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  • 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
  • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
  • C12N5/0662—Stem cells
  • C12N5/0668—Mesenchymal stem cells from other natural sources
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
  • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
  • A61K35/28—Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
• • C—CHEMISTRY; METALLURGY
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  • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
  • C12N2501/40—Regulators of development
  • C12N2501/415—Wnt; Frizzeled
• • 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
  • C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
  • C12N2506/45—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from artificially induced pluripotent stem 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
  • C12N2533/00—Supports or coatings for cell culture, characterised by material
  • C12N2533/50—Proteins
  • C12N2533/52—Fibronectin; Laminin

Definitions

• the present invention relates generally to mesenchymal stromal cells (MSCs), and more specifically to methods of generating MSCs from pluripotent stem cells (PSCs).
• MSCs mesenchymal stromal cells
• PSCs pluripotent stem cells
• Pluripotent stem cells are cells that are capable of self-renewing and to giving rise to all cells of the three primary groups of cells that make up a human body, including: ectoderm (skin and nervous system cells), endoderm (including gastrointestinal and respiratory tracts cells, endocrine glands cells, liver cells, and pancreas cells), and mesoderm (including bone, cartilage, most of the circulatory system cells, muscles cells, connective tissue cells, and more).
• Pluripotent stem cells can be induced pluripotent stem cells (iPSCs) or embryonic stems cells (ESCs). Because they can propagate indefinitely and give rise to every cell type in the body, they represent a potential source for the development of therapeutic cells.
• iPSCs induced pluripotent stem cells
• ESCs embryonic stems cells
• MSCs Mesenchymal stromal cells
• ICT International Society for Cellular Therapy
• MSCs have been differentiated to MSCs (i) with a temporal induction of neural ectoderm using CHIR-99021 and SB-431542 in chemically defined media, followed by the exposure to the conventional MSC growth medium, containing bovine serum; (ii) by directly exposing the cells to the conventional medium that is used for growing MSCs: the medium containing 10% bovine serum; or (iii) using the TGF/ACTIVIN/NODAL inhibitor, SB431542 for the initial differentiation of hPSCs to MSCs.
• the present invention is based on the seminal discovery that the sole use of a WNT signaling pathway activator, such as a GSK3
• a WNT signaling pathway activator such as a GSK3
• the present invention provides a method of generating mesenchymal stromal/stem cells (MSCs) including: (a) contacting an adherent culture of pluripotent stem cells (PSCs) with a WNT signaling pathway activator for about 4-8 days; and (b) expanding the cells in culture from (a) in the absence of the WNT signaling pathway inhibitor, thereafter, thereby generating MSCs.
• MSCs mesenchymal stromal/stem cells
• the WNT signaling pathway activator is a GSK3
• the GSK3P inhibitor is CHIR99021.
• the PSCs are contacted with about 4 pM of CHIR99021.
• the PSCs are contacted with CHIR99021 for about 6 days.
• the PSCs are contacted with about 4 pM of CHIR99021 for about 6 days.
• contacting comprises incubating the PSCs with fewer than three WNT signaling pathway activators.
• contacting comprises incubating the PSCs with a single WNT signaling pathway activator.
• the PSCs are maintained in a culture media including a ROCK inhibitor.
• the ROCK inhibitor is Y- 27632.
• the PSCs are cultured on a laminin-coated surface.
• expanding the cells includes culturing the cells on a non-coated surface.
• the method produces an enriched culture of MSCs.
• the enriched culture includes at least about 90% MSCs.
• the enriched culture includes at least about 95% MSCs.
• the MSCs are CD73, CD44 and CD90 positive.
• the MSCs are CD14, CD31, TRA 1-60, CD34, HLA-DR, SOX2, miR-302 HT, PECAM1, CDH5, CD45, MYH6 and SOXIO negative.
• the MSCs generated are multipotent stem cells with osteogenic, adipogenic and chondrogenic differentiation potential.
• expanding the cells includes culturing the cells in a MSCs supporting culture media.
• contacting and expanding the cells include contacting and expanding in serum-free culture conditions.
• the culture of PSCs does not include embryoid bodies.
• the WNT signaling pathway activator is a WNT ligand, a recombinant protein, an inhibitor of a WNT signaling pathway inhibitor or an indirect WNT activator.
• the PSCs are human pluripotent stem cells (hPSCs).
• the hPSCs are induced pluripotent stem cells (hiPSCs) or human embryonic stems cells (hESCs).
• the PSCs are iPSCs.
• the invention provides a method of generating mesenchymal stromal/stem cells (MSCs) including: (a) contacting an adherent culture of iPSCs with about 4 pM of a single GSK3P inhibitor for about 6 days; and (b) expanding the cells in culture from (a) in the absence of the GSK30 inhibitor thereafter, wherein after (b), a population of at least about 90% pure CD73+, CD44+ and CD90+ MSCs is obtained, thereby obtaining MSCs.
• MSCs mesenchymal stromal/stem cells
• FIG. 1 is a schematic representation of a method of the invention.
• FIG. 2 shows photographs illustrating MSCs obtained by a method of the invention in continuous culture (left) and after being frozen thawed (right).
• FIG. 3 is a graph illustrating MSCs growth kinetics in culture.
• FIGS. 4A-4C illustrate characterization of the MSCs.
• FIG. 4A shows histograms illustrating CD73, CD90, CD44 and CD105 expression on MSCs as evaluated by flow cytometry.
• FIG. 4B shows histograms illustrating CD14, CD45, CD31, TRA 1-60, CD34 and HLA-DR expression in MSCs as evaluated by flow cytometry.
• FIG. 4C shows histograms illustrating negative controls staining on MSCs as evaluated by flow cytometry.
• FIGS. 5A-5E illustrate the expression of various alternative markers on iPSCs, MSCs, EPC, HSCs, and CMs, as measured by RT-qPCR.
• FIG. 5A is a graph bar illustrating the expression of pluripotency genes.
• FIG. 5B is a graph bar illustrating the expression of endothelial markers.
• FIG. 5C is a graph bar illustrating the expression of hematopoietic marker.
• FIG. 5D is a graph bar illustrating the expression of cardiac marker.
• FIG.5E is a graph bar illustrating the expression of neural crest marker.
• FIG. 6 shows illustrative photographs of adipogenic, osteogenic and chondrogenic cells differentiated from MSCs.
• FIGS. 7A-7B illustrate the differentiation of MSCs in various culture media.
• FIG. 7A shows histograms illustrating CD73, CD44 and CD90 expression on MSCs differentiated in MSC expansion medium 1.
• FIG. 7B shows histograms illustrating CD73, CD44 and CD90 expression on MSCs differentiated in MSC expansion medium 2.
• FIG. 8 shows photographs illustrating MSCs derived from 3 independent iPSCs lines.
• FIG. 9 shows histograms illustrating CD73, CD90, CD44 and CD105 expression on 3 independent MSC lines as evaluated by flow cytometry.
• FIG. 10 shows histograms illustrating TRA 1-60, CD31, CD45, CD14, CD34 and HLA-DR expression on MSCs as evaluated by flow cytometry.
• the present invention is based on the seminal discovery that that the sole use of a
• WNT signaling pathway activator such as a GSK3P inhibitor on PSCs yields enriched cultures of MSCs that can further yield terminally differentiated osteogenic, adipogenic and chondrogenic cells.
• the term “about” in association with a numerical value is meant to include any additional numerical value reasonably close to the numerical value indicated.
• the value can vary up or down by 5-10%.
• the value for a value of about 100, means 90 to 110 (or any value between 90 and 110).
• the present invention provides a method of generating mesenchymal stromal/stem cells (MSCs) including: (a) contacting an adherent culture of pluripotent stem cells (PSCs) with a WNT signaling pathway activator for about 4-8 days; and (b) expanding the cells in culture from (a) in the absence of the a WNT signaling pathway activator, thereafter, thereby generating MSCs.
• MSCs mesenchymal stromal/stem cells
• the methods described herein provide cellular culture conditions in which human pluripotent stem cells are grown, that yield the generation of a population of mesenchymal stromal/stem cells (MSCs).
• MSCs mesenchymal stromal/stem cells
• Stem cells are undifferentiated cells that have the ability to self-renew indefinitely and to remain in said undifferentiated state.
• embryonic stem cells which can only be isolated from the inner mass of a blastocyst
• the term “pluripotent stem cells”, as used herein refers to cells that are capable of generating all the cell types of an organism, i.e., cells derived from any of the three germ layers.
• multipotent stem cells can differentiate into several cell type, but only those of a closely related family of cells, generally the cell types of the organ from which they originate. Most adult stem cells are multipotent but small amounts of pluripotent adult stem cells can be retrieved from umbilical cord or other tissues.
• the sources of cells used for cell therapy include stem cells such as embryonic stem cells (ESCs), adult stem cells, and induced pluripotent stem cells (iPSCs).
• ESCs embryonic stem cells
• iPSCs induced pluripotent stem cells
• the PSCs used in the methods described herein are human (hPSCs), and in some instances the human PSCs are induced pluripotent stem cells (hiPSCs) or human embryonic stems cells (hESCs). In various aspects, the PSCs are human induced pluripotent stem cells (hiPSCs).
• MSCs By “generating” or “producing” MSCs, it is meant that the present methods provide physical and chemical culture conditions that have been optimized to induce the differentiation of iPSCs into MSCs.
• the differentiation method described herein yields a cell population that is enriched for MSCs. For example, greater than 80%, greater than 85%, greater than 90%, greater than 95%, 96%, 97%, 98% or 99% MSCs are obtained in short times and using convenient cultures conditions.
• Physical culture conditions include but are not limited to the culture environment of the cell (e.g., adherent versus suspension culture, or in two-dimensional versus in three- dimensional culture systems), the pH of the culture media, the gas concentration in the incubator (e.g., CO2 concentration, 02 concentration), and the temperature.
• the culture environment of the cell e.g., adherent versus suspension culture, or in two-dimensional versus in three- dimensional culture systems
• the pH of the culture media e.g., the pH of the culture media
• the gas concentration in the incubator e.g., CO2 concentration, 02 concentration
• hematopoietic cell lines Most of the cells derived from vertebrates, except for hematopoietic cell lines and a few others, are anchorage-dependent and have to be cultured on a suitable substrate that is specifically treated to allow cell adhesion and spreading (i.e., tissue-culture treated). However, many cell lines can also be adapted for suspension culture.
• the culture of PSCs is an adherent layer of cells.
• the layer of cells is grown in a two-dimensional culture system or on microcarriers.
• PSCs can be cultured in a three-dimensional culture system, by relying on nonadherent conditions and the formation of embryoid bodies.
• the culture of PSCs does not include embryoid bodies.
• Physical culture conditions include the gas concentration in the incubator. Incubation of cell cultures is typically performed in normal atmosphere with 15-22% oxygen and 5% CO2 for expansion and seeding.
• the PSCs are grown in a humidified atmosphere including about 5% CO2 concentration, and normoxic conditions (non-hypoxic O2 concentration). While hypoxic culture conditions are thought to support stem cell performance in general, in the present methods, the PSCs are cultured under conditions that are not hypoxic conditions.
• normoxic refer to culture conditions including atmospheric O2 concentration (e.g., about 15-25% O2 concentration).
• hypoxic conditions are characterized by a lower oxygen concentration as compared to the oxygen concentration of ambient air (approximately 15%-25% oxygen).
• Chemical culture conditions include but are not limited to the agents or molecules that are added to the culture medium to achieve the desired effects sought after (i.e., differentiation of PSCs into MSCs).
• agent and “molecule” are used interchangeably and include, but are not limited to, small molecules (including small molecules that do not have optimal cell-permeability), lipids, nucleosides, nucleotides, nucleic acids, polynucleotides, oligonucleotides, antibodies, toxins, negatively charged polymers and other polymers, for example proteins, peptides, hormones, carbohydrates, or polyamines.
• the PSCs are contacted with an agent that is a “a WNT signaling pathway activator”.
• a WNT signaling pathway activator By “contacting” it is meant that the cells are cultured with the one or more agent of interest. That is the cells are cultured in their regular culture media, in which a desired concentration of one or more agent of interest is added. For examples, the cells are cultured with a WNT signaling pathway activator.
• a “pathway signaling activator” as used herein refers to any molecule that can activate, enhancing, or inducing a signaling pathway of interest.
• a signaling pathway is a series of chemical reactions in which a group of molecules in a cell work together to control a cell function, such as cell differentiation.
• a cell receives signals from its environment when a molecule, such as a hormone or growth factor, binds to a specific protein receptor on or in the cell. After the first molecule in the pathway receives a signal, it activates another molecule. This process is repeated through the entire signaling pathway until the last molecule is activated and the cell function is carried out.
• molecule includes, but is not limited to, small molecules (including small molecules that do not have optimal cellpermeability), lipids, nucleosides, nucleotides, nucleic acids, polynucleotides, oligonucleotides, antibodies, toxins, negatively charged polymers and other polymers, for example proteins, peptides, hormones, carbohydrates, or polyamines.
• Non- limiting examples of polynucleotides include short interfering nucleic acid (siNA), antisense, enzymatic nucleic acid molecules, 2',5'-oligoadenylate, triplex forming oligonucleotides, aptamers, and decoys.
• siNA short interfering nucleic acid
• antisense antisense
• enzymatic nucleic acid molecules 2',5'-oligoadenylate
• triplex forming oligonucleotides aptamers
• decoys decoys
• Biologically active molecules include antibodies (e.g., monoclonal, chimeric, humanized etc.), cholesterol, hormones, antivirals, peptides, proteins, chemotherapeutics, small molecules, vitamins, co-factors, nucleosides, nucleotides, oligonucleotides, enzymatic nucleic acids, antisense nucleic acids, triplex forming oligonucleotides, 2,5-A chimeras, allozymes, aptamers, decoys and analogs thereof, and small nucleic acid molecules, such as short interfering nucleic acid (siNA), short interfering RNA (siRNA), double-stranded RNA (dsRNA), micro-RNA (miRNA), antagomirs, and short hairpin RNA (shRNA) molecules.
• siNA short interfering nucleic acid
• siRNA short interfering RNA
• dsRNA double-stranded RNA
• miRNA micro-RNA
• the WNT signaling pathways are a group of signal transduction pathways which begin with proteins that pass signals into a cell through cell surface receptors. Wnt signaling pathways use either nearby cell-cell communication (paracrine) or same-cell communication (autocrine). Three Wnt signaling pathways have been characterized: the canonical Wnt pathway, the noncanonical planar cell polarity pathway, and the noncanonical Wnt/calcium pathway. All three pathways are activated by the binding of a Wnt-protein ligand to a Frizzled family receptor, which passes the biological signal to the Dishevelled protein inside the cell. The canonical Wnt pathway leads to regulation of gene transcription and is thought to be negatively regulated in part by the SPATS 1 gene.
• the noncanonical planar cell polarity pathway regulates the cytoskeleton that is responsible for the shape of the cell.
• the noncanonical Wnt/calcium pathway regulates calcium inside the cell.
• Wnt signaling was first identified for its role in carcinogenesis, then for its function in embryonic development. The embryonic processes it controls include body axis patterning, cell fate specification, cell proliferation and cell migration. These processes are necessary for proper formation of important tissues including bone, heart, and muscle. Its role in embryonic development was discovered when genetic mutations in Wnt pathway proteins produced abnormal fruit fly embryos. Later research found that the genes responsible for these abnormalities also influenced breast cancer development in mice. Wnt signaling also controls tissue regeneration in adult bone marrow, skin, and intestine.
• WNT signaling pathway activator include WNT ligands, GSK3 inhibitors, Axin inhibitors, APC inhibitors.
• WNT activators also include:
• WNT3a is the most commonly used WNT ligand.
• Wntl, WNT2, Wnt3a, and Wnt7a are other available options.
• Another protein, Norrin is not related to Wnt family proteins, but it induces activation of the canonical Wnt signaling pathway.
• Indirect WNT activators GSK-3P inhibitors: a. LiCl is commonly used. b. Indirubins and derivatives: 6-bromo-indirubin-30-oxime (6-BIO) is commonly used. c. Small molecules: CHIR99021 is commonly used. Some others include SB- 216763 and SB-415286.
• Acceptable concentrations range for the application described herein is dependent upon the WNT signaling pathway activator, and one of skill in the art would easily determine such concentration range.
• an appropriate concentration range is about 1-500 ng/ml, preferably up to 200 ng/ml.
• WNT3a is usually used in a range of about 1 - 200 ng/ml, preferably above 10 ng/ml;
• LiCl is usually used in a range of about 1-
• CFUR99021 is usually used in a range of about 0.1-10 pM
• R-spondin is usually used in a range of about 1-200 ng/ml
• 6-bromoindirubin-3 '-oxime (BIO) is usually used in a range of about 0.1-10 pM
• Norrin is usually used in an range of about 1 - 200 ng/ml
• SB415286 is usually used in a range of about 1-100 pM.
• the PSCs are contacted with the GSK3P inhibitor for about 4-8 days.
• the cells are contacted with the GSK3P inhibitor for about 2, 3, 4, 5, 6, 7, 8, 9 or 10 days.
• the PSCs are contacted with the GSK3P inhibitor for about 6 days.
• GSK3P inhibitors There are many GSK3P inhibitors available. The methods described herein includes the use of a combination of GSK3P inhibitors.
• GSK3P inhibitors include: 3F8, A 1070722, Alsterpaullone, AR-A 014418, AZD 2858, BIO, BlO-acetoxime, CHIR 98014, CHIR 99021, CHIR 99021 trihydrochloride, Indirubin-3 '-oxime, Kenpaullone, Lithium carbonate, Lithium Chloride, SB 216763, SB 415286, TC-G 24, TCS 2002, TCS 21311, TDZD 8 and TWS 119.
• contacting comprises incubating the PSCs with fewer than three GSK3P inhibitors.
• the method includes contacting PSCs with a combination of two different GSK3P inhibitors.
• contacting comprises incubating the PSCs with a single GSK3P inhibitor.
• the GSK3P inhibitor is CHIR99021.
• the PSCs are contacted with CHIR99021 for about 4-8 days.
• the cells are contacted with CHIR99021for about 2, 3, 4, 5, 6, 7, 8, 9 or 10 days.
• the PSCs are contacted with CHIR99021for about 6 days.
• the PSCs are contacted with about 4 pM of CHIR99021for about 6 days.
• the PSCs are cultured on scaffold composed of microcarriers, which are beads or particles.
• the beads may be microscopic or macroscopic and may further be dimensioned to permit penetration into tissues or compacted to form a particular geometry.
• the framework for the cell cultures comprises particles that, in combination with the cells, form a three-dimensional tissue. The cells attach to the particles and to each other to form a three-dimensional tissue. Beads or microcarriers are typically considered a two- dimensional system or scaffold.
• a “microcarriers” refers to a particle having size of nanometers to micrometers, where the particles may be any shape or geometry, being irregular, non-spherical, spherical, or ellipsoid.
• the size of the microcarriers suitable for the purposes herein can be of any size suitable for the particular application. In some embodiments, the size of microcarriers suitable for the three-dimensional tissues may be those administrable by injection.
• microcarriers are made of biodegradable materials.
• microcarriers comprising two or more layers of different biodegradable polymers may be used.
• at least an outer first layer has biodegradable properties for forming the three-dimensional tissues in culture, while at least a biodegradable inner second layer, with properties different from the first layer, is made to erode when administered into a tissue or organ.
• the microcarriers are porous microcarriers.
• Porous microcarriers refer to microcarriers having interstices through which molecules may diffuse in or out from the microparticle.
• the microcarriers are non-porous microcarriers.
• a nonporous microparticle refers to a microparticle in which molecules of a select size do not diffuse in or out of the microparticle.
• the microcarriers comprise degradable scaffolds. These include microcarriers made from naturally occurring polymers, non-limiting example of which include, among others, fibrin, casein, serum albumin, collagen, gelatin, lecithin, chitosan, alginate, or poly-amino acids such as poly-lysine.
• the degradable microcarriers are made of synthetic polymers, non-limiting examples of which include, among others, polylactide (PLA), polyglycolide (PGA), poly(lactide-co-glycolide) (PLGA), poly(caprolactone), polydioxanone trimethylene carbonate, polyhybroxyalkonates (e.g., poly(hydroxybutyrate), poly (ethyl glutamate), poly (DTH iminocarbony (bisphenol A iminocarbonate), poly (ortho ester), and poly cyanoacrylates.
• PHA polylactide
• PGA polyglycolide
• PLGA poly(lactide-co-glycolide)
• poly(caprolactone) polydioxanone trimethylene carbonate
• polyhybroxyalkonates e.g., poly(hydroxybutyrate), poly (ethyl glutamate), poly (DTH iminocarbony (bisphenol A iminocarbonate), poly
• the microcarriers comprise hydrogels, which are typically hydrophilic polymer networks filled with water. Hydrogels have the advantage of selective trigger of polymer swelling. Depending on the composition of the polymer network, swelling of the microparticle may be triggered by a variety of stimuli, including pH, ionic strength, thermal, electrical, ultrasound, and enzyme activities.
• Non-limiting examples of polymers useful in hydrogel compositions include, among others, those formed from polymers of poly(lactide-co-glycolide); poly(N-isopropylacrylamide); poly (methacrylic acid-g- polyethylene glycol); polyacrylic acid and poly(oxypropylene-co-oxyethylene) glycol; and natural compounds such as chrondroitan sulfate, chitosan, gelatin, fibrinogen, or mixtures of synthetic and natural polymers, for example chitosan-poly (ethylene oxide).
• the polymers may be crosslinked reversibly or irreversibly to form gels adaptable for forming three dimensional tissues.
• microcarriers or beads for use in the present invention are composed wholly or composed partly of dextran.
• cells can require to be grown on coated surfaces, to enhance or improve their adhesion and/or spreading (i.e., using a coating).
• Coating as an additional surface treatment stands for all additional modifications made to increase cell adhesion in addition to the standard plasma or corona treatment which is performed on all cell culture plastic by manufacturer. Usually, coating is done with proteins or peptides.
• expanding the cells includes culturing the cells on a non-coated surface. That is, after the initial differentiation step of the cells (i.e., while the PSCs are contacted with a GSK3 inhibitor), the cells are expanded in the absence of the GSK3P inhibitor, and on a surface that is not coated.
• the enriched culture includes at least about 95% CD73, CD44 and CD90 positive and CD14, CD31, TRA 1-60, CD34, HLA-DR, SOX2, miR-302 HT, PECAM1, CDH5, CD45, MYH6 and SOX10 negative MSCs.
• the MSCs generated are multipotent stem cells with osteogenic, adipogenic and chondrogenic differentiation potential.
• differentiation potential it is meant that the MSCs obtained by the methods described herein can be terminally differentiated (using differentiation protocol well known in the art) into osteogenic, adipogenic and chondrogenic cells.
• the cells are expanded in a MSCs supporting culture media.
• MSCs supporting culture media There are several culture media that are known in the art as appropriate to support MSCs culture and expansion in vitro.
• the MSCs obtained with the methods described herein can be expanded in any MSCs supporting culture media.
• contacting and expanding the cells include contacting and expanding in serum-free culture conditions.
• the invention provides a method of generating mesenchymal stromal/stem cells (MSCs) including: (a) contacting an adherent culture of iPSCs with about 4 pM of a single GSK3P inhibitor for about 6 days; and (b) expanding the cells in culture from (a) in the absence of the GSK3P inhibitor thereafter, wherein after (b), a population of at least about 90% pure CD73+, CD44+ and CD90+ MSCs is obtained, thereby obtaining MSCs.
• MSCs mesenchymal stromal/stem cells
• the invention provides a method of generating terminally differentiated osteogenic, adipogenic and/or chondrogenic from iPSCs including: a) generating MSCs cells by:(i) contacting an adherent culture of iPSCs with about 4 pM of a single GSK3P inhibitor for about 6 days; and (ii) expanding the cells in culture from (a) in the absence of the GSK3P inhibitor thereafter, wherein after (b), a population of at least about 90% pure CD73+, CD44+ and CD90+ MSCs is obtained; and b) contacting the MSCs cells of a) with a mixture of agents to induce differentiation of the MSCs into terminally differentiated osteogenic, adipogenic and/or chondrogenic cells, thereby generating terminally differentiated osteogenic, adipogenic and/or chondrogenic cells.
• the protocol involves the addition of a single factor, a GSK3b inhibitor, CHIR99021 during the initial days under adherent conditions either with or without serum.
• the hiPSCs were seeded on Laminin (iMatrix 511) in iPS-brew medium with Rock inhibitor. The next day, the medium was replaced with MSC induction medium (MSCI medium: iPS Brew medium with a GSK3b inhibitor, CHIR99021 (4pM)). This treatment was continued for 6 days, with MSCI medium replacement every day.
• MSC growth medium which can be serum-free (e.g., Milteny MSC medium) or with serum components (hPL or FCS) - an MSC-supportive medium in general.
• the hiPSC-MSCs generated by the method described herein contained the more than >90% of the cells expressing the MSC markers, CD73, CD90, CD105 and CD44 and less than 1-2% of the cells expressing endothelial cell markers (CD31), hematopoietic stem/progenitor markers (CD45 and CD34), immunogenic marker HLA-G, or the pluripotency marker TRA-1-60.
• the hiPSCs were thawed on Thursdays and passaged on Monday mornings and Thursday afternoons at a density of 200,000-250,000 per well of a 6-well plate. For the experiments, the hiPSCs were seeded on Thursdays and differentiations initiated on Fridays.
• Pre- warm Accutase and the required volume of pre-warmed iPS Brew XF medium (XF Medium).
• PS to the XF medium at 1:100 ratio (XF medium+PS).
• Rock inhibitor Y- 27632 (Y)
• Y Rock inhibitor
• the hiPSCs should be 5070-100% confluent and undifferentiated, before passaging or seeding for the experiment. Aspirate and completely remove the medium and wash the cells with 2 ml PBS per well. After removing PBS, add 1 ml of pre-warmed Accutase+Y and place the plate in the CO2 incubator for 10 min. Most cells should come-off by gently agitating the plate. If this is not the case, prolong digestion for 2 more minutes and so forth, until the cells lift-off virtually by themselves.
• the hiPSC-induced (iMSCs) are seeded on plastic. Maintain a passaging ratio of 1 :2 for 3 passages, before diluting them.
• the cells can be diluted at 200K cells per well of a 6-well plate with 2ml MSCG medium. Medium is replaced the next day after seeding and then, every 2 days. Every Friday, the MSCs are fed with 3-4 ml MSCG medium.
• iPSC-MSCs For the passaging of iPSC-MSCs, aspirate and discard the medium. For 1 well of a 6- well plate, rinse the cells with 1 ml PBS (-/-). After discarding the PBS, add 1 ml of 0.5 mM EDTA (prepared in PBS (-/-)) and incubate the cells in the incubator for 4 minutes. Then aspirate, and discard EDTA. Add 1ml of TrypLE per well (6-well plate) and place the plate in the CO2 incubator for 4 minutes. Followinged by this, use a 1 ml pipette to bring the cells into solution by pipetting up and down. Add in minimum 3 ml MSCG medium in a 15 ml tube.
• iPSC-MSCs Cryopreservation and long-term storage of the iPSC-derived MSCs (iPSC-MSCs) [0105] The iPSC-MSCs are stored for long-term at -196°C, in the freezing medium.

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