Method for differentiating pluripotent stem cells into mesenchymal stromal cells

Abstract

The present invention relates to CD73 + CD44 + , CD90 + A method for generating a population of mesenchymal stromal cells (MSCs) is provided. + CD44 + , CD90 + MSCs are used in methods to generate terminally differentiated osteogenic, adipogenic, and chondrogenic cells from pluripotent stem cells (PSCs), which involve the use of a single agent, a WNT signaling pathway activator (e.g., a GSK3β inhibitor), in adherent cultures of PSCs.

Description

[Technical Field]

[0001] (CROSS-REFERENCE TO RELATED APPLICATIONS) This application claims the benefit of priority under 35 U.S.C. §119(e) of U.S. Provisional Patent Application No. 63 / 449,513, filed March 2, 2023, the entire contents of which are incorporated herein by reference in their entirety.

[0002] BACKGROUND OF THE INVENTION FIELD OF THE INVENTION The present invention relates generally to mesenchymal stromal cells (MSCs), and more particularly to methods for generating MSCs from pluripotent stem cells (PSCs). [Background technology]

[0003] (Background information) Pluripotent stem cells are cells capable of self-renewal and generating all three major cell groups that make up the human body: ectoderm (skin cells and nervous system cells), endoderm (including digestive and respiratory tract cells, endocrine gland cells, liver cells, and pancreatic cells), and mesoderm (including bone cells, chondrocytes, most circulatory system cells, muscle cells, connective tissue cells, etc.). Pluripotent stem cells can be induced pluripotent stem cells (iPSCs) or embryonic stem cells (ESCs). Because they can proliferate indefinitely and give rise to every cell type in the body, they represent a promising source for the development of therapeutic cells. Among many other cell types, PSCs can be differentiated into mesenchymal stromal cells and have the potential to generate osteogenic, adipogenic, and chondrogenic cells.

[0004] Mesenchymal stromal cells (MSCs) are self-renewing, adult multipotent stem cells present in small numbers in various tissues (e.g., bone marrow, adipose tissue, placenta, teeth, and umbilical cord), and possibly in all vascularized human tissues. MSCs have been shown to have therapeutic value for several diseases. The International Society for Cellular Therapy (ISCT) has published the following minimum criteria for defining multipotent MSCs: plastic adherence; expression of the cell surface markers CD73, CD90, and CD105; lack of expression of other lineage markers; and trilineage differentiation potential in vitro.

[0005] Currently, bone marrow-derived MSCs are used to treat diseases. Currently, over 1,400 clinical trials using MSCs for the treatment of various human diseases and conditions are registered on the clinicaltrials.gov website. However, the limited supply of bone marrow-derived MSCs is an obstacle to their successful use in cell replacement therapy. This shortcoming can be met by pluripotent stem cells (PSCs), which have considerable advantages over MSCs due to their unlimited proliferation and multilineage differentiation potential. The use of human PSCs (hPSCs) in either regenerative medicine or disease modeling / drug discovery requires their directed differentiation into a pure population of a specific cell type, avoiding other cell types. Differentiation of PSCs into MSCs could be a valuable method for generating sufficient cells for applications such as regenerative medicine, disease modeling, or drug screening for musculoskeletal disorders.

[0006] Various methods have been employed to induce MSCs from hPSCs. These protocols typically involve differentiating hiPSCs to a mesoderm-like state and then further differentiating them into MSCs. For example, human PSCs have been differentiated into MSCs by (i) transient induction of neuroectoderm using CHIR-99021 and SB-431542 in a chemically defined medium, followed by exposure to conventional bovine serum-containing MSC growth medium; (ii) direct exposure of the cells to 10% bovine serum-containing medium, the conventional medium used to grow MSCs; or (iii) use of the TGF / ACTIVIN / NODAL inhibitor SB431542 for initial differentiation of hPSCs into MSCs.

[0007] The protocols used to differentiate hPSCs into MSCs use either non-adherent (embryoid body) or adherent methods. Most create embryoid bodies from hiPSCs before differentiating them into MSCs, and use complex procedures involving several small molecules and / or recombinant proteins. Summary of the Invention [Problem to be solved by the invention]

[0008] There remains a need in the art for simpler and more efficient protocols for MSC derivation. [Means for solving the problem]

[0009] (Summary of the Invention) The present invention is based on the promising discovery that the use of a WNT signaling pathway activator (e.g., a GSK3β inhibitor) solely on PSCs results in enriched cultures of MSCs that can further give rise to terminally differentiated osteogenic, adipogenic, and chondrogenic cells.

[0010] In one embodiment, the present invention provides a method for producing mesenchymal stromal cells / mesenchymal stem cells (MSCs), the method comprising: (a) contacting an adherent culture of pluripotent stem cells (PSCs) with a WNT signaling pathway activator for about 4 days to about 8 days; and (b) thereafter expanding the cells in culture derived from (a) in the absence of the WNT signaling pathway inhibitor, thereby producing MSCs.

[0011] In one embodiment, the WNT signaling pathway activator is a GSK3β inhibitor. In some embodiments, the GSK3β inhibitor is CHIR99021. In one embodiment, the PSCs are contacted with about 4 μM CHIR99021. In another embodiment, the PSCs are contacted with CHIR99021 for about 6 days. In various embodiments, the PSCs are contacted with about 4 μM CHIR99021 for about 6 days. In one embodiment, the contacting step comprises incubating the PSCs with fewer than three WNT signaling pathway activators. In another embodiment, the contacting step comprises incubating the PSCs with a single WNT signaling pathway activator. In one embodiment, prior to (a), the PSCs are maintained in a culture medium comprising a ROCK inhibitor. In some embodiments, the ROCK inhibitor is Y-27632. In another embodiment, the PSCs are cultured on a laminin-coated surface. In one embodiment, the step of expanding the cells comprises culturing the cells on an uncoated surface. In another embodiment, the method produces an enrichment culture of MSCs. In some embodiments, the enrichment culture comprises at least about 90% MSCs. In other embodiments, the enrichment culture comprises at least about 95% MSCs. In one embodiment, the MSCs are positive for CD73, CD44, and CD90. In another embodiment, the MSCs are negative for CD14, CD31, TRA 1-60, CD34, HLA-DR, SOX2, miR-302 HT, PECAM1, CDH5, CD45, MYH6, and SOX10. In one embodiment, the generated MSCs are multipotent stem cells with osteogenic, adipogenic, and chondrogenic differentiation potential. In another embodiment, the step of expanding the cells comprises culturing the cells in an MSC-supporting culture medium. In one embodiment, the contacting and expanding steps comprise contacting and expanding in serum-free culture conditions. In another embodiment, said culture of PSCs does not comprise embryoid bodies.In one embodiment, 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. In one embodiment, the PSC is a human pluripotent stem cell (hPSC). In some embodiments, the hPSC is an induced pluripotent stem cell (hiPSC) or a human embryonic stem cell (hESC). In one embodiment, the PSC is an iPSC.

[0012] In another embodiment, the present invention provides a method of producing mesenchymal stromal cells / mesenchymal stem cells (MSCs), the method comprising: (a) contacting an adherent culture of iPSCs with about 4 μM of a single GSK3β inhibitor for about 6 days; and (b) thereafter expanding the cells in culture derived from (a) in the absence of said GSK3β inhibitor, wherein after (b), a population of at least about 90% pure CD73+, CD44+ and CD90+ MSCs is obtained, thereby yielding MSCs. [Brief explanation of the drawings]

[0013] [Figure 1] FIG. 1 is a schematic diagram of the method of the present invention.

[0014] [Figure 2] FIG. 2 shows a photograph showing MSCs during continuous culture (left) and after being frozen and thawed (right), both obtained by the method of the present invention.

[0015] [Figure 3] FIG. 3 is a graph showing the growth kinetics of MSCs in culture.

[0016] [Figure 4A]Figures 4A-4C show the characterization of MSCs. Figure 4A shows histograms demonstrating the expression of CD73, CD90, CD44, and CD105 in MSCs assessed by flow cytometry. Figure 4B shows histograms demonstrating the expression of CD14, CD45, CD31, TRAIL-1-60, CD34, and HLA-DR in MSCs assessed by flow cytometry. Figure 4C shows histograms demonstrating negative control staining in MSCs assessed by flow cytometry. [Figure 4B] Figures 4A-4C show the characterization of MSCs. Figure 4A shows histograms demonstrating the expression of CD73, CD90, CD44, and CD105 in MSCs assessed by flow cytometry. Figure 4B shows histograms demonstrating the expression of CD14, CD45, CD31, TRAIL-1-60, CD34, and HLA-DR in MSCs assessed by flow cytometry. Figure 4C shows histograms demonstrating negative control staining in MSCs assessed by flow cytometry. [Figure 4C] Figures 4A-4C show the characterization of MSCs. Figure 4A shows histograms demonstrating the expression of CD73, CD90, CD44, and CD105 in MSCs assessed by flow cytometry. Figure 4B shows histograms demonstrating the expression of CD14, CD45, CD31, TRAIL-1-60, CD34, and HLA-DR in MSCs assessed by flow cytometry. Figure 4C shows histograms demonstrating negative control staining in MSCs assessed by flow cytometry.

[0017] [Figures 5A-5E] Figures 5A-5E show the expression of various surrogate markers in iPSCs, MSCs, EPCs, HSCs, and CMs as measured by RT-qPCR. Figure 5A is a bar graph showing the expression of pluripotency genes. Figure 5B is a bar graph showing the expression of endothelial markers. Figure 5C is a bar graph showing the expression of hematopoietic markers. Figure 5D is a bar graph showing the expression of cardiac markers. Figure 5E is a bar graph showing the expression of neural crest markers.

[0018] [Figure 6] FIG. 6 shows exemplary photographs of adipogenic, osteogenic, and chondrogenic cells differentiated from MSCs.

[0019] [Figures 7A-7B] Figures 7A-7B show the differentiation of MSCs in various culture media. Figure 7A shows histograms showing the expression of CD73, CD44, and CD90 in MSCs differentiated in MSC Expansion Medium 1. Figure 7B shows histograms showing the expression of CD73, CD44, and CD90 in MSCs differentiated in MSC Expansion Medium 2.

[0020] [Figure 8] FIG. 8 shows photographs showing MSCs derived from three independent iPSC lines.

[0021] [Figure 9-1] FIG. 9 shows histograms showing the expression of CD73, CD90, CD44 and CD105 in three independent MSC lines assessed by flow cytometry. [Figure 9-2] FIG. 9 shows histograms showing the expression of CD73, CD90, CD44 and CD105 in three independent MSC lines assessed by flow cytometry.

[0022] [Figure 10-1] FIG. 10 shows histograms showing the expression of TRA 1-60, CD31, CD45, CD14, CD34 and HLA-DR in MSCs as assessed by flow cytometry. [Figure 10-2] FIG. 10 shows histograms showing the expression of TRA 1-60, CD31, CD45, CD14, CD34 and HLA-DR in MSCs as assessed by flow cytometry. [Figure 10-3]FIG. 10 shows histograms showing the expression of TRA 1-60, CD31, CD45, CD14, CD34 and HLA-DR in MSCs as assessed by flow cytometry. [Figure 10-4] FIG. 10 shows histograms showing the expression of TRA 1-60, CD31, CD45, CD14, CD34 and HLA-DR in MSCs as assessed by flow cytometry. DETAILED DESCRIPTION OF THE INVENTION

[0023] (Detailed Description of the Invention) The present invention is based on the promising discovery that the use of a WNT signaling pathway activator (e.g., a GSK3β inhibitor) alone in PSCs results in enriched cultures of MSCs that can further give rise to terminally differentiated osteogenic, adipogenic, and chondrogenic cells.

[0024] Before the present compositions and methods are described, it is to be understood that the present invention is not limited to the particular compositions, methods, and experimental conditions described, as such compositions, methods, and conditions may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.

[0025] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "the method" includes one or more methods, and / or steps of the type described herein that will become apparent to those skilled in the art upon reading this disclosure and so forth.

[0026] As used herein, the term "and / or" includes any and all combinations of one or more of the associated listed items.

[0027] As used herein, the term "about" in connection with a numerical value is intended to include any additional numerical value reasonably close to the indicated numerical value. For example, depending on the context, the value may vary by 5% to 10% higher or lower. For example, a value of about 100 means 90 to 110 (or any value between 90 and 110).

[0028] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

[0029] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention belongs. Any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, but it is understood that modifications and variations are included within the spirit and scope of this disclosure. Preferred methods and materials are described herein.

[0030] In one embodiment, the present invention provides a method for producing mesenchymal stromal cells / mesenchymal stem cells (MSCs), the method comprising: (a) contacting an adherent culture of pluripotent stem cells (PSCs) with a WNT signaling pathway activator for about 4 days to about 8 days; and (b) thereafter expanding the cells in culture derived from (a) in the absence of the WNT signaling pathway activator, thereby producing MSCs.

[0031] The methods described herein provide cell culture conditions in which human pluripotent stem cells are grown, said cell culture conditions resulting in the generation of a population of mesenchymal stromal cells / mesenchymal stem cells (MSCs).

[0032] Stem cells are undifferentiated cells that have the ability to self-renew and remain in their undifferentiated state indefinitely. In contrast to embryonic stem cells, which can only be isolated from the inner cell mass of a blastocyst, there are three known sources of adult stem cells available: bone marrow (which requires bone drilling), adipose tissue (which can be obtained by liposuction), and blood (from which adult stem cells can be extracted, among other cells). As used herein, the term "pluripotent stem cells" refers to cells that can generate all of an organism's cell types, i.e., cells derived from any of the three germ layers. On the other hand, multipotent stem cells can differentiate into several cell types, but only into cell types of closely related cell families, generally only into cell types of the organ from which they originated. Although most adult stem cells are multipotent, small amounts of pluripotent adult stem cells can be recovered from the umbilical cord or other tissues. 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).

[0033] In some embodiments, the PSCs used in the methods described herein are human PSCs (hPSCs), and in some cases, the human PSCs are induced pluripotent stem cells (hiPSCs) or human embryonic stem cells (hESCs). In various embodiments, the PSCs are human induced pluripotent stem cells (hiPSCs).

[0034] By "producing" or "generating" MSCs, it is intended that the present methods provide optimized physical and chemical culture conditions for inducing differentiation of iPSCs into MSCs. The differentiation methods described herein result in cell populations enriched for MSCs. For example, greater than 80%, greater than 85%, greater than 90%, greater than 95%, 96%, 97%, 98%, or 99% MSCs can be obtained in a short time using conventional culture conditions.

[0035] Physical culture conditions include, but are not limited to, the cell culture environment (e.g., adherent culture or suspension culture, or two-dimensional culture system or three-dimensional culture system), the pH of the culture medium, gas concentration in the incubator (e.g., CO2 concentration, O2 concentration), and temperature.

[0036] Two basic systems exist for growing cells in culture as monolayers on an artificial substrate (i.e., adherent culture) or in suspension in culture medium (suspension culture). The majority of cells derived from vertebrates (with the exception of hematopoietic cell lines and a few others) are anchorage-dependent and must be cultured on a suitable substrate that has been specially treated (i.e., tissue culture-treated) to allow cell attachment and spreading. However, many cell lines can also be adapted for suspension culture.

[0037] In various embodiments, the culture of PSCs is an adherent monolayer of cells. In some embodiments, the monolayer of cells is grown in a two-dimensional culture system or on microcarriers.

[0038] PSCs can be cultured in a three-dimensional culture system by relying on non-adherent conditions and the formation of embryoid bodies. In one embodiment, in the methods described herein, the culture of PSCs does not include embryoid bodies.

[0039] Physical culture conditions include gas concentrations in the incubator. Cell culture incubation is typically performed in a standard atmosphere containing 15%-22% oxygen and 5% CO for expansion and seeding. In various embodiments, the PSCs are grown in a humidified atmosphere containing approximately 5% CO and normoxic conditions (non-hypoxic O concentrations). While hypoxic culture conditions are generally believed to support stem cell performance, in the present method, the PSCs are cultured under conditions that are not hypoxic. As used herein, "normoxic" conditions refer to culture conditions that include atmospheric O concentrations (e.g., approximately 15%-25% O concentrations). As used herein, hypoxic conditions are characterized by a low oxygen concentration compared to the oxygen concentration of ambient air (approximately 15%-25% oxygen).

[0040] Chemical culture conditions include, but are not limited to, agents or molecules added to the culture medium to achieve the desired effect (i.e., differentiation of PSCs to MSCs). The terms "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 (e.g., proteins, peptides, hormones, carbohydrates, or polyamines).

[0041] In the methods described herein, the PSCs are contacted with an agent that is a "WNT signaling pathway activator." By "contacting," it is meant that the cells are cultured with one or more agents of interest. That is, the cells are cultured in their normal culture medium, to which a desired concentration of one or more agents of interest has been added. For example, the cells are cultured with a WNT signaling pathway activator.

[0042] As used herein, a "pathway signaling activator" refers to any molecule that can activate, enhance, or induce 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 cellular function (e.g., cell differentiation). A cell receives a signal from its environment when a molecule (e.g., a hormone or growth factor) binds to a specific protein receptor on or in the cell. After the first molecule in the pathway receives the signal, it activates another molecule. This process is repeated throughout the signaling pathway until the final molecule is activated and the cellular function is carried out. Abnormal activation of a signaling pathway or inhibition of a signaling pathway can result in disease or, in the case of pluripotent cells, can result in a change in the pluripotent state and thus differentiation. The term "molecule" includes, but is 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 (e.g., proteins, peptides, hormones, carbohydrates, or polyamines). Non-limiting examples of polynucleotides include small interfering nucleic acids (siNAs), antisense, enzymatic nucleic acid molecules, 2',5'-oligoadenylates, triplex-forming oligonucleotides, aptamers, and decoys. Bioactive molecules include antibodies (e.g., monoclonal antibodies, chimeric antibodies, humanized antibodies, etc.), cholesterol, hormones, antivirals, peptides, proteins, chemotherapeutic agents, small molecules, vitamins, cofactors, nucleosides, nucleotides, oligonucleotides, enzymatic nucleic acids, antisense nucleic acids, triplex-forming oligonucleotides, 2,5-A chimeras, allozymes, aptamers, decoys and their analogs, and small nucleic acid molecules (e.g., small interfering nucleic acids (siNA), small interfering RNA (siRNA), double-stranded RNA (dsRNA), microRNA (miRNA), antagomir, and short hairpin RNA (shRNA) molecules).

[0043] The Wnt signaling pathway is a group of signaling pathways that initiate with proteins that transmit signals to cells through cell surface receptors. Wnt signaling pathways utilize either paracrine signaling between neighboring cells or autocrine signaling within the same cell. Three Wnt signaling pathways have been characterized: the canonical Wnt pathway, the non-canonical planar cell polarity pathway, and the non-canonical Wnt / calcium pathway. All three pathways are activated by the binding of Wnt protein ligands to Frizzled family receptors, which transmit biological signals to the Dishevelled protein within the cell. The canonical Wnt pathway regulates gene transcription, and the canonical Wnt pathway is thought to be negatively regulated in part by the SPATS1 gene. The non-canonical planar cell polarity pathway regulates the cytoskeleton, which is responsible for cell shape. The non-canonical Wnt / calcium pathway regulates intracellular calcium. Wnt signaling was first identified for its role in carcinogenesis and subsequently for its function in embryonic development. Embryonic processes regulated by Wnt signaling include axial patterning, cell fate specification, cell proliferation, and cell migration. These processes are necessary for the proper formation of critical 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. Subsequent studies revealed that the genes responsible for these abnormalities also affect the development of breast cancer in mice. Wnt signaling also controls tissue regeneration in adult bone marrow, skin, and intestine.

[0044] In one embodiment, said WNT signaling pathway activator is a WNT ligand, a recombinant protein, an inhibitor of the WNT signaling pathway inhibitor, or an indirect WNT activator.

[0045] Many substances can activate the WNT signaling pathway (e.g., activate β-catenin): non-limiting examples of WNT signaling pathway activators include WNT ligands, GSK3 inhibitors, Axin inhibitors, and APC inhibitors.

[0046] WNT activators also include:

[0047] 1. Recombinant Proteins. There are several WNT ligands that can activate WNT signaling. WNT3a is the most commonly used WNT ligand. Recombinant Wnt1, WNT2, Wnt3a, and Wnt7a are other available options. Another protein, Norrin, is not related to the Wnt family proteins, but it induces activation of the canonical Wnt signaling pathway.

[0048] 2. Inhibition of WNT inhibitors. A commonly used option is R-spondin.

[0049] 3. Indirect WNT activators: GSK-3β inhibitors: -a.LiCl is commonly used. -b. Indirubin and derivatives: 6-bromo-indirubin-30-oxime (6-BIO) is commonly used. -c. Small molecules: CHIR99021 is commonly used. Others include SB-216763 and SB-415286.

[0050] The acceptable concentration range for the applications described herein depends on the WNT signaling pathway activator, and one skilled in the art can easily determine such concentration range. Generally, for WNT ligands, a suitable concentration range is from about 1 ng / ml to about 500 ng / ml, preferably up to 200 ng / ml. For example, WNT3a is typically used in the range of about 1 ng / ml to about 200 ng / ml, preferably greater than 10 ng / ml; LiCl is typically used in the range of about 1 mM to about 10 mM; CHIR99021 is typically used in the range of about 0.1 μM to about 10 μM; R-spondin is typically used in the range of about 1 ng / ml to about 200 ng / ml; 6-bromoindirubin-3'-oxime (BIO) is typically used in the range of about 0.1 μM to about 10 μM; Norrin is typically used in the range of about 1 ng / ml to about 200 ng / ml; and SB415286 is typically used in the range of about 1 μM to about 100 μM.

[0051] In some embodiments, the WNT signaling pathway activator is a GSK3 inhibitor.

[0052] In one embodiment, the PSCs are contacted with about 2 μM to about 6 μM of the GSK3β inhibitor. For example, the cells are contacted with about 1 μM, about 2 μM, about 3 μM, about 4 μM, about 5 μM, about 6 μM, about 7 μM, or about 8 μM of the GSK3β inhibitor. In various embodiments, the PSCs are contacted with about 4 μM of the GSK3β inhibitor.

[0053] In another embodiment, the PSCs are contacted with the GSK3β inhibitor for about 4 days to about 8 days. For example, the cells are contacted with the GSK3β inhibitor for about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, or about 10 days. In various embodiments, the PSCs are contacted with the GSK3β inhibitor for about 6 days.

[0054] In many embodiments, the PSCs are contacted with about 4 μM of the GSK3β inhibitor for about 6 days.

[0055] There are many GSK3β inhibitors available.The methods described herein comprise the use of a combination of GSK3β inhibitors.Non-limiting examples of GSK3β inhibitors include: 3F8, A 1070722, Alsterpaullone, AR-A 014418, AZD 2858, BIO, BIO-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.

[0056] In one embodiment, the contacting step comprises incubating the PSCs with fewer than three GSK3β inhibitors. For example, the method comprises contacting the PSCs with a combination of two different GSK3β inhibitors. In another embodiment, the contacting step comprises incubating the PSCs with a single GSK3β inhibitor. In various embodiments, the GSK3β inhibitor is CHIR99021.

[0057] In one embodiment, the PSCs are contacted with about 2 μM to about 6 μM CHIR99021. For example, the cells are contacted with about 1 μM, about 2 μM, about 3 μM, about 4 μM, about 5 μM, about 6 μM, about 7 μM, or about 8 μM CHIR99021. In various embodiments, the iPSCs are contacted with about 4 μM CHIR99021.

[0058] In another embodiment, the PSCs are contacted with CHIR99021 for about 4 days to about 8 days. For example, the cells are contacted with CHIR99021 for about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, or about 10 days. In various embodiments, the PSCs are contacted with CHIR99021 for about 6 days.

[0059] In many embodiments, the PSCs are contacted with about 4 μM CHIR99021 for about 6 days.

[0060] In one embodiment, prior to (a), the PSCs are maintained in a culture medium containing a ROCK inhibitor. In some embodiments, the ROCK inhibitor is Y-27632.

[0061] In the methods described herein, the PSCs are cultured as adherent layers of cells, which are grown in two-dimensional culture systems or on microcarriers.

[0062] For example, the PSCs are cultured in a scaffold composed of microcarriers (beads or particles). The beads may be microscopic or macroscopic, and may be sized to allow penetration into tissue or compressed to form a particular shape. In some embodiments, the cell culture framework includes particles that combine with the cells to form a three-dimensional tissue. The cells attach to the particles and to each other to form the three-dimensional tissue. Beads or microcarriers are typically considered two-dimensional systems or scaffolds. As used herein, "microcarriers" refers to particles having sizes ranging from nanometers to micrometers, and the particles may be of any shape or form (irregular, non-spherical, spherical, or ellipsoidal). Microcarriers suitable for purposes herein may be of any size suitable for a particular application. In some embodiments, microcarriers suitable for the three-dimensional tissue may be of a size that allows administration by injection. In some embodiments, the microcarriers have a particle size range of at least about 1 μm, at least about 10 μm, at least about 25 μm, at least about 50 μm, at least about 100 μm, at least about 200 μm, at least about 300 μm, at least about 400 μm, at least about 500 μm, at least about 600 μm, at least about 700 μm, at least about 800 μm, at least about 900 μm, or at least about 1000 μm.

[0063] In some embodiments, the microcarriers are made of biodegradable materials. In some embodiments, microcarriers comprising two or more different biodegradable polymer layers can be used. In some embodiments, at least a first outer layer has biodegradable properties for forming the three-dimensional tissue during culture, while at least a second inner biodegradable layer, having properties different from those of the first layer, is made to degrade when administered to a tissue or organ.

[0064] In some aspects, the microcarriers are porous microcarriers. Porous microcarriers refer to microcarriers that have pores through which molecules can diffuse into or out of the microparticle. In other embodiments, the microcarriers are non-porous microcarriers. Non-porous microparticles refer to microparticles in which molecules of a selected size cannot diffuse into or out of the microparticle.

[0065] Microcarriers for use in the compositions are biocompatible and have low or no toxicity to cells. The microcarriers can comprise a variety of polymers (natural or synthetic, charged (i.e., anionic or cationic) or uncharged, biodegradable or non-biodegradable). The polymers can be homopolymers, random copolymers, block copolymers, graft copolymers, and branched polymers.

[0066] In some embodiments, the microcarriers comprise non-biodegradable microcarriers, including, but not limited to, those made of polysulfone, poly(acrylonitrile-co-vinyl chloride), ethylene vinyl acetate, and hydroxyethyl methacrylate-methyl-methacrylate copolymers, which are useful for providing tissue bulking properties or in embodiments where the microcarriers are eliminated by the body.

[0067] In some embodiments, the microcarriers comprise degradable scaffolds, including microcarriers made from naturally occurring polymers, non-limiting examples of which include fibrin, casein, serum albumin, collagen, gelatin, lecithin, chitosan, alginate, or polyamino acids (e.g., polylysine), among others. In other embodiments, the degradable microcarriers are made of synthetic polymers, non-limiting examples of which include 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(orthoesters), and polycyanoacrylates, among others.

[0068] In some embodiments, the microcarriers comprise hydrogels, which are typically hydrophilic polymer networks filled with water. Hydrogels have the advantage of being selective inducers of polymer swelling. Depending on the composition of the polymer network, swelling of the microparticles can be induced by various stimuli, including pH, ionic strength, heat, electricity, ultrasound, and enzyme activity. Non-limiting examples of polymers useful in hydrogel compositions include, among others, polymers formed from the polymer poly(lactide-co-glycolide); polymers formed from the polymer poly(N-isopropylacrylamide); polymers formed from the polymer poly(methacrylic acid-g-polyethylene glycol); polymers formed from polyacrylic acid and poly(oxypropylene-co-oxyethylene) glycol; and polymers formed from natural compounds (e.g., chondroitin sulfate, chitosan, gelatin, fibrinogen) or blends of synthetic and natural polymers (e.g., chitosan-poly(ethylene oxide)). The polymers can be reversibly or irreversibly crosslinked to form gels that are compatible for forming three-dimensional tissues.

[0069] In exemplary embodiments, microcarriers or beads for use in the present invention are composed entirely of dextran or partially of dextran.

[0070] In addition to treating the tissue culture surface, cells may need to be grown on a coated surface (i.e., using a coating) to enhance or improve cell adhesion and / or spreading. "Coating" as an additional surface treatment refers to any additional modifications made to increase cell adhesion beyond the standard plasma or corona treatment performed by manufacturers on all cell culture plastics. Typically, coatings are made with proteins or peptides. Various proteins can be used to coat tissue culture-treated dishes, including poly-L-lysine, poly-D-lysine, poly-ornithine, gelatin, collagen I, collagen IV, fibronectin, laminin, vitronectin, osteopontin, fibronectin domains, Matrigel™ (several components of the extracellular matrix to which growth factors and the like are bound), collagen gel, alginate gel, and lactate gel.

[0071] In one embodiment, the two-dimensional culture system or microcarriers are coated. In various embodiments, the PSCs are cultured on a surface coated with laminin.

[0072] In another embodiment, the step of expanding the cells comprises culturing the cells on an uncoated surface, i.e., after an initial differentiation step of the cells (i.e., during which the PSCs are contacted with a GSK3β inhibitor), the cells are expanded in the absence of the GSK3β inhibitor and on an uncoated surface.

[0073] In the method described herein, induced pluripotent stem cells are differentiated into MSCs.As used herein, " MSCs " refers to the multipotent cells that express a combination of specific markers, do not express the markers specific to other differentiated cells or the markers specific to undifferentiated cells, and can be further terminally differentiated into osteogenic cells, adipogenic cells or chondrogenic cells.For example, MSCs are usually characterized as the adhesion cells that are positive for CD73, CD44 and CD90.MSCs are usually characterized as the adhesion cells that are positive for CD73, CD44 and CD90, and negative for CD14, CD31, TRA 1-60, CD34, HLA-DR, SOX2, miR-302HT, PECAM1, CDH5, CD45, MYH6 and SOX10. After approximately 6 days of culture under the conditions described herein, the MSCs are adherent cells that are positive for CD73, CD44, and CD90 and negative for CD14, CD31, TRA 1-60, CD34, HLA-DR, SOX2, miR-302 HT, PECAM1, CDH5, CD45, MYH6, and SOX10.

[0074] In one embodiment, the methods described herein produce an enrichment culture of MSCs. In some embodiments, the enrichment culture comprises at least about 80% MSCs. For example, the enrichment culture comprises at least about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or more MSCs that are positive for CD73, CD44, and CD90. In one embodiment, the enrichment culture comprises at least about 95% MSCs that are positive for CD73, CD44, and CD90 and negative for CD14, CD31, TRAIL-1-60, CD34, HLA-DR, SOX2, miR-302 HT, PECAM1, CDH5, CD45, MYH6, and SOX10.

[0075] In one embodiment, the generated MSCs are multipotent stem cells with osteogenic, adipogenic and chondrogenic differentiation potential.

[0076] By "differentiation potential" it is intended that the MSCs obtained by the methods described herein are capable of terminal differentiation into osteogenic, adipogenic and chondrogenic cells (using differentiation protocols well known in the art).

[0077] In another embodiment, once differentiated into MSCs, the cells are expanded in an MSC-supporting culture medium. There are several culture media known in the art that are suitable for supporting the in vitro culture and expansion of MSCs. The MSCs obtained using the methods described herein can be expanded in any MSC-supporting culture medium. In one embodiment, the contacting step and the expanding step of the cells comprise contacting and expanding in serum-free culture conditions.

[0078] In another embodiment, the present invention provides a method of producing mesenchymal stromal cells / mesenchymal stem cells (MSCs), the method comprising: (a) contacting an adherent culture of iPSCs with about 4 μM of a single GSK3β inhibitor for about 6 days; and (b) thereafter expanding the cells in culture derived from (a) in the absence of said GSK3β inhibitor, wherein after (b), a population of at least about 90% pure CD73+, CD44+ and CD90+ MSCs is obtained, thereby yielding MSCs.

[0079] In one embodiment, the present invention provides a method for producing terminally differentiated osteogenic, adipogenic and / or chondrogenic cells from iPSCs, the method comprising the steps of: a) producing MSC cells by: (i) contacting an adherent culture of iPSCs with about 4 μM of a single GSK3β inhibitor for about 6 days; (ii) thereafter expanding the cells in culture from (a) in the absence of said GSK3β inhibitor, wherein after (b), a population of at least about 90% pure CD73+, CD44+ and CD90+ MSCs is obtained; and b) contacting the MSC cells of a) with a mixture of agents to induce differentiation of the MSCs into terminally differentiated osteogenic, adipogenic and / or chondrogenic cells, thereby producing terminally differentiated osteogenic, adipogenic and / or chondrogenic cells.

[0080] Below, examples are presented that discuss the method of preparing MSCs from iPSCs that are intended for the application discussed.The following examples are provided to further illustrate the embodiments of the present invention, but are not intended to limit the scope of the present invention.These are typical of those that can be used, but other procedures, methodologies or techniques known to those skilled in the art can be used instead. [Example]

[0081] Example 1 (Design of a protocol for differentiating iPSCs into MSCs) In a stepwise iterative development process, a novel protocol that promotes MSC differentiation with high efficiency was identified using a simple, GMP-compatible workflow (see Figure 1).

[0082] This protocol involves the addition of a single factor, the GSK3b inhibitor CHIR99021, for the first few days under either serum-containing or serum-free adherence conditions. hiPSCs were seeded on laminin (iMatrix 511) in iPS-brew medium containing the Rock inhibitor. The following day, the medium was replaced with MSC induction medium (MSCI medium: iPS-brew medium containing the GSK3b inhibitor CHIR99021 (4 μM)). This treatment was continued for 6 days, with daily changes of MSCI medium. On day 7, the medium was replaced with MSC growth medium (MSCG), which may be serum-free (e.g., Milteny MSC medium) or contain serum components (hPL or FCS) (commonly known as MSC support medium). The hiPSC-MSCs generated by the methods described herein contained greater than 90% cells expressing the MSC markers CD73, CD90, CD105, and CD44, and less than 1-2% cells expressing the endothelial cell marker (CD31), hematopoietic stem / progenitor cell markers (CD45 and CD34), the immunogenic marker HLA-G, or the pluripotency marker TRA-1-60.

[0083] Following this protocol, MSCs were maintained in culture for extended periods of time, and they maintained their phenotypic characteristics both in continuous culture and after being frozen and thawed (see Figures 2 and 3).

[0084] Example 2 (material and method) [Table 1-1] [Table 1-2] [Table 1-3]

[0085] [Table 2-1] [Table 2-2]

[0086] (method):

[0087] (hiPSC thawing and maintenance):

[0088] hiPSCs were thawed on Thursday and passaged at a density of 200,000-250,000 cells per well of a 6-well plate on Monday morning and Thursday afternoon. For these experiments, hiPSCs were seeded on Thursday and differentiation was initiated on Friday.

[0089] (Thawing hiPSCs): -PS was added to iPS Brew XF medium (XF medium) at a ratio of 1:100 (XF medium + PS). Rock inhibitor (Y-27632(Y)) was added to XF medium + PS at a concentration of 1:1000 and mixed. - Coat each well of a 6-well plate with 3 μl of iMatrix-511 in 2 ml of XF medium + PS + Y per well for at least 1 hour at 37° C. Preheat the required volume of iPS Brew XF medium (XF medium). - Add 5 ml of XF medium + PS + Y medium to a 15 ml tube. - Transfer frozen cryovials to the work area, ideally using a liquid nitrogen dewar, or alternatively on dry ice, if the walking distance is short (less than approximately 2 minutes) and within the same laboratory area. - Thaw a frozen cryovial of a given cell line by hand or in a 37°C water bath. Once thawed, wipe the tube with a disinfectant wipe and transfer it to a cell culture hood. The duration of the thawing process should be no more than 4 minutes maximum. -Open the cryotube and gently pipette up and down once using a 1 ml pipette, then transfer the cell suspension to the 15 ml tube containing 5 ml of XF medium + PS + Y medium. Close the 15 ml tube and centrifuge at 300 x g for 1 minute. Meanwhile, label the lid of a new plate with the cell line ID, passage number, date, operator's initials, and any other desired information. - Completely remove the supernatant by pipetting / aspiration. Using a 1 ml pipette, aspirate approximately 300 μl of XF medium + PS + Y medium from the newly prepared 6 well(s) and gently resuspend the cell pellet by pipetting up and down twice. Transfer the cells dropwise into the new well(s) containing XF medium + PS + Y medium, place the plate in an incubator, and then shake the plate several times in an infinity symbol ("∞") fashion to evenly distribute the cell aggregates in the culture well(s). - The next day (Friday), the medium is replaced with 4 ml per well of XF medium + PS medium.

[0090] (hiPSC maintenance):

[0091] (Monday) -Preheat Accutase and the required volume of preheated iPS Brew XF medium (XF medium). Add PS to XF medium at a ratio of 1:100 (XF medium + PS). Add Rock inhibitor (Y-27632(Y)) at a concentration of 1:1000 to both Accutase and XF medium + PS and mix. - Coat the wells of a 6-well plate with 3 μl of iMatrix-511 in 2 ml of XF medium + PS + Y per well for at least 1 hour at 37°C. - Treat the hiPSCs with 1 ml of Accutase+Y per well. - The hiPSCs should be 50-70%-100% confluent and undifferentiated before passage or plating for this experiment. Aspirate the medium completely and wash the cells with 2 ml of PBS per well. After removing the PBS, add 1 ml of prewarmed Accutase+Y and place the plate in a CO2 incubator for 10 minutes. Most cells should detach by gently agitating the plate. If not, extend the digestion for an additional 2 minutes, and so on, until the cells have essentially detached on their own. - Add 1 ml (equal to the volume of Accutase+Y added) of pre-warmed XF medium+PS+Y to a 15 ml tube. Transfer cells from all wells to this 15 ml tube. Centrifuge at 300 g for 3 minutes. The supernatant should be clear and the cells should form a small pellet. - Remove the supernatant and resuspend in 2 ml of pre-warmed XF medium + PS + Y per collected well of a 6-well plate by pipetting up and down three times with a 1 ml pipette. Immediately collect 10 μl of the suspension into a counting chamber or cell counter and quantify the cell titer. - Seed 200,000 cells per well into each well of a pre-coated 6-well plate. Transfer the plate to a CO2 incubator and slowly move the plate in an infinity symbol pattern inside the incubator.

[0092] (Tuesday) - Feed the cells by replacing the medium with 2.5 ml per well of pre-warmed XF medium + PS.

[0093] (Wednesday) - Feed the cells by replacing the medium with 3 ml per well of pre-warmed XF medium + PS.

[0094] (Thursday) - Ensure the cells are subconfluent and completely undifferentiated. In the afternoon, coat the wells with iMatrix-511 and passage the cells as above at a density of 30,000 cells per well of a 6-well plate.

[0095] (differentiation of hiPSCs to MSCs)

[0096] (Friday (Day 0)) - Check for even distribution of hiPSCs in the wells to be used for differentiation. Cells should be flat and form sparse colonies. - Prepare and / or preheat MSCI medium. Thaw the required aliquot of CHIR99021 (CHIR) at room temperature (2-5 min) and then mix by flicking the tube. - Rinse the cells once with 1 ml of PBS at room temperature. - Aspirate the PBS and add 4 ml of MSCI medium. Place the plate back in the CO2 incubator over the weekend.

[0097] (Monday (3rd day)) - Replace the medium with freshly prepared pre-warmed MSCI medium at a rate of 3 ml per well.

[0098] (Tuesday (4th day)) - Replace the medium with freshly prepared pre-warmed MSCI medium at a rate of 3 ml per well.

[0099] (Wednesday (5th day)) - Replace the medium with freshly prepared pre-warmed MSCI medium at a rate of 3 ml per well. - Collect the medium from the wells into a 15 ml tube. Add 1 ml of pre-warmed MSCI medium to each well to avoid starving the cells. Centrifuge the tube containing the collected medium at 300 × G for 1 minute to collect any floating MSC progenitor cells at the bottom of the tube. Carefully aspirate the supernatant from the centrifuged tube, retaining only the cell pellet. Then, add a sufficient volume of pre-warmed MSCI medium (volume = 2 × (number of wells from which medium was collected)) down the side of the tube. Following this, gently resuspend the cells by pipetting up and down twice with a 5 ml pipette, attempting to preserve clumps without disturbing them. Then, very gently add 2 ml of the resuspended cells to each well. (Thursday (6th day)) - Collect the medium from the wells into a 15 ml tube. Add 1 ml of freshly pre-warmed MSCG medium to each well to avoid starving the cells. Centrifuge the tube containing the collected medium at 300 × G for 1 minute to collect any floating MSC progenitor cells at the bottom of the tube. Carefully aspirate the supernatant from the centrifuged tube, retaining only the cell pellet. Then, add a sufficient volume of pre-warmed MSCG medium (volume = 2 × (number of wells from which medium was collected)) down the side of the tube. Following this, gently resuspend the cells by pipetting up and down twice with a 5 ml pipette, attempting to preserve clumps without disturbing them. Then, very gently add 2 ml of the resuspended cells to each well.

[0100] (Friday (7th day)) - Replace the medium with 4 ml of pre-warmed MSCG medium.

[0101] (Monday (10th day)) - Replace the medium with pre-warmed MSCG medium (3 ml per well).

[0102] (Tuesday (11th day)) -Replace the medium.

[0103] (Wednesday (12th day)) - Replace the medium with prewarmed MSCG medium (3 ml per well). The wells will be approximately 80% confluent between 12 and 20 days, depending on the cell line. Continue this schedule of treating the cells with MSCG medium until that point. Once the cells are greater than 90% confluent, passage the cells at a 1:2 ratio onto laminin-coated 6-well plates (laminin: 3 μl iMatrix-511 in 2 ml MSCG medium) (P1). From the next passage (P2) onwards, hiPSC-induced (iMSCs) are seeded onto plastic. A 1:2 passage ratio is maintained for three passages, after which they are diluted. From the fourth passage onwards, cells can be diluted to 200K cells per well of a 6-well plate in 2 ml of MSCG medium. The medium is replaced the day after seeding and every two days thereafter. Every Friday, MSCs are fed with 3-4 ml of MSCG medium. - For passaging iPSC-MSCs, aspirate and discard the medium. Rinse the cells with 1 ml of PBS(- / -) for one well of a 6-well plate. 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 the EDTA. Add 1 ml of TrypLE per well (6-well plate) and place the plate in a CO2 incubator for 4 minutes. Next, use a 1 ml pipette to bring the cells into solution by pipetting up and down. Add at least 3 ml of MSCG medium to a 15 ml tube. When the cells are in solution, pipette them up and down twice and transfer them to a 15 ml tube. Centrifuge at 300 x G for 3 minutes, discard the supernatant, and resuspend the cell pellet in MSC medium by pipetting up and down 4 to 5 times. After cell counting, add the correct volume of the cell suspension to plates or flasks at a 1:2 ratio (up to the third passage) or 20K cells / cm. 2(After the 4th passage)

[0104] (Cryopreservation and long-term storage of iPSC-derived MSCs (iPSC-MSCs))

[0105] iPSC-MSCs are stored in freezing medium at -196°C for long-term storage.

[0106] (iPSC-MSC freezing): - Prepare the freezing medium and pre-cool it to 2-8°C. - Prepare appropriate labels for the cryovials. - MSCs are harvested and counted as done during standard passaging. - Place the correct number of cells to be frozen into a tube and centrifuge at 300 x G for 3 minutes. If the volume of the cell suspension is larger, increase the centrifugation period accordingly. After centrifugation, pipette / aspirate the supernatant and discard. - Resuspend the cell pellet in a sufficient volume of pre-chilled freezing medium. Attempt to prepare a homogenous single-cell suspension by pipetting up and down once or twice 5-6 times, and dispense the correct volume of the cell suspension in freezing medium into pre-labeled cryovials. 500,000 MSCs can be resuspended in 1 ml of freezing medium. - Transfer the cryovials into Mr. Frosty™ freezing containers and store them in a -80°C freezer, slowly cooling the temperature (close to -1°C / min). It is recommended to transfer the cryovials into a liquid nitrogen tank 24 hours to one week after the freezing procedure.

[0107] (iPSC-MSC thawing): -Prepare in advance a tube containing the correct volume of pre-warmed MSCG medium. - Remove the cryovial from the liquid nitrogen and immediately place it in a water bath at 37°C with intermittent shaking until approximately 80% of the cells are thawed (this takes less than 2 minutes). - The cell suspension in the cryovial is quickly transferred by pipetting into the MSCG medium in the tube and centrifuged at 300 g for 3 minutes. - The supernatant is aspirated and discarded, and the pellet is resuspended in 1-2 ml of MSCG medium and transferred into a plate or flask. -The flasks are then incubated and maintained at 37°C in a CO2 incubator for further MSC culture.

[0108] (Analysis method):

[0109] (Flow cytometry) - To perform flow cytometry, the cells are rinsed with PBS (1 ml per well of a 6-well plate). -1 ml of 0.5 mM EDTA is added and the plate is placed in a CO2 incubator for 4 minutes. - Discard the supernatant, add 1 ml of TrypLE and place the plate again in the CO2 incubator for 4 minutes. - Add 2-3 ml of medium into a 15 ml tube and transfer the cells into this tube. -Transfer to a 15 ml tube and centrifuge at 300 x G for 2.0 to 3.0 minutes. - Aspirate and discard the medium and add 1 ml of PBS to each tube. - Vortex briefly and centrifuge at 300 x G for 2.0 minutes. - Remove the supernatant and resuspend the cells in a sufficient volume of PBS so that 1 million cells are contained in 100 μl of PBS. Dispense 100 μl into each 1.5 ml tube. If fewer cells are used, a minimum of 200,000 cells should be contained in 100 μl of PBS for each antibody. - 1.0 μl to 5.0 μl of each staining antibody (depending on the antibody concentration) is added to the 100 μl cell suspension, each tube is briefly vortexed for 5 seconds, and incubated in the dark at room temperature for 20 minutes. After incubation, add 300 μl of PBS and centrifuge at 400×g for 1 minute. Discard the supernatant. - If the primary antibody was not pre-stained, add 100 μl of PBS to the pellet and a sufficient volume of secondary antibody (final concentration according to the manufacturer's instructions). Then, vortex the tubes briefly for 5 seconds each and incubate for 20 minutes. After incubation, add 300 μl of PBS and centrifuge at 400 × g for 1 minute. Discard the supernatant. Add 300 μl of PBS to each tube, resuspend by vortexing briefly for 5 seconds, and perform flow cytometry. - If the primary antibody was pre-stained, add 300 μl of PBS to each tube and resuspend by briefly vortexing for 5 seconds before proceeding with flow cytometry. - After the detection is completed, save the file to G-drive and analyze the flow cytometry results with analysis software.

[0110] (MSC differentiation)

[0111] (osteogenic differentiation): - The iPSC-derived MSCs were plated in a 12-well plastic plate at 10,000 cells / cm in XF medium + PS. 2 and incubate overnight in a CO2 incubator. The next day, the medium is replaced with pre-warmed StemPro Osteogenic Medium (22.25 ml StemPro® Osteocyte / Chondrocyte Differentiation Basal Medium + 2.5 ml StemPro Osteogenic Supplement + 250 μl PS) and the incubation is continued in a CO2 incubator. MSCs expand as they differentiate in the osteogenic medium. - Replace with fresh pre-warmed osteogenic medium every 3-4 days. After 21 days or more, bone formation can be observed using Alizarin Red S staining analysis.

[0112] (Alizarin Red S staining analysis) - Remove the medium from the wells of the 12-well plate. - Rinse once with PBS (1 ml). - Remove the PBS from the wells - Fix the cells with a 4% formaldehyde solution (diluted in PBS) for 30 minutes. After fixing the cells, rinse them twice with distilled water and stain them with 2% Alizarin Red S solution for 2-3 minutes at room temperature on a plate shaker. - Rinse the wells 3-4 times with 2 ml of deionized water, visualize under a light microscope and capture images.

[0113] (adipogenic differentiation): - The iPSC-derived MSCs were plated in a 12-well plastic plate at 80K cells / cm in XF medium + PS. 2 and incubate overnight in a CO2 incubator. The next day, the medium is replaced with pre-warmed StemPro Adipogenic Medium (22.25 ml StemPro® Adipocyte Differentiation Basal Medium + 2.5 ml StemPro Adipogenic Supplement + 250 μl PS) and incubation is continued in a CO2 incubator. The MSCs expand as they differentiate under adipogenic conditions. -Replace with fresh pre-warmed adipogenic medium every 3-4 days. After 21 days or more, adipogenesis can be observed using Oil Red O staining analysis.

[0114] (Oil Red O staining analysis) - Remove the medium from the wells of the 12-well plate. - Rinse once with PBS (1 ml). - Remove the PBS from the wells - Fix the cells with a 4% formaldehyde solution (diluted in PBS) for 30 minutes. After fixation, the cells are rinsed twice with distilled water and stained with Oil Red O solution (6 ml of Oil Red O stock solution + 4 ml of deionized water) for 20 minutes at room temperature on a plate shaker. - Rinse the wells 3-4 times with 2 ml of deionized water, visualize under a light microscope and capture images.

[0115] (chondrogenic differentiation): - The iPSC-derived MSCs are resuspended in XF medium + PS at a cell concentration of 1.6 x 107 viable cells / ml. - Micromass cultures are created by seeding 5 μl droplets of cell solution into the center of the wells of a multi-well plate (5 droplets in 12 wells). After culturing the micromass cultures for at least 2 hours in a 37°C incubator with 5% CO2, warmed chondrogenic medium is added to the culture vessels and re-incubated in a 37°C incubator with 5% CO2. - Re-feed the cultures every 2-3 days - After 14 days or more, cartilage formation can be observed using Alcian blue staining analysis.

[0116] (Alcian blue staining analysis) - After the differentiation time is complete, carefully remove the medium. - Gently wash the spheroids with PBS (1 ml). - Remove the PBS from the tube. - Fix the cells with a 4% formaldehyde solution (diluted in PBS) for 3 hours. - After fixation, the spheroids are transferred to plates labeled with wells, which makes it easier to handle the spheroids. - Aspirate the fixative and wash twice with distilled water - The cells are stained with Alcian Blue staining solution and incubated in the dark for 45 minutes. - Carefully remove the Alcian Blue staining solution with a 1 ml pipette, retaining the spheroids. - Wash twice with deionized water for 10 minutes each. - PBS is added to the spheroids, cartilage spheroids are analyzed and images are captured. The cartilage spheroids stain dark blue and the negative control stains light blue.

[0117] Example 3 Generation and characterization of natural killer cells differentiated from human pluripotent stem cells The present invention is broadly relevant to the field of iPSC-based applications (e.g., cell therapy) and targets MSCs as a differentiation product. The procedure facilitates their derivation and enables a more robust manufacturing process under cleanroom conditions, particularly in cell therapy, because it avoids complex handling procedures and is based on a simple, defined iPSC processing protocol. The novel approach developed for differentiating MSC cells from iPSCs allows for higher yields, higher purity, and increased simplicity compared to existing methods. This is achieved by replacing the labor-intensive, undefined EB stage with directed iPSC differentiation into MSCs that further differentiate into osteogenic, adipogenic, and chondrogenic cells. The protocol was developed with GMP-compatible processes in mind. The protocol allows for off-the-shelf MSCs derived from HLA-homozygous iPSC banks.

[0118] iPSCs were maintained and expanded as described in Examples 1-2 and differentiated into MSCs according to the method described in Example 2 and shown in FIG.

[0119] From day 6 onwards, a culture medium suitable for MSCs is used without the GSK3β inhibitor. As shown in Figures 2 and 3, MSCs obtained by the method described herein exhibited continued exponential growth and retained their phenotype even after freezing and thawing.

[0120] Characterization of the MSCs included analysis of MSC-specific biomarkers by flow cytometry. As shown in Figure 4A, more than 99% of the resulting MSCs expressed CD73, CD90, and CD44, and approximately 82% expressed CD105. As further shown in Figure 4B, it was demonstrated that the MSCs did not express non-MSC markers (e.g., CD14, CD45, CD31, CD34, TRA 1-60, or HLA-DR). The specificity of this analysis was confirmed by negative controls (Figure 4C).

[0121] Characterization of the cells further included analysis of the expression of pluripotency genes (e.g., miR-302 HT and SOX2); endothelial markers (e.g., PECAM and CDH5); hematopoietic markers (e.g., CD45); cardiac markers (e.g., MYH6); and neural crest markers (e.g., SOX10) by RTqPCR. As shown in Figures 5A-5E, MSCs were negative for all of these non-MSC markers.

[0122] MSCs were further analyzed to assess whether the culture medium in which they were expanded could affect their phenotype. As shown in Figures 7A-7B, the expression of CD73, CD44, and CD90 was assessed in MSCs maintained and expanded in two different MSC culture media. There was no effect of the culture media on the expression of these MSC markers.

[0123] The reproducibility of the method was assessed, and the phenotype and expression of MSC markers were analyzed in MSCs derived from several independent iPSC lines. As shown in Figures 8, 9, and 10, regardless of the iPSC line used, the resulting MSCs retained their phenotype (adhering to plastic in culture) and their strong expression of CD44, CD73, and CD90, while remaining negative for non-MSC markers (e.g., TRA 1-60, CD31, CD45, Cd14, Cd34, and HLA-DR).

[0124] (References) Jiang Y, Jahagirdar BN, Reinhardt RL, Schwartz RE, Keene CD, Ortiz-Gonzalez XR, Reyes M, Lenvik T, Lund T, Blackstad M, Du J, Aldrich S, Lisberg A, Low WC, Largaespada DA, Verfaillie CM. Pluripotency of mesenchymal stem cells derived from adult marrow. Nature. 2002 Jul 4;418(6893):41-9. Pittenger MF, Mackay AM, Beck SC, Jaiswal RK, Douglas R, Mosca JD, Moorman MA, Simonetti DW, Craig S, Marshak DR. Multilineage potential of adult human mesenchymal stem cells. Science. 1999 Apr 2;284(5411):143-7. Lalu MM, Mazzarello S, Zlepnig J, Dong YYR, Montroy J, McIntyre L, Devereaux PJ, Stewart DJ, David Mazer C, Barron CC, McIsaac DI, Fergusson DA. Safety and Efficacy of Adult Stem Cell Therapy for Acute Myocardial Infarction and Ischemic Heart Failure (SafeCell Heart): A Systematic Review and Meta-Analysis. Stem Cells Transl Med. 2018 Dec;7(12):857-866. Eom YW, Shim KY, Baik SK. Mesenchymal stem cell therapy for liver fibrosis. Korean J Intern Med. 2015 Sep;30(5):580-9. Jin MC, Medress ZA, Azad TD, Doulames VM, Veeravagu A. Stem cell therapies for acute spinal cord injury in humans: a review. Neurosurg Focus. 2019 Mar 1;46(3): E10. Cortez-Toledo E, Rose M, Agu E, Dahlenburg H, Yao W, Nolta JA, et al. Enhancing retention of human bone marrow mesenchymal stem cells with prosurvival factors promotes angiogenesis in a mouse model of limb ischemia. Stem Cells Dev. 2019;28(2):114-9. Dominici M, Le Blanc K, Mueller I, Slaper-Cortenbach I, Marini F, Krause D, Deans R, Keating A, Prockop Dj, Horwitz E. Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement. Cytotherapy. 2006;8(4):315- Gomez-Salazar M, Gonzalez-Galofre ZN, Casamitjana J, Crisan M, James AW, Peault B. Five Decades Later, Are Mesenchymal Stem Cells Still Relevant? Front Bioeng Biotechnol. 2020 Feb 28; 8:148. Jin W, He Y, Li T, Long F, Qin X, Yuan Y, Gao G, Shakhawat HM, Liu X, Jin G, Zhou Z. Rapid and robust derivation of mesenchymal stem cells from human pluripotent stem cells via temporal induction of neuralized ectoderm. Cell Biosci. 2022 Mar 15;12(1):31. Sanchez L, Gutierrez-Aranda I, Ligero G, Rubio R, Munoz-Lopez M, Garcia-Perez JL, Ramos V, Real PJ, Bueno C, Rodriguez R, Delgado M, Menendez P. Enrichment of human ESC-derived multipotent mesenchymal stem cells with immunosuppressive and anti-inflammatory properties capable to protect against experimental inflammatory bowel disease. Stem Cells. 2011 Feb;29(2):251-62. doi: 10.1002 / stem.569. Zhao Q, Gregory CA, Lee RH, Reger RL, Qin L, Hai B, Park MS, Yoon N, Clough B, McNeill E, Prockop DJ, Liu F. MSCs derived from iPSCs with a modified protocol are tumor-tropic but have much less potential to promote tumors than bone marrow MSCs. Proc Natl Acad Sci U S A. 2015 Jan 13;112(2):530-5. Hawkins KE, Corcelli M, Dowding K, Ranzoni AM, Vlahova F, Hau KL, Hunjan A, Peebles D, Gressens P, Hagberg H, de Coppi P, Hristova M, Guillot PV. Embryonic Stem Cell-Derived Mesenchymal Stem Cells (MSCs) Have a Superior Neuroprotective Capacity Over Fetal MSCs in the Hypoxic-Ischemic Mouse Brain. Stem Cells Transl Med. 2018 May;7(5):439-449. doi: 10.1002 / sctm.17-0260. Leyendecker Junior A. TGF-β Inhibitor SB431542 Promotes the Differentiation of Induced Pluripotent Stem Cells and Embryonic Stem Cells into Mesenchymal-Like Cells. Stem Cells Int. 2018 Jul 2; 2018:7878201. doi: 10.1155 / 2018 / 7878201. Karlsson C, Emanuelsson K, Wessberg F, Kajic K, Axell MZ, Eriksson PS, Lindahl A, Hyllner J, Strehl R. Human embryonic stem cell-derived mesenchymal progenitors--potential in regenerative medicine. Stem Cell Res. 2009 Jul;3(1):39-50. Sheyn D, Ben-David S, Shapiro G, De Mel S, Bez M, Ornelas L, Sahabian A, Sareen D, Da X, Pelled G, Tawackoli W, Liu Z, Gazit D, Gazit Z. Human Induced Pluripotent Stem Cells Differentiate into Functional Mesenchymal Stem Cells and Repair Bone Defects. Stem Cells Transl Med. 2016 Nov;5(11):1447-1460. Giuliani M, Oudrhiri N, Noman ZM, Vernochet A, Chouaib S, Azzarone B, Durrbach A, Bennaceur-Griscelli A. Human mesenchymal stem cells derived from induced pluripotent stem cells down-regulate NK-cell cytolytic machinery. Blood. 2011 Sep 22;118(12):3254-62. Liu Y, Goldberg AJ, Dennis JE, Gronowicz GA, Kuhn LT. One-step derivation of mesenchymal stem cell (MSC)-like cells from human pluripotent stem cells on a fibrillar collagen coating. PLoS One. 2012;7(3): e33225.

[0125] While the invention has been described with reference to the above examples, it will be understood that modifications and variations are encompassed within the spirit and scope of the invention. Accordingly, the invention is limited only by the appended claims.

Claims

1. 1. A method for producing mesenchymal stromal cells / mesenchymal stem cells (MSCs), comprising: (a) contacting an adherent culture of pluripotent stem cells (PSCs) with a WNT signaling pathway activator for about 4 days to about 8 days; and (b) then expanding the cells in culture from (a) in the absence of the WNT signaling pathway activator, thereby generating MSCs; method.

2. The method of claim 1 , wherein the WNT signaling pathway activator is a GSK3β inhibitor.

3. 3. The method of claim 2, wherein the GSK3β inhibitor is CHIR99021.

4. 4. The method of claim 3, wherein the PSCs are contacted with about 2 μM to about 6 μM CHIR99021.

5. 4. The method of claim 3, wherein the PSCs are contacted with about 4 μM CHIR99021.

6. 4. The method of claim 3, wherein the PSCs are contacted with CHIR99021 for about 6 days.

7. 4. The method of claim 3, wherein the PSCs are contacted with about 4 μM CHIR99021 for about 6 days.

8. 10. The method of claim 1, wherein the contacting step comprises incubating the PSCs with fewer than three WNT signaling pathway activators.

9. 2. The method of claim 1, wherein the contacting step comprises incubating the PSCs with a single WNT signaling pathway activator.

10. The method of claim 1 , wherein prior to (a), the iPSCs are maintained in a culture medium containing a ROCK inhibitor.

11. The method of claim 8, wherein the ROCK inhibitor is Y-27632.

12. The method of claim 1 , wherein the PSCs are cultured on a laminin-coated surface.

13. 10. The method of claim 1, wherein the step of expanding the cells comprises culturing the cells on an uncoated surface.

14. The method of claim 1 , wherein an enrichment culture of MSCs is produced.

15. The method of claim 12, wherein the enrichment culture comprises at least about 90% MSCs.

16. The method of claim 12, wherein the enrichment culture comprises at least about 95% MSCs.

17. The method of claim 1, wherein the MSCs are positive for CD73, CD44, and CD90.

18. 2. The method of claim 1, wherein the MSCs are negative for CD14, CD31, TRA 1-60, CD34, HLA-DR, SOX2, miR-302 HT, PECAM1, CDH5, CD45, MYH6, and SOX10.

19. The method of claim 1, wherein the generated MSCs are multipotent stem cells with osteogenic, adipogenic and chondrogenic differentiation potentials.

20. 10. The method of claim 1, wherein the step of expanding the cells comprises culturing the cells in an MSC-supporting culture medium.

21. 10. The method of claim 1, wherein the contacting and expanding the cells comprises contacting and expanding in serum-free culture conditions.

22. The method of claim 1 , wherein said culture of PSCs does not contain embryoid bodies.

23. 2. The method of claim 1, wherein 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.

24. 2. The method of claim 1, wherein the PSCs are human pluripotent stem cells (hPSCs).

25. 25. The method of claim 24, wherein the hPSCs are induced pluripotent stem cells (hiPSCs) or human embryonic stem cells (hESCs).

26. 2. The method of claim 1, wherein the PSCs are iPSCs.

27. 1. A method for producing mesenchymal stromal cells / mesenchymal stem cells (MSCs), comprising: (a) contacting an adherent culture of induced pluripotent stem cells (iPSCs) with about 4 μM of a single GSK3β inhibitor for about 6 days; and (b) then expanding the cells in culture from (a) in the absence of said GSK3β inhibitor; (b) after which a population of at least about 90% pure CD73+, CD44+ and CD90+ MSCs is obtained; thereby obtaining MSCs A method comprising:

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