cartilage and gut epithelium after injection into mice.
  • iPS cells typically require the expression of or exposure to at least one member from Sox family and at least one member from Oct family.
  • Sox and Oct are thought to be central to the transcriptional regulatory hierarchy that specifies ES cell identity.
  • Sox may be Sox-1, Sox-2, Sox-3, Sox-15, or Sox-18; Oct may be Oct-4.
  • Additional factors may increase the reprogramming efficiency, like Nanog, Lin28, Klf4, or c-Myc; specific sets of reprogramming factors may be a set comprising Sox-2, Oct-4, Nanog and, optionally, Lin-28; or comprising Sox-2, Oct4, Klf and, optionally, c-Myc.
  • IPS cells like ES cells, have characteristic antigens that can be identified or confirmed by immunohistochemistry or flow cytometry. Pluripotency of embryonic stem cells can be confirmed by injecting approximately 0.5-10 X 10 6 cells into the rear leg muscles of 8-12 week old male SCID mice. Teratomas develop that demonstrate at least one cell type of each of the three germ layers. III. SSEA-4 and Agents that bind thereto
  • Stage-specific embryonic antigen-4 is a glycolipid carbohydrate antigen expressed on the surface of human embryonic stem cells (hESC) and induced pluripotent stem (iPS) cells.
  • hESC human embryonic stem cells
  • iPS induced pluripotent stem
  • the SSEA-4 negative cells may be able to effectively and appropriately differentiate into all the required cell types that are necessary for proper development, while a primed SSEA-4 positive cell may represent a cell with a more limited differentiation potential or a cell that is not capable of appropriately differentiating into a particular cell type, which may be problematic for research applications and for therapeutic applications. Therefore, methods for identifying the naive stem cell have valuable applications.
  • SSEA-4 The structure of SSEA-4 is known in the art, and antibodies directed to SSEA-4 are commercially available.
  • the structure of SSEA-4 is shown in FIG. 5.
  • Antibodies that bind to SSEA-4 are commercially available and include, for example, anti-SSEA4 antibody [MC813] (ab 16287) from Abeam ®, anti -human SSEA-4 Antibody, Clone MC-813-70 from Stemcell TechnologiesTM, SSEA-4 Antibody (MC-813-70) from ThermoFisher ScientificTM, for example. Since the structure of SSEA-4 is known, and SSEA-4 binding agents are known in the art, one could easily envision other binding agents that are within the scope of the current disclosure.
  • the stem cells are cultured in an environment that includes a nutrient medium or culture medium that is capable of supporting the attachment, survival and/or proliferation of a stem cell in vitro or ex vivo.
  • a nutrient medium or culture medium that is capable of supporting the attachment, survival and/or proliferation of a stem cell in vitro or ex vivo.
  • a person skilled in the art of culturing stem cells would be familiar with techniques for culturing these cells, for instance, as evidenced in U.S. Pat. No. 6,875,607, the entire contents of which are incorporated herein by reference.
  • nutrient medium refers to a nutritive solution for culturing or growing cells, preferably stem cells (whether differentiated or undifferentiated) that contain nutrients that are capable of supporting the survival and/or proliferation of the cells, and/or their attachment a support surface.
  • the nutrient medium may contain any of the following in an appropriate combination: isotonic saline, buffer, amino acids, serum or serum replacement, and other exogenously added factors.
  • the culture media may be one that denotes conditions that result in a measurable amount of cell attachment, survival and/or proliferation. Effective conditions can be readily determined and/or optimized by a skilled worker using conventional methods. Among the factors to be varied include, for example, the seeding density, the vessel, the culture medium, the temperature, the O 2 /CO 2 concentrations, and the like.
  • a variety of culture medium capable of supporting the attachment, survival and/or proliferation of stem cells may be used in conjunction with the methods of the disclosure.
  • Commercially available culture medium such as DMEM, F12, aMEM, Hepatostim.TM., RPMI, or combinations thereof, may be used, either in the presence or absence or serum.
  • the serum is human serum.
  • the serum is non- human. Suitable sera include calf serum, fetal calf serum, horse serum, or the like. The skilled addressee would also know that a serum supplement could be used in place of serum in the culture media.
  • the stem cell may be brought into contact with the support surface by any suitable means.
  • a stem cell in a culture medium may be poured, pipetted or dispensed into a culture vessel comprising the support surface, or a medical device or scaffold comprising the support surface may be submerged in culture medium in which the stem cell is suspended.
  • the medium in certain aspects can be prepared using a medium used for culturing animal cells as its basal medium, such as any of AIM V, X-VIVO-15, NeuroBasal, EGM2, TeSR, BME, BGJb, CMRL 1066, Glasgow MEM, Improved MEM Zinc Option, FMDM, Medium 199, Eagle MEM, aMEM, DMEM, Ham, RPMI- 1640, and Fischer's media, as well as any combinations thereof, but the medium may not be particularly limited thereto as far as it can be used for culturing animal cells. Particularly, the medium may be xeno-free or chemically defined.
  • a medium used for culturing animal cells as its basal medium, such as any of AIM V, X-VIVO-15, NeuroBasal, EGM2, TeSR, BME, BGJb, CMRL 1066, Glasgow MEM, Improved MEM Zinc Option, FMDM, Medium 199, Eagle MEM, aMEM, DMEM, Ham,
  • the medium can be a serum-containing or serum-free medium, or xeno-free medium. From the aspect of preventing contamination with heterogeneous animal-derived components, serum can be derived from the same animal as that of the stem cell(s).
  • the serum-free medium refers to medium with no unprocessed or unpurified serum and accordingly, can include medium with purified blood-derived components or animal tissue- derived components (such as growth factors).
  • the medium may contain or may not contain any alternatives to serum.
  • the alternatives to serum can include materials which appropriately contain albumin (such as lipid-rich albumin, bovine albumin, albumin substitutes such as recombinant albumin or a humanized albumin, plant starch, dextrans and protein hydrolysates), transferrin (or other iron transporters), fatty acids, insulin, collagen precursors, trace elements, 2-mercaptoethanol, 3'- thiolgiycerol, or equivalents thereto.
  • the alternatives to serum can be prepared by the method disclosed in International Publication No. 98/30679, for example (incorporated herein in its entirety). Alternatively, any commercially available materials can be used for more convenience.
  • the commercially available materials include knockout Serum Replacement (KSR), Chemically-defined Lipid concentrated (Gibco), and Glutamax (Gibco).
  • the medium may be a serum-free medium that is suitable for neural cell development.
  • the medium may comprise B-27 ® supplement, xeno-free B-27 ® supplement (available at world wide web at http://www.thermofisher.com/us/en/home/technical-resources/media-formulation.250.html), NS21 supplement (Chen et al., J Neurosci Methods, 2008 Jun 30; 171(2): 239-247, incorporated herein in its entirety), GS21TM supplement (available at world wide web at amsbio.com/B-27.aspx), or a combination thereof at a concentration effective for producing T cells from the 3D cell aggregate.
  • the medium may comprise one, two, three, four, five, six, seven, eight, nine, ten, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more of the following B-27 supplement ingredients: Vitamins such as biotin; DL Alpha Tocopherol Acetate; DL Alpha- Tocopherol; Vitamin A (acetate); proteins such as BSA (bovine serum albumin) or human albumin, fatty acid free Fraction V; Catalase; Human Recombinant Insulin; Human Transferrin; Superoxide Dismutase; Other Components such as Corticosterone; D-Galactose; Ethanolamine HC1; Glutathione (reduced); L-Carnitine HC1; Linoleic Acid; Linolenic Acid; Progesterone; Putrescine 2HC1; Sodium Selenite; and T3 (triodo-I-thyronine).
  • Vitamins such as biotin; DL Alpha Tocopherol Acetate; DL Alpha- Tocophe
  • the medium may comprise externally added ascorbic acid.
  • the medium can also contain externally added fatty acids or lipids, amino acids (such as nonessential amino acids), vitamin(s), growth factors, cytokines, antioxidant substances, 2- mercaptoethanol, pyruvic acid, buffering agents, and inorganic salts.
  • One or more of the medium components may be added at a concentration of at least, at most, or about 0.1, 0.5, 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 180, 200, 250 ng/L, ng/ml, ⁇ g/ml, mg/ml, or any range derivable therein.
  • the medium used may be supplemented with at least one externally added cytokine at a concentration from about 0.1 ng/mL to about 500 ng/mL, more particularly 1 ng/mL to 100 ng/mL, or at least, at most, or about 0.1, 0.5, 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 180, 200, 250 ng/L, ng/ml, ⁇ g/ml, mg/ml, or any range derivable therein.
  • Suitable cytokines include but are not limited to, FLT3 ligand (FLT3L), interleukin 7 (IL-7), stem cell factor (SCF), thrombopoietin (TPO), IL-2, IL-4, IL-6, IL-15, IL-21, TNF-alpha, TGF-beta, interferon-gamma, interferon-lambda, TSLP, thymopentin, pleotrophin, midkine.
  • the culture medium may include at least one of FLT3L and IL-7. More particularly, the culture may include both FLT3L and IL- 7.
  • human stem cells may be grown in aggregates, or colonies.
  • Standard culture of human stem cells involves exposure to media enriched with growth factors found in fetal bovine serum (FBS) or defined serum replacements.
  • FBS fetal bovine serum
  • standard hPSC culture systems utilize support cells such as an inactivated mouse embryonic fibroblast (MEF) feeder layer to support growth and prevent differentiation. These cells provide necessary intercellular interactions, extracellular scaffolding and factors creating a robust and stable hPSC culture environment.
  • FBS fetal bovine serum
  • MEF mouse embryonic fibroblast
  • starting cells of a selected population may comprise at least, at most, or exactly 10 4 , 10 5 , 10 6 , 10 7 , 10 8 , 10 9 , 10 10 , 10 11 , 10 12 , 10 13 cells or any range derivable therein.
  • the starting cell population may have a seeding density of at least, at most, or exactly 10, 10 1 , 10 2 , 10 3 , 10 4 , 10 5 , 10 6 , 10 7 , 10 8 cells/ml, or any range derivable therein.
  • a culture vessel used for culturing methods and cells of the disclosure can include, but is particularly not limited to: flask, flask for tissue culture, dish, petri dish, dish for tissue culture, multi dish, micro plate, micro-well plate, multi plate, multi-well plate, micro slide, chamber slide, tube, tray, Cell STACK® Chambers, culture bag, and roller bottle, as long as it is capable of culturing the stem cells therein.
  • the stem cells may be cultured in a volume of at least, at most, or exactly 0.2, 0.5, 1, 2, 5, 10, 20, 30, 40, 50 ml, 100 ml, 150 ml, 200 ml, 250 ml, 300 ml, 350 ml, 400 ml, 450 ml, 500 ml, 550 ml, 600 ml, 800 ml, 1000 ml, 1500 ml, or any range derivable therein, depending on the needs of the culture.
  • the culture vessel may be a bioreactor, which may refer to any device or system that supports a biologically active environment.
  • the bioreactor may have a volume of at least, at most, or exactly 2, 4, 5, 6, 8, 10, 15, 20, 25, 50, 75, 100, 150, 200, 500 liters, 1, 2, 4, 6, 8, 10, 15 cubic meters, or any range derivable therein.
  • the culture vessel can be cellular adhesive or non-adhesive and selected depending on the purpose.
  • the cellular adhesive culture vessel can be coated with any of substrates for cell adhesion such as extracellular matrix (ECM) to improve the adhesiveness of the vessel surface to the cells.
  • the substrate for cell adhesion can be any material intended to attach stem cells or feeder cells (if used).
  • the substrate for cell adhesion includes collagen, gelatin, poly-L-lysine, poly-D-lysine, laminin, and fibronectin and mixtures thereof for example MatrigelTM, and lysed cell membrane preparations.
  • the invention provides methods for cryo-preserving (i.e. freezing) the cells described herein.
  • the invention provides compositions comprising cryopreserved cells, wherein the cell is one or more cells described herein.
  • the disclosure provides a composition comprising a cryopreserved naive hESC or naive hiPSC.
  • the composition may comprise at least 1, 10, 100, 1000, 10000, 100000, 1000000, or 10000000 cells.
  • the cryopreserved cell may include a suitable media containing one or more cryoprotectants, such as DMSO or FBS to facilitate freezing the cells.
  • the disclosure provides a composition comprising a cryo- preserved in vitro differentiated progeny of a cell described herein.
  • cryopreserved compositions may be used in research and therapeutic applications.
  • a subject in need of cell therapy may be treated with the cryopreserved composition described herein.
  • label intends a directly or indirectly detectable compound or composition that is conjugated directly or indirectly to the agent to be detected, e.g., agent that binds to SSEA4.
  • the label may be detectable by itself (e.g. radioisotope labels or fluorescent labels) or, in the case of an enzymatic label, may catalyze chemical alteration of a substrate compound or composition which is detectable.
  • the labels can be suitable for small scale detection or more suitable for high-throughput screening. As such, suitable labels include, but are not limited to radioisotopes, fluorochromes, chemiluminescent compounds, dyes, and proteins, including enzymes. The label may be simply detected or it may be quantified.
  • a response that is simply detected generally comprises a response whose existence merely is confirmed
  • a response that is quantified generally comprises a response having a quantifiable (e.g., numerically reportable) value such as an intensity, polarization, and/or other property.
  • the detectable response may be generated directly using a luminophore or fluorophore associated with an assay component actually involved in binding, or indirectly using a luminophore or fluorophore associated with another (e.g., reporter or indicator) component.
  • luminescent labels that produce signals include, but are not limited to bioluminescence and chemiluminescence. Detectable luminescence response generally comprises a change in, or an occurrence of, a luminescence signal. Suitable methods and luminophores for luminescently labeling assay components are known in the art and described for example in Haugland, Richard P. (1996) Handbook of Fluorescent Probes and Research Chemicals (6.sup.th ed.). Examples of luminescent probes include, but are not limited to, aequorin and luciferases.
  • fluorescent labels include, but are not limited to, fluorescein, rhodamine, tetramethylrhodamine, eosin, erythrosin, coumarin, methyl-coumarins, pyrene, Malacite green, stilbene, Lucifer Yellow, Cascade Blue.TM., and Texas Red.
  • suitable optical dyes are described in the Haugland, Richard P. (1996) Handbook of Fluorescent Probes and Research Chemicals (6.sup.th ed.).
  • the fluorescent label is functionalized to facilitate covalent attachment to a cellular component present in or on the surface of the cell or tissue such as a cell surface marker.
  • Suitable functional groups including, but not are limited to, isothiocyanate groups, amino groups, haloacetyl groups, maleimides, succinimidyl esters, and sulfonyl halides, all of which may be used to attach the fluorescent label to a second molecule.
  • the choice of the functional group of the fluorescent label will depend on the site of attachment to either a linker, the agent, the marker, or the second labeling agent.
  • Attachment of the fluorescent label may be either directly to the cellular component or compound or alternatively, can by via a linker.
  • Suitable binding pairs for use in indirectly linking the fluorescent label to the intermediate include, but are not limited to, antigens/antibodies, e.g., rhodamine/anti-rhodamine, biotin/avidin and biotin/strepavi Examples of suitable labels are also described in the table below:
  • kits comprising the products disclosed herein such as the cell culture medium, cells, and/or SSEA-4 binding agents. Further, the current disclosure provides kits for isolating and/or purifying stem cells, and kits for culturing and/or identifying the stem cells.
  • kits according the present invention can comprise as their constituents, for example, SSEA-4 binding agents, culture media or solutions and such for culturing cells. VIII. Methods and Therapeutic Applications
  • the methods and compositions described herein are useful for purification, isolation, and identification of naive stem cells.
  • the methods include detection or isolation of naive SSEA-4-negative stem cells on the basis of their SSEA-4- negative status.
  • Such methods may include methods known in the art such as fluorescent activated cell sorting (FACS), immunostaining, immunohistochemistry, histological methods, precipitation, chromatography, solid-support assays, magnetic activated cell sorting (MACS), and panning.
  • human embryonic stem cell therapy comprises first identifying and isolating SSEA-4 negative cells.
  • Treatment aspects relate to treating a patient with a stem cell, wherein the stem cell is a SSEA-4-negative cell.
  • the method may further comprise differentiation of the SSEA-4 negative naive stem cell. The differentiation may be done in vitro or in vivo. Therefore, treatment aspects relate to the administration of the SSEA-4 negative stem cell or its progeny.
  • Treatment aspects relate to treating a patient with a stem cell, wherein the stem cell is a SSEA-4-negative cell.
  • the therapy is for the treatment of conditions of the blood and immune system.
  • the therapy is for the treatment of leukemias and lymphomas, including: acute myelogeneous leukemia (AML), acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), chronic myelogeneous leukemia (CML), juvenile myelomonocytic leukemia, Hodgkin lymphoma, and non-Hodgkin lymphoma (NHL); for bone marrow diseases and other diseases when bone marrow fails to work, including: severe aplastic anemia, fanconi anemia, paroxysmal nocturnal hemoglobinuria (PNH), pure red cell aplasia, and amegakaryocytosis/congenital thrombocytopenia; for inherited immune system disorders, including: severe combined immunodeficiency (SCID, all types) and Wiskott- Aldrich syndrome (WAS); for hemoglobinopathies (diseases with poorly functioning red blood cells), including: beta thalassemia
  • FIG. 1 For embodiments, the in vitro fertilized embryo is first screened for SSEA-4 expression and wherein the individual is implanted with a embryo (cleavage or blastocyst) that has been first screened and determined to be SSEA-4-negative.
  • Example 1 Naive human pluripotent cells feature a methylation landscape devoid of blastocyst or germline memory
  • hESCs Human embryonic stem cells
  • SSEA4 Stage Specific Embryonic Antigen 4
  • hESCs Human embryonic stem cells
  • EpiSCs murine epiblast stem cells
  • hESCs and EpiSCs are said to exhibit a "primed pluripotent state” while, mouse ESCs derived from the pre-implantation blastocyst exhibit a "naive pluripotent state” corresponding to an earlier stage of development (Nichols and Smith, 2009).
  • hESCs are transfected with KLF2 and NANOG and cultured in media with titrated two inhibitors plus leukemia inhibitory factor and G56983 (t2iL+Go) (Takashima et al., 2014).
  • primed cells can be reverted by transferring to a media containing a cocktail of five inhibitors plus LIF, Activin and/or Fibroblast Growth Factor 2 (5iLA/F)(Theunissen et al., 2014).
  • Applicants reverted primed hESCs to the naive state using 5iLAF to evaluate gene expression and DNA methylation and also derived a new hESC line called UCLA20n entirely under 5iLAF naive culture conditions. It was discovered that reversion and maintenance in 5iLAF creates a heterogeneous mixture of cells in which the Stage Specific Embryonic Antigen 4 (SSEA4) negative cells are enriched in the transcriptional program of the pre- implantation epiblast. In contrast UCLA20n hESCs are almost entirely devoid of SSEA4 surface expression.
  • SSEA4 Stage Specific Embryonic Antigen 4
  • Applicants also performed RNA-Seq of UCLA20n at passage 20 after derivation. Applicants did not analyze UCLA19n as it was found to be 70% polyploid by passage 14. Consistent with the expression patterns of genes associated with naive pluripotency in mice, the 5 iLAF SSEA4 negative cells and UCLA20n had elevated levels of NANOG as well as a dramatic up-regulation of KRUPPLE LIKE FACTOR (KLF) family transcription factors and reduced expression primed state master regulators such as ZINC FINGER OF THE CEREBELLUM (ZIC) family transcription factors and OTX2 (Buecker et al., 2014; Tang et al., 2011; Yang et al., 2014) ( Figure IE).
  • KLF KRUPPLE LIKE FACTOR
  • the methylation pattern of the blastocyst is shaped by events during gametogenesis and early embryogenesis.
  • the male pronucleus is selectively demethylated in early embryonic development, with only a few regions such as paternally methylated imprinted loci protected from DNA demethylation (Okae et al., 2014; Smith et al., 2014; Smith et al., 2012).
  • the methylation pattern of the blastocyst strongly resembles Metaphase II human oocytes ( Figure 2B, C).
  • RNA and protein levels of DNA methylation and demethylation machinery were analyzed. Applicants found that the RNA and protein levels of the de novo DNA methyltransferase DNMT3B dropped sharply in the 5iLAF SSEA4 negative cells, while DNMT3A was unchanged and DNMT3L increased dramatically relative to primed hESCs. UHRF1 RNA levels were slightly elevated in naive hESCs. However, at the protein level, Applicants observe a 65% loss of UHRFl, and both DNMT1 RNA and protein levels were reduced by 50% in the naive state. Furthermore, expression of the 5mC oxidases TETl and TET2 increased substantially in the naive state. ( Figure 2G, 2H).
  • murine embryonic germ cell lines are transcriptionally similar to murine ESCs but have widespread loss of imprints and contribute poorly to chimeras (Leitch et al., 2013; Oliveros-Etter et al., 2015; Tada et al., 1998), demonstrating the importance of imprints in correct differentiation of pluripotent cells in vivo.
  • Applicants observed extensive karyotypic abnormalities in cells after prolonged culture in 5iLAF. Loss of DNA methylation has been linked to karyotypic instability (Haaf, 1995).
  • methylation at the imprinted loci is clearly depressed relative to surrounding regions. This may reflect the observation that many imprinted loci are promoters or regulatory elements that are active in the blastocyst (Rugg-Gunn et al., 2007). Thus if methylation is partially eroded at the imprint, the relevant transcription factors bind and cause further demethylation (as is generally the case at these genetic elements). In other words, methylation may be a very weak barrier to locus activation in 5iLAF. Similar dynamics may be at work at L1HS elements.
  • Tada T.
  • Tada M.
  • Hilton K.
  • Barton S.C.
  • Sado T.
  • Takagi N.
  • Surani M.A.

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Abstract

La présente invention concerne des méthodes d'isolement et de culture de cellules souches embryonnaires naïves qui, du point de vue du phénotype, ressemblent à des cellules souches issues de la pré-implantation. L'invention porte également sur des méthodes et compositions thérapeutiques comprenant ces cellules.
PCT/IB2017/050613 2016-02-03 2017-02-03 Méthodes et compositions de culture de cellules souches WO2017134628A1 (fr)

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