Methods and compositions for generating human induced mesenchymal stem cells

Abstract

Methods are provided for generating human induced mesenchymal stem cells (iMSCs) from human pluripotent stem cells (e.g., embryonic stem cells). Progenitor cells for iMSCs are first generated in a two-step protocol, and differentiation into iMSCs is achieved by a third step culture. iMSCs express mesenchymal surface markers and exhibit tri-lineage differentiation into adipocytes, osteocytes, and chondrocytes. Also provided are culture media, methods for isolating extracellular vesicles from iMSCs, and kits.

Description

[Technical field]

[0001] Related Applications This application claims priority to U.S. Provisional Application No. 63 / 307,368, filed February 7, 2022. The contents of the prior application are incorporated herein by reference in their entirety. [Background technology]

[0002] Mesenchymal stem cells (MSCs) are non-hematopoietic adult stem cells that have the ability to self-renew and also exhibit multilineage differentiation. MSCs can be isolated from various tissues (e.g., umbilical cord, bone marrow, and adipose tissue, as well as amniotic fluid, menstrual blood, and endometrium). MSCs are multipotent stromal cells that can differentiate into various cell types, including bone cells, chondrocytes, muscle cells, and adipocytes. MSCs have also been shown to have immunomodulatory effects, including the ability to produce immunomodulatory molecules (e.g., cytokines). The multipotent properties of MSCs make them an attractive option for potential development in clinical applications.

[0003] However, there are numerous constraints to the vigorous in vitro expansion of ex vivo isolated adult MSCs, including a decline in plasticity and potency over time, as well as the accumulation of DNA aberrations and replicative senescence (see, e.g., Rombouts and Ploemacher (2003) Leukemia 17:160-170; Miura et al. (2006) Stem Cells 24:1095-1103; Kyriakou et al. (2008) Haematologica 93:1457-1465; Liu et al. (2012) PLoS One 7:e33225). Furthermore, reports of the in vivo efficacy of MSCs have been inconsistent due to variability in cell quality across donors and tissue sources (see, e.g., Wagner and Ho (2007) Stem Cell Rev. 3:239-248; Galipeau (2013) Cytotherapy 15:2-8; Kimbrel (2014) Stem Cells and Develop. 23:1611-1624; Tyndall (2014) Nat. Rev. Rheumatol. 10:117-124). Thus, there remains a need in the art for more reliable and consistent sources of MSCs.

[0004] Attempts have also been made to generate MSCs in vitro from other stem cells. For example, approaches to generate MSCs from human induced pluripotent stem cells (iPSCs) have been described (e.g., Hynes et al. (2014) Stem Cells. Dev. 23:1084-1096; Yang et al. (2014) PLoS One 9:e100285; Kang et al. (2015) Stem Cell Res. Ther. 6:144; Lin et al. (2016) Curr. Stem Cell Res. Ther. 11:122-130; Spitzhorn et al. (2018) Stem Cells Dev. 27:1702-1714; Wang et al. (2018) Stem Cells 36:903-914; Yang et al. (2019) Cell Death & Disease 10:718; Xu et al. (2019) Stem Cells 37:754-765; Spitzhorn et al. (2019) Stem Cell Res. Ther. 10:100. Such cells are referred to in the art as iPSC-MSCs.

[0005] Although some progress has been made, there remains a need for efficient and robust methods and compositions for generating human induced mesenchymal stem cells from human pluripotent stem cells in culture. Summary of the Invention

[0006] The present disclosure provides a method for generating human induced mesenchymal stem cells (iMSCs) and their progenitors from human pluripotent stem cells (e.g., human embryonic stem cells) using a three-step protocol that generates iMSCs in as little as 21 days of culture. The present disclosure first provides a two-step protocol that allows for obtaining iMSC progenitors in as little as 14 days of culture. The third step allows for at least 7 more days of further culture to obtain mature iMSCs that express mesenchymal surface markers and have tri-lineage differentiation potential into adipocytes, osteocytes, and chondrocytes upon further culture under appropriate cell-specific differentiation conditions. iMSCs also exhibit immunomodulatory capabilities. The methods of the present disclosure can be used to obtain iMSC progenitors, as well as mature iMSCs and further differentiated cells obtained from iMSCs.

[0007] Thus, in one aspect, the present disclosure provides a method of generating human mesenchymal stem cell progenitors, comprising: (a) culturing human pluripotent stem cells in a culture medium comprising a WNT pathway agonist and a BET pathway antagonist for at least 2 days (or at least 3 or 4 days), e.g., from day 0 to day 4, to generate inducible cells; (b) culturing the induced cells from step (a) in a culture medium comprising a PDGF pathway agonist, an IGF1 pathway agonist, and an FGF-beta pathway agonist for at least 10 days (or at least 11, 12, 13, or 14 days), e.g., from day 4 to day 14, to generate human mesenchymal stem cell progenitor cells.

[0008] In embodiments, the human pluripotent stem cells are human embryonic stem cells.

[0009] In some embodiments, the WNT pathway agonist is a glycogen synthase kinase 3 (Gsk3) inhibitor (e.g., CHIR98014). In some embodiments, the WNT pathway agonist is selected from the group consisting of CHIR98014, CHIR99021, SB 216763, SB 415286, LY2090314, 3F8, A 1070722, AR-A 014418, BIO, AZD1080, WNT3A, and combinations thereof. In some embodiments, the WNT pathway agonist is present in the culture medium at a concentration in the range of 0.25-0.75 μM. In some embodiments, the WNT pathway agonist is present in the culture medium at a concentration of 0.5 μM. In some embodiments, the WNT pathway agonist is CHIR98014 at a concentration of 0.5 μM.

[0010] In some embodiments, the BET pathway antagonist is a triazolo-diazepine compound (e.g., (+)-JQ1). In some embodiments, the BET pathway antagonist is selected from the group consisting of (+)-JQ1, TEN-010, OTX015, I-BET762, I-BET151, BAY1238097, ABBV-744, ABBV-075, iBET-BD1, iBET-BD2, SJ432, RVX-208, MS417, AZD5153, and combinations thereof. In some embodiments, the BET pathway antagonist is present in the culture medium at a concentration in the range of 25-75 nM. In one embodiment, the BET pathway antagonist is (+)-JQ1 at a concentration of 50 nM.

[0011] In some embodiments, the PDGF pathway agonist is PDGF-BB. In some embodiments, the PDGF-BB is present in the culture medium at a concentration in the range of 7.5-12.5 ng / ml. In one embodiment, the PDGF pathway agonist is PDGF-BB, which is present in the culture medium at a concentration of 10 ng / ml.

[0012] In some embodiments, the IGF1 pathway agonist is IGF1. In some embodiments, the IGF1 is present in the culture medium at a concentration in the range of 15-25 ng / ml. In one embodiment, the IGF1 pathway agonist is IGF1, which is present in the culture medium at a concentration of 20 ng / ml.

[0013] In some embodiments, the FGF-beta pathway agonist is FGF-beta. In some embodiments, the FGF-beta is present in the culture medium at a concentration in the range of 7.5-12.5 ng / ml. In one embodiment, the FGF-beta pathway agonist is FGF-beta, which is present in the culture medium at a concentration of 10 ng / ml.

[0014] In various embodiments, the culture medium in step (a) and step (b) of this method comprises a serum-containing basal medium. In one embodiment, the serum-containing basal medium is a DMEM / F12 medium containing 10-15% fetal bovine serum.

[0015] In one embodiment, the method further comprises culturing the human mesenchymal stem cell progenitor cell in a culture medium comprising a basal medium containing L-glutamine supplement for at least 7 days (e.g., 14 days or more). In one embodiment, the L-glutamine supplement is L-alanine-L-glutamine dipeptide. In one embodiment, the basal medium containing L-glutamine supplement is DMEM / F12 medium containing 10% fetal bovine serum supplemented with GlutaMAX™.

[0016] In another aspect, the present disclosure provides a method of generating human induced mesenchymal stem cells (iMSCs), comprising: (a) culturing human pluripotent stem cells in a culture medium comprising a WNT pathway agonist and a BET pathway antagonist for at least 2 days (or at least 3 days, or 4 days), e.g., from day 0 to day 4, to generate inducible cells; (b) culturing the induced cells from step (a) in a culture medium comprising a PDGF pathway agonist, an IGF1 pathway agonist, and an FGF-beta pathway agonist for at least 10 days (or at least 11, 12, 13, or 14 days), e.g., from day 4 to day 14, to generate human mesenchymal stem cell progenitor cells; (c) culturing the human mesenchymal stem cell progenitor cells from step (b) in a culture medium comprising a basal medium and an L-glutamine supplement for at least 7 days to generate iMSCs.

[0017] In embodiments, step (c) comprises culturing the human mesenchymal stem cell progenitor cells in basal medium and L-glutamine supplement for more than 7 days, such as at least 14 days, at least 21 days, or at least 28 days or more.

[0018] In embodiments, the human pluripotent stem cells are human embryonic stem cells.

[0019] In embodiments, the iMSCs generated in step (c) express one or more surface markers selected from the group consisting of CD73, CD90, CD105, CD29, CD44, and combinations thereof.

[0020] In embodiments, the iMSCs generated in step (c) are capable of further differentiation into adipocytes, osteocytes, or chondrocytes.

[0021] Suitable WNT pathway agonists, BET pathway antagonists, PDGF pathway agonists, IGF1 pathway agonists, and FGF-beta pathway agonists, and concentrations thereof, are described above and further herein.

[0022] In some embodiments, the culture medium in steps (a), (b), and (c) comprises a serum-containing basal medium. In some embodiments, the serum-containing basal medium is a DMEM / F12 medium containing 10-15% fetal bovine serum.

[0023] In some embodiments, the L-glutamine supplement used in step (c) is L-alanine-L-glutamine dipeptide. In one embodiment, the basal medium containing the L-glutamine supplement in step (c) is DMEM / F12 medium containing 10% fetal bovine serum supplemented with GlutaMAX™.

[0024] In yet another embodiment, the method includes isolating extracellular vesicles produced by the iMSCs from the culture after step (c). Additionally or alternatively, extracellular vesicles produced by the iMSC progenitor cells may be isolated after step (b).

[0025] In yet another aspect, the present disclosure relates to a two-stage culture medium for obtaining human mesenchymal stem cell progenitors, comprising: (i) a first stage medium comprising a WNT pathway agonist and a BET pathway antagonist; and (ii) a second stage medium comprising a PDGF pathway agonist, an IGF1 pathway agonist, and an FGF-beta pathway agonist. Suitable WNT pathway agonists, BET pathway antagonists, PDGF pathway agonists, IGF1 pathway agonists, and FGF-beta pathway agonists, and concentrations thereof, are described above and further herein.

[0026] Other features and advantages of the invention will be apparent from the following detailed description, and from the claims. [Brief description of the drawings]

[0027] [Figure 1] FIG. 1 is a schematic diagram of an exemplary three-stage iMSC differentiation protocol of the present disclosure. [Diagram 2] Photographs showing cell morphology during iMSC differentiation protocol. A (left and right panels) shows cells at mesodermal stage. B shows cells at mesenchymal stage at low density (10x) (left panel) or high density (5x) (right panel). [Diagram 3]Figure 1 shows the results of FACS analysis of positive (A) and negative (B) MSC markers. Markers tested are indicated above the graph. The negative cocktail included anti-CD11b, anti-CD19, anti-CD34, anti-CD45, and anti-HLA-DR antibodies. [Figure 4] Photographs showing the morphology of iMSCs after differentiation into adipocytes (A), osteocytes (B), or chondrocytes (C). [Diagram 5] 1 is a bar graph showing that treatment with immune stimulants (ds-RNA or IFNr) increases the expression of immune regulatory genes in iMSCs. [Figure 6] Graph showing the results of a mixed lymphocyte reaction using hPBMCs alone (left panel), hPBMCs + inactivated iMSCs (middle panel), or hPBMCs + activated MSCs (right panel). The results show proliferation of hPBMCs. [Figure 7] 1 is a bar graph showing the results of an ELISA study on IL-10 secretion by iMSCs treated with low or high doses of IFNr. [Figure 8] Graph showing the results of a FACS-based analysis quantifying extracellular vesicle production by iMSCs targeting tetraspanin proteins. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0028] Herein, methodologies and compositions are described that allow for the generation of human induced mesenchymal stem cells (iMSCs) under defined culture conditions using a multi-step protocol that can first generate iMSC precursor cells and then further differentiate into mature iMSCs. As described in Example 1, a three-step protocol (schematically shown in FIG. 1) for the generation of iMSCs from human pluripotent stem cells (e.g., human embryonic stem cells) was developed. As described in Example 2, iMSCs express mesenchymal surface markers and lack expression of pluripotent and hematopoietic markers. As described in Example 3, iMSCs are capable of tri-lineage differentiation and mature into adipocytes, osteocytes, or chondrocytes when cultured under appropriate cell-specific differentiation conditions. As described in Example 4, iMSCs exhibit immunomodulatory capabilities, including upregulation of immunomodulatory genes when treated with immune stimulants, the ability to modulate mixed lymphocyte reactions, and the ability to secrete anti-inflammatory factors when stimulated with interferon-gamma (IFNr). Additionally, as described in Example 5, the iMSCs and their progenitor cells described herein are a source of extracellular vesicles (EVs) (e.g., exosomes) that can be harvested from cell cultures.

[0029] Various aspects of the invention are described in further detail in the following subsections.

[0030] I.Cells The starting cells used in the culture of the present disclosure are human pluripotent stem cells. As used herein, the term "human pluripotent stem cells" (abbreviated as hPSC) refers to human stem cells that have the ability to differentiate into a variety of different cell types. As used herein, the term "pluripotent" refers to cells that have the ability to differentiate into cell types characteristic of all three germ layers (endoderm, mesoderm, and ectoderm) under different conditions. Pluripotent cells are primarily characterized by their ability to differentiate into all three germ layers, for example, using nude mice and teratoma formation assays. Although pluripotency can also be demonstrated by the expression of embryonic stem cell (ES cell) markers, the preferred test of pluripotency is to demonstrate the ability to differentiate into cells of each of the three germ layers.

[0031] Human pluripotent stem cells include, for example, human embryonic stem cells (e.g., ES cell lines) and induced pluripotent stem cells (iPSCs). Non-limiting examples of human embryonic stem cell lines include ES03 cells (WiCell Research Institute) and H9 cells (Thomson, JA et al. (1998) Science 282:1145-1147), as well as low-immunity embryonic stem cells (including hESC SKO-CIITA, SKO-B2M, or DKO) (Petrus-Reurer et al. (2020) Stem Cell Reports 14:648-662). Non-limiting examples of induced pluripotent stem cells (iPSCs) include 19-11-1, 19-9-7, or 6-9-9 cells (e.g., as described in Yu, J. et al. (2009) Science 324:797-801), Foreskin (clone 1 to clone 4), and IMR90 (clone 1 to clone 4) (Yu et al. (2007) Science 318:1917-20). Human pluripotent stem cells (PSCs) express cell markers that can be used to identify cells as PSCs. Non-limiting examples of pluripotent stem cell markers include TRA-1-60, TRA-1-81, TRA-2-54, SSEA1, SSEA3, SSEA4, CD9, CD24, OCT3, OCT4, NANOG, and / or SOX2. In embodiments, mature iMSCs generated by the methods of the present disclosure lack expression of the pluripotent stem cell marker TRA-1-60.

[0032] The pluripotent stem cells are subjected to culture conditions that induce cell differentiation, as described herein. As used herein, the term "differentiation" refers to the development of cells from a more primitive stage to more mature (i.e., less primitive) cells, typically exhibiting phenotypic characteristics of commitment to a particular cell lineage. In the iMSC differentiation method described herein, progenitor cells of iMSC are first generated, which are then further differentiated into mature iMSCs.

[0033] As described in Example 2, mature iMSCs generated by the differentiation method express mesenchymal surface markers (e.g., CD73, CD90, CD105, CD29, and / or CD44). In embodiments, the iMSCs express one or more mesenchymal surface markers selected from the group consisting of CD73, CD90, CD105, CD29, CD44, and combinations thereof. In embodiments, in a population of iMSCs, at least 90% (or at least 95%, or at least 98%, or at least 99%) of the viable cells express one or more mesenchymal surface markers selected from the group consisting of CD73, CD90, CD105, CD29, CD44, and combinations thereof.

[0034] Also, as described in Example 2, the mature iMSCs generated by the differentiation method lack expression of at least one pluripotent stem cell marker (e.g., TRA-1-60) and / or at least one hematopoietic cell marker (e.g., CD34, CD45, and / or HLA-DR). In embodiments, the mature iMSCs lack expression of one or more markers selected from the group consisting of TRA-1-60, CD11b, CD19, CD34, CD45, HLA-DR, and combinations thereof. In embodiments, in a population of iMSCs, less than 5% (or less than 3%, or less than 2%, or less than 1%) of the viable cells express one or more surface markers selected from the group consisting of TRA-1-60, CD11b, CD19, CD34, CD45, HLA-DR, and combinations thereof.

[0035] II. Culture medium components The disclosed methods for generating human induced mesenchymal stem cells include culturing human pluripotent stem cells (e.g., human ESCs) in a culture medium containing specific agonists and / or antagonists of cell receptors and / or signaling pathways.

[0036] In the first step (referred to herein as step (a)) of the multi-step protocol (shown diagrammatically in FIG. 1), human pluripotent stem cells are cultured for at least 2 days, or at least 3 days, or 4 days (days 0-4) in induction medium comprising a WNT pathway agonist and a BET pathway antagonist. In the second step (referred to herein as step (b)), the induced cells from step (a) are further cultured for at least 10 days (or at least 11, 12, 13, or 14 days), e.g., from day 4 to day 14 (after the 4-day induction step), in enriched medium comprising a PDGF pathway agonist, an IGF1 pathway agonist, and an FGF-beta pathway agonist, to generate iMSC progenitor cells. Finally, in the third step (referred to herein as step (c)), the iMSC progenitor cells from step (b) are further cultured for at least 7 more days in maintenance medium comprising an L-glutamine supplement to generate mature iMSCs.

[0037] As used herein, an "agonist" of a cell receptor or signaling pathway refers to an agent that stimulates (upregulates) a cell receptor or signaling pathway. Stimulation of a cell signaling pathway can be initiated outside the cell, for example, by using an agonist (e.g., an agonist can be a receptor ligand) that activates a cell surface receptor involved in the signaling pathway. Additionally or alternatively, stimulation of intracellular signaling can be initiated inside the cell, for example, by using a small molecule agonist that interacts with a component(s) of the signaling pathway inside the cell.

[0038] As used herein, an "antagonist" of a cell signaling pathway is intended to refer to an agent that inhibits (downregulates) the cell signaling pathway. Inhibition of an intracellular signaling pathway can be initiated extracellularly, for example, by using an antagonist that blocks a cell surface receptor involved in the signaling pathway. Additionally or alternatively, inhibition of intracellular signaling can be initiated intracellularly, for example, by using a small molecule antagonist that interacts intracellularly with a component(s) of the signaling pathway.

[0039] Agonists of the WNT pathway include drugs, molecules, compounds, or substances that can stimulate (upregulate) the canonical Wnt / β-catenin signaling pathway. This pathway is biologically activated by the binding of Wnt protein ligands to Frizzled family receptors. In one embodiment, the WNT pathway agonist is a glycogen synthase kinase 3 (Gsk3) inhibitor (e.g., CHIR98014). In one embodiment, the WNT pathway agonist is selected from the group consisting of CHIR98014, CHIR99021, SB 216763, SB 415286, LY2090314, 3F8, A 1070722, AR-A 014418, BIO, AZD1080, WNT3A, and combinations thereof. In one embodiment, the WNT pathway agonist is present in the culture medium at a concentration in the range of 0.25-0.75 μM, 0.3-0.7 μM, 0.4-0.6 μM, or 0.45-0.55 μM. In one embodiment, the WNT pathway agonist is present in the culture medium at a concentration of 0.5 μM. In one embodiment, the WNT pathway agonist is CHIR98014. In one embodiment, the WNT pathway agonist is CHIR98014, which is present in the culture medium at a concentration in the range of 0.25-0.75 μM, 0.3-0.7 μM, 0.4-0.6 μM, or 0.45-0.55 μM. In one embodiment, the WNT pathway agonist is CHIR98014, which is present in the culture medium at a concentration of 0.5 μM.

[0040] Antagonists of the BET pathway include drugs, molecules, compounds, or substances capable of inhibiting (downregulating) BET proteins ("bromodomain and extraterminal motif" proteins) that contain bromodomain(s). Bromodomains are protein interaction modules that selectively recognize eN-acetylated lysine residues (Kac). The human BET family (BRD2, BRD3, BRD4, and BRDT), which all contain two conserved bromodomains per target, play a key role in regulating the transcription of growth stimulatory genes.

[0041] In one embodiment, the BET pathway antagonist is a triazolo-diazepine compound, non-limiting examples of which include (+)-JQ1 (e.g., as described in Filippakopoulos et al. (2010) Nature 468:1067-1073), TEN-010 (e.g., as described in Finley and Copeland (2014) Chem. Biol. 21:1196-1210), OTX015 (e.g., as described in Seal et al. (2012) Bioorg. Med. Chem. Lett. 22:2968-2972), and structurally related compounds. In one embodiment, the triazolo-diazepine compound is (+)-JQ1 (Sigma Aldrich; catalog number SML1524). Various other BET pathway antagonists have been described (e.g., as reviewed in Cochran et al. (2019) Nat. Rev. Drug Disc. 18:609-628; and Zaware et al. (2019) Nat. Struct. Mol. Biol. 26:870-879). In some embodiments, the BET pathway antagonist is selected from the group consisting of (+)-JQ1, TEN-010, OTX015, I-BET762, I-BET151, BAY1238097, ABBV-744, ABBV-075, iBET-BD1, iBET-BD2, SJ432, RVX-208, MS417, AZD5153, and combinations thereof. In embodiments, the BET pathway antagonist is present in the culture medium at a concentration in the range of 25-75 nM, 30-70 nM, 40-60 nM, or 45-55 nM. In one embodiment, the BET pathway antagonist is present in the culture medium at a concentration of 50 nM. In embodiments, the BET pathway antagonist is (+)-JQ1, which is present in the culture medium at a concentration in the range of 25-75 nM, 30-70 nM, 40-60 nM, or 45-55 nM. In one embodiment, the BET pathway antagonist is (+)-JQ1 at a concentration of 50 nM.

[0042] PDGF (Platelet-Derived Growth Factor) pathway agonists include agents, molecules, compounds, or substances capable of stimulating (upregulating) a signaling pathway initiated by binding of PDGF (e.g., PDGF-AA, PDGF-AB, or PDGF-BB) to its receptor. In one embodiment, the PDGF pathway agonist is PDGF-BB (e.g., R&D Systems; Catalog No. 220-BB). In one embodiment, the PDGF agonist is 740Y-P (Tocris; Catalog No. 1983). In one embodiment, the PDGF pathway agonist is PDGF-BB, which is present in the culture medium at a concentration in the range of 7.5-12.5 ng / ml, 8.0-12.0 ng / ml, 9.0-11.0 ng / ml, 9.5-10.5 ng / ml, or 10 ng / ml.

[0043] IGF1 (insulin-like growth factor 1) pathway agonists include drugs, molecules, compounds, or substances capable of stimulating (upregulating) the signaling pathway initiated by the binding of IGF1 to its receptor. In one embodiment, the IGF1 pathway agonist is IGF1 (e.g., R&D Systems; Catalog No. 291-G1). Other examples of IGF1 pathway agonists include agonist peptides (e.g., IGF1 30-41 peptide and IGF1 24-41 peptide). In one embodiment, the IGF1 pathway agonist is IGF1, which is present in the culture medium at a concentration in the range of 15-25 ng / ml, 16.5-23.5 ng / ml, 17.5-22.0 ng / ml, 19.0-21.0 ng / ml, or 20 ng / ml.

[0044] FGF-beta (fibroblast growth factor-beta) pathway agonists include agents, molecules, compounds, or substances capable of stimulating (upregulating) a signaling pathway initiated by binding of FGF-beta to its receptor. In one embodiment, the FGF-beta pathway agonist is FGF-beta (e.g., R&D Systems; Catalog No. 3718-FB). In one embodiment, the FGF-beta pathway agonist is FGF-beta, which is present in the culture medium at a concentration in the range of 7.5-12.5 ng / ml, 8.0-12.0 ng / ml, 9.0-11.0 ng / ml, 9.5-10.5 ng / ml, or 10 ng / ml.

[0045] In embodiments, the culture medium in step (a) and step (b) of the iMSC differentiation protocol comprises a basal medium. In one embodiment, the basal medium is DMEM / F12 medium, although other media composed of components similar to DMEM / F12 are suitable for use as the basal medium. In embodiments, the basal medium comprises serum. In embodiments, the serum is selected from fetal bovine serum and human serum. In one embodiment, the basal medium (the basal medium of step (a), step (b), or both steps (a) and (b)) is DMEM / F12 medium containing 10% fetal bovine serum.

[0046] The iMSC precursor cells generated by steps (a) and (b) of the differentiation protocol are further cultured in a third step, referred to herein as step (c), to generate mature iMSCs. This step comprises culturing the human mesenchymal stem cell precursor cells from step (b) in a culture medium comprising a basal medium and an L-glutamine supplement for at least 7 days to generate iMSCs. In embodiments, the precursor cells are cultured for at least 14 days, at least 21 days, at least 28 days, at least 1 month, or at least 2 months to generate iMSCs.

[0047] In some embodiments, the L-glutamine supplement is L-alanine-L-glutamine dipeptide.In one embodiment, the L-alanine-L-glutamine dipeptide is GlutaMAX™ (e.g., Thermo Fisher; catalog number 35050-061).In one embodiment, the L-glutamine supplement is L-glutamine (e.g., Thermo Fisher; catalog number 25030081).

[0048] In one embodiment, the basal medium is DMEM / F12 medium, although other media composed of components similar to DMEM / F12 are suitable for use as the basal medium. In various embodiments, the basal medium comprises serum. In various embodiments, the serum is selected from fetal bovine serum and human serum. In one embodiment, the basal medium of step (c) is DMEM / F12 medium containing 10-15% fetal bovine serum (e.g., 10% FBS). In one embodiment, the culture medium used in step (c) comprises DMEM / F12 medium containing 10-15% fetal bovine serum (e.g., 10% FBS) supplemented with GlutaMAX™.

[0049] III.Culture conditions In combination with the defined culture medium described in subsection II above, the methods of generating iMSCs of the present disclosure utilize standard culture conditions established in the art for cell culture. For example, cells are cultured at 37° C. and 20% O 2 and 5% CO 2The cells can be cultured under the conditions of. The base medium can be used as the starting medium, to which supplements can be added. For example, in one embodiment, commercially available DMEM / F12 medium containing L-glutamine (Thermo Fisher; Catalog No. 11320033) can be used as the base medium. Alternatively, DMEM / F12 medium without L-glutamine (Thermo Fisher; Catalog No. 21331046) can be used as the base medium and supplemented with L-glutamine or a stabilized form of glutamine supplement (e.g., GlutaMAX™). Other examples of similar base media include Advanced DMEM / F12 medium (Thermo Fisher; Catalog No. 12364-010), HPLM medium (Thermo Fisher; Catalog No. A4899101), and aMEM medium (Thermo Fisher; Catalog No. 22571020).

[0050] Other media known in the art suitable for use as basal media for culturing human pluripotent stem cells include, but are not limited to, mTeSR1 (STEMCELL Technologies), Essential 8™ Medium (Thermo Fisher), PeproGrow™ hESC Basal Media (PeproTech), and StemFlex Medium (Thermo Fisher). Cells can be cultured in standard culture vessels or plates (e.g., culture dishes, culture flasks, or 96-well plates). Starting human pluripotent cells can be obtained as cell lines (e.g., human ESCs or iPSCs) established in the art.

[0051] In various embodiments of the disclosed method, the cells obtained from the three-step protocol described in Example 1 are cultured for a sufficient time such that at least 90% (more preferably at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) of the cells in the cell population express at least one, and preferably a plurality (e.g., at least two, three, four, or five) mesenchymal cell surface markers. Non-limiting examples of mesenchymal cell surface markers include CD73, CD90, CD105, CD29, CD44, and combinations thereof. In various embodiments, the cells obtained from the three-step protocol described in Example 1 are cultured for a sufficient time such that less than 5% (more preferably less than 4%, less than 3%, less than 2%, less than 1%, or less than 0.5%) of the cells in the cell population express one or more markers selected from the group consisting of TRA-1-60, CD34, CD45, HLA-DR, and combinations thereof.

[0052] In embodiments, iMSCs generated by the protocol described in Example 1 can be further treated for immunomodulatory effects (see Example 4). For example, to upregulate immunomodulatory genes in iMSCs, the cells can be treated with immune stimulants (e.g., double-stranded RNA (ds-RNA) or interferon-gamma (IFN-r)). iMSCs can be stimulated to secrete anti-inflammatory factors (e.g., interleukin-10 (IL-10)) by treatment with IFN-r.

[0053] In embodiments, iMSCs generated by the protocol described in Example 1 can be subjected to further differentiation under conditions to generate specific cell types, including but not limited to adipocytes, osteocytes, or chondrocytes. Conditions for generating such cells from iMSCs are well established in the art (see, e.g., Example 3).

[0054] IV.Applications The disclosed methods and compositions for generating human induced mesenchymal stem cells and their progenitors allow for efficient and robust utilization of these cell populations in a variety of applications. For example, iMSCs generated using the disclosed methods and compositions can be used to study human mesenchymal cell development and differentiation, including differentiation into a variety of other cell types. Thus, iMSCs generated by the disclosed methods also serve as a source of stem cells for generating a variety of further differentiated cell types that have applications for both research and clinical purposes.

[0055] The iMSCs generated by the methods of the present disclosure can be cultured in vitro and used as a source for other agents, such as a source of immunomodulatory agents (e.g., IL-10) that can be secreted by the iMSCs, or a source of extracellular vesicles (EVs) (e.g., exosomes) that are produced by the iMSCs. Such agents can be purified from the iMSCs or from the culture medium in which the cells are grown, by standard methods known in the art.

[0056] Additionally, iMSCs generated by the disclosed methods can be used for therapeutic purposes, e.g., in clinical settings where mesenchymal stem cells derived by other methods are used or are being considered or tested for use. iMSCs used for therapeutic purposes can be unmodified cells or cells that have been modified (e.g., to express a therapeutic agent, e.g., a recombinant secreted protein or an mRNA agent). Non-limiting examples of clinical settings where mesenchymal stem cells have been used and are currently being tested include cardiac injury (e.g., ischemic cardiomyopathy) and autoimmune diseases (e.g., Crohn's disease, multiple sclerosis, systemic lupus erythematosus, systemic sclerosis).

[0057] V. Composition In another aspect, the disclosure provides a composition related to a method for generating human induced mesenchymal stem cells, the composition comprising a culture medium and a kit.

[0058] In one aspect, the present disclosure provides a two-stage culture medium for obtaining human mesenchymal stem cell progenitors, comprising: (i) a first stage medium comprising a WNT pathway agonist and a BET pathway antagonist; and (ii) a second stage medium comprising a PDGF pathway agonist, an IGF1 pathway agonist, and an FGF-beta pathway agonist. Non-limiting examples of suitable agents and their concentrations include those described in subsection II above. In one embodiment, the first stage medium comprises CHIR98014 (e.g., 0.5 uM) and (+)-JQ1 (e.g., 50 nM), and the second stage medium comprises PDGF-BB (e.g., 10 ng / ml), IGF1 (e.g., 20 ng / ml), and FGF-beta (e.g., 10 ng / ml). The first and second stage media can include basal media (eg, DMEM / F12 with 10% FBS) as described in Sections II and III above.

[0059] In another aspect, the disclosure provides a kit for generating human induced mesenchymal stem cells, comprising the two-step culture medium described above and a sample of human pluripotent stem cells (e.g., hESC or iPSC lines), along with instructions for culturing the human pluripotent stem cells using the two-step culture medium (e.g., according to a protocol described herein) to generate iMSCs.

[0060] The present invention is further illustrated by the following examples, which should not be construed as further limiting. The contents of the figures and all references, patents, and published patent applications cited throughout this application are expressly incorporated herein by reference. EXAMPLES

[0061] Example 1: Culture protocol for generating inducible mesenchymal stem cells We developed a three-step protocol for generating induced mesenchymal stem cells (iMSCs) from human embryonic stem cells (hESCs). The protocol is shown diagrammatically in Figure 1. The protocol involves an initial 4-day induction phase using induction medium, followed by a 10-day enrichment phase using enrichment medium. These first two steps result in cultures of human iMSC progenitor cells that can be further differentiated into mature iMSCs. The third step involves further differentiating the iMSC progenitor cells into mature iMSCs by culturing them in maintenance medium for an additional period (e.g., at least 1-2 weeks).

[0062] To maintain hESCs in culture prior to the initiation of the iMSC protocol, hESCs were grown in mTeSR1 medium (STEMCELL Technologies; Cat. No. 85850) in 6-well plates coated with 1% Matrigel (Corning; Cat. No. 354277). The medium was replaced daily. When cells reached 80-90% confluency, the medium was aspirated and 1mL / well of Versene (Thermo Fisher; Cat. No. 15040066) was added. The culture plates were incubated at 37°C for 6-7 minutes, the Versene was gently aspirated, and the cells were resuspended in mTeSR1 medium (supplemented with 5uM Y-27632 (Tocris; Cat. No. 1254)). Cells were counted and seeded at 100k viable cells / well in a new plate. Cells were maintained in mTeSR1 medium until use in the iMSC protocol, and the medium was replaced daily.

[0063] Phase 1 Implementation Protocol In the first stage of the iMSC protocol, on day 1 (the day before the start of the differentiation protocol), 60–100k / well of live hESCs were seeded (approximately 8000 cells / cm) onto 1% Geltrex (Thermo Fisher; catalog no. 1413301)-coated 6-well plates in 2 mL / well of mTeSR1 medium supplemented with 5 μM Y-27632. 2). This passage was designated as passage 0 (P0). Then on day 0 (the start of differentiation), the medium was replaced with 2 mL / well of induction medium. The recipe for induction medium is as follows: DMEM / F12 (Thermo Fisher; Cat. No. 11320033) + 10% fetal bovine serum (FBS; Thermo Fisher; Cat. No. 10082147) supplemented with 50 nM (+)-JQ1 (Sigma Aldrich; Cat. No. SML1524) and 0.5 μM Chir98014 (Tocris; Cat. No. 6695). (+)-JQ1 is an inhibitor (antagonist) of the BET (bromodomain and extraterminal motif) family. Chir98014 is an activator (agonist) of the WNT pathway (through GSK inhibition). Cells were maintained in induction medium for 2 days (days 0 to 2), then the culture medium was replaced and cultured in fresh induction medium for an additional 2 days (days 2 to 4), for a total of 4 days of culture in induction medium in the first stage.

[0064] Phase 2 Enhancement Protocol On day 4, the induction medium was aspirated from the cultures and 2 mL / well of enrichment medium was added. The recipe for enrichment medium was as follows: DMEM / F12 medium + 10% FBS supplemented with 10 ng / mL PDGF-BB (R&D Systems; Catalog No. 220-BB), 20 ng / mL IGF1 (R&D Systems; Catalog No. 291-G1), and 10 ng / mL FGF-beta (R&D Systems; Catalog No. 3718-FB). Cells were maintained in enrichment medium from day 4 to day 14, with medium being changed every 2 days.

[0065] Once the cells reached 90% confluence in enriched medium (typically between days 6 and 8 (depending on the initial seeding density)), the cells were passaged to P1. For passaging, the enriched medium was aspirated and the cells were washed once with phosphate-buffered saline (PBS). The cells were treated with 1mL / well of Accutase (Thermo Fisher; Cat. No. A1110501) and incubated in a 37°C incubator for 5-6 minutes. Cell detachment was stopped by adding DMEM / F12 medium containing 10% FBS. The cells were suspended, transferred to a centrifuge tube, and spun down at 300g for 3 minutes. After centrifugation, the supernatant was discarded and the cell pellet was resuspended in enriched medium supplemented with 5uM Y-27632. The cells were seeded at approximately 150k viable cells / well into a new 6-well plate coated with 1% Geltrex (approximately 15,000 cells / cm). 2 ). Cells were maintained in enriched medium until day 14, with fresh medium replaced every 2 days. If cells again reached 90% confluence before day 14, cells were passaged again in enriched medium as above.

[0066] Phase 3 Maintenance Protocol The culture medium was replaced with maintenance medium on day 14 and then every 4 days. The recipe for maintenance medium was as follows: DMEM / F12 + 10% FBS supplemented with 2 mM GlutaMax (Thermo Fisher; Cat. No. 35050-061). Cells were passaged when they reached 90% confluence.

[0067] If cells were not passaged to P2 during the enrichment phase (i.e., before day 14), a P2 passage was performed on day 14 using maintenance medium as follows: Cell culture medium was aspirated and cells were washed once with PBS. Accutase (1 mL / well) was added and cells were incubated in a 37 °C incubator for 5–6 min. Dissociation was stopped by adding DMEM / F12 + 10% FBS and suspended cells were transferred to a centrifuge tube. Cells were centrifuged at 300 g for 3 min, the supernatant was discarded and the cell pellet was resuspended in maintenance medium supplemented with 5 μM Y-27632. Cells were transferred from the wells to 10 cm dishes coated with 0.1% gelatin (Millipore; catalogue no. ES-006) (approximately 10,000 cells / cm). 2 Additional medium was added to reach a total volume of 8-10 mL / dish.

[0068] The cells were then maintained in maintenance medium (changed every 4 days) and whenever the cell density reached 90% confluence, the cells were split for the next passage as follows: The cell culture medium was aspirated and the cells were washed once with PBS. TrypLE Select (5mL / dish) was added and the cells were incubated in a 37°C incubator for 8-10 minutes. DMEM / F12+10% FBS was added to stop the separation and the suspended cells were transferred to a centrifuge tube. The cells were centrifuged at 300g for 3 minutes, the supernatant was discarded and the cell pellet was resuspended in maintenance medium supplemented with 5μM Y-27632. The cells were seeded at 300k / dish of viable cells onto 10cm dishes coated with 0.1% gelatin (approximately 5,000 cells / cm). 2 Additional medium was added to reach a total volume of 8-10 mL / dish. Mesenchymal stem cell-like cells began to appear from day 21 of culture.

[0069] Cell morphology was examined microscopically at different stages of the differentiation protocol. The results are shown in Figure 2A-B. From day 4 to day 14, the cells were at the mesoderm stage. Representative examples are shown in Figure 2A (left and right panels). Cells at this stage showed early mesoderm-like morphology with relatively large nuclei, and some cells had filopodia. From day 21 onwards, the cells began to acquire mesenchymal / fibroblast-like morphology, as shown in Figure 2B (left panel) (low density 10x). At high density, the cells became spindle-like in shape, and a wavy pattern could be observed, as shown in Figure 2B (right panel) (high density 5x).

[0070] The inducible MSCs maintained a consistent proliferation rate in culture for at least three months, and the cells were expanded and used in validation studies (described further below).

[0071] Example 2: Surface marker expression of induced mesenchymal stem cells Surface marker expression of induced mesenchymal stem cells (iMSCs) was examined by standard FACS analysis. Positive markers of MSCs examined were CD73, CD90, CD105, CD29, and CD44. As shown in Figure 3A, iMSCs generated by the differentiation protocol described in Example 1 showed surface expression of each of these mesenchymal markers. Negative markers examined included TRA-1-60, CD11b, CD19, CD34, CD45, and HLA-DR. As shown in Figure 3B, iMSCs did not express any of the negative markers tested (the "negative cocktail" included anti-CD11b, anti-CD19, anti-CD34, anti-CD45, and anti-HLA-DR antibodies).

[0072] Example 3: Tri-lineage differentiation of induced mesenchymal stem cells To examine the differentiation potential of iMSCs generated according to the protocol described in Example 1, the cells were further treated using adipocyte, osteocyte, or chondrocyte differentiation protocols.

[0073] For adipogenesis, iMSCs were cultured using the Adipogenesis Differentiation Kit (Thermo Fisher; Catalog No. A1007001) according to the manufacturer's instructions. After 21 days of adipocyte differentiation, cells were stained with Oil Red solution (Sigma Aldrich; Catalog No. O1391), which stains lipid particles in adipocytes red. The results are shown in Figure 4A. This figure shows the ability of iMSCs to differentiate into adipocytes.

[0074] For osteogenesis, iMSCs were cultured using the Osteogenesis Differentiation Kit (Thermo Fisher; Catalog No. A1007201) according to the manufacturer's instructions. After 14 days of osteocyte differentiation, the cells were stained with Alizarin Red solution (Sigma Aldrich; Catalog No. TMS-008-C), which stains osteocytes red. The results are shown in Figure 4B. This figure shows the ability of iMSCs to differentiate into osteocytes.

[0075] For chondrogenesis, iMSCs were cultured using the Chondrogenesis Differentiation Kit (Thermo Fisher; Catalog No. A1007101) according to the manufacturer's instructions. After 14 days of chondrocyte differentiation, cells were stained with Alcian Blue solution (Sigma Aldrich; Catalog No. B8438), which stains chondrocytes blue. The results are shown in Figure 4C. This figure shows the ability of iMSCs to differentiate into chondrocytes.

[0076] Overall, the results in Figures 4A-C demonstrate the capacity of iMSCs for tri-lineage differentiation.

[0077] Example 4: Immune modulation of induced mesenchymal stem cells In this example, various tests were performed to investigate the immunomodulatory potential of iMSCs generated according to the differentiation protocol described in Example 1.

[0078] In the first set of tests, iMSCs were treated with immune stimulants (double-stranded RNA (dsRNA) or recombinant human interferon gamma (IFNr) (R&E Systems; Cat. No. 35-IF-100)). iMSCs (100k cells / sample) were cultured with dsRNA (500ng / sample) or IFNr (10ng / ml) for 24 hours. After treatment, the immune-modulatory capacity of iMSCs was analyzed by quantitative real-time PCR of marker genes that could be upregulated by the tested immune stimulants. The immune-stimulatory marker genes examined were: RIG1, TLR3, COX2, IDO1, HGF, and TGF-beta. The control genes (for MSC surface markers) were: CD73, CD90, and CD105. The results are shown in FIG. 5. The results showed that treatment of iMSCs with immune stimulants increased the expression of several genes associated with immune stimulation (especially IDO1, TLR3, and HGF), whereas control marker genes were not upregulated.

[0079] In a second set of experiments, iMSCs were used in a mixed lymphocyte reaction (MLR). Human peripheral blood mononuclear cells (hPBMCs) (Lonza; Cat. No. cc-2702) were labeled with CellTrack Violet (Thermo Fisher; Cat. No. C34557) to indicate cell proliferation. iMSCs (50k cells / well of a 24-well plate; approximately 25,000 cells / cm) were cultured at 100°C for 1 h. 2iMSCs were pretreated with 10 ng / ml IFNr for 3 days. These are referred to as “activated iMSCs”, whereas iMSCs without IFNr pretreatment are referred to as “inactivated iMSCs”. For MLR, hPBMCs were added on top of iMSCs and co-cultured for 3 days in co-culture medium supplemented with recombinant human IL-2 (R&D Systems; Cat. No. 202-IL) and anti-CD3 (eBioscience; Cat. No. 16-0037-81) to promote T cell proliferation. After 3 days, hPBMCs were harvested and analyzed by flow cytometry. As shown in Figure 6, five peaks were observed in the “hPBMC only” group, indicating that the cells had proliferated four times. In the “+inactivated iMSC” group, the peaks were indistinguishable. This suggests that co-culture with inactivated iMSCs hindered the proliferation of hPBMCs. The single dominant peak in the "+activated iMSC" group indicates that cell proliferation occurred only once and then ceased during co-culture with activated iMSC.

[0080] In a third set of experiments, we investigated the production of the anti-inflammatory factor IL-10 by iMSCs after IFNr treatment. iMSCs (50k cells / well in a 24-well plate; approximately 25,000 cells / cm) were cultured in vitro. 2 ) were treated with low (20 ng / mL) or high (50 ng / mL) concentrations of IFNr for 3 days to stimulate the secretion of IL-10. The control group had the same culture conditions but no IFNr treatment. After 3 days, the culture supernatants were collected and the levels of IL-10 secretion were quantified by standard ELISA analysis (human IL-10 Quantikine ELISA kit; R&D Systems; Cat. No. D1000B). As shown in Figure 7, the secretion of IL-10 by iMSCs was dose-dependent with respect to the concentration of IFNr treatment.

[0081] Example 5: Production of extracellular vesicles by induced mesenchymal stem cells In this example, the production of extracellular vesicles by iMSCs was quantified. Cells were first washed with PBS and then cultured in maintenance medium (described in Example 1) containing exosome-depleted FBS. After 4 days of culture, the medium was collected and the total volume was measured. iMSCs were separated from the culture vesicles and the cell number was counted. Extracellular vesicles were quantified using FACS-based analysis targeting tetraspanin proteins on the vesicle membrane. The results of three representative samples are shown in Figure 8 (with PBS and medium only controls), demonstrating the production of extracellular vesicles by iMSCs.

[0082] Equivalent Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments described herein which equivalents are intended to be encompassed by the following claims.

Claims

1. 1. A method for generating human mesenchymal stem cell progenitors, comprising: (a) culturing human pluripotent stem cells in a culture medium containing a canonical WNT pathway agonist and a BET pathway antagonist for at least two days to generate inducible cells; (b) culturing the induced cells from step (a) in a culture medium comprising a PDGF pathway agonist, an IGF1 pathway agonist, and an FGF-beta pathway agonist for at least 10 days to generate human mesenchymal stem cell progenitor cells.

2. 1. A method for generating human induced mesenchymal stem cells (iMSCs), comprising: (a) culturing human pluripotent stem cells in a culture medium containing a canonical WNT pathway agonist and a BET pathway antagonist for at least two days to generate inducible cells; (b) culturing the induced cells from step (a) in a culture medium comprising a PDGF pathway agonist, an IGF1 pathway agonist, and an FGF-beta pathway agonist for at least 10 days to generate human mesenchymal stem cell progenitor cells; (c) culturing the human mesenchymal stem cell progenitor cells from step (b) in a culture medium comprising a basal medium and an L-glutamine supplement for at least 7 days to generate iMSCs.

3. 3. The method of claim 1 or 2, wherein step (a) comprises culturing the human pluripotent stem cells in the culture medium comprising a canonical WNT pathway agonist and a BET pathway antagonist from day 0 to day 4 to generate inducible cells.

4. 3. The method of claim 1 or 2, wherein step (b) comprises culturing the induced cells from step (a) in a culture medium comprising a PDGF pathway agonist, an IGF1 pathway agonist, and an FGF-beta pathway agonist from day 4 to day 14 to generate human mesenchymal stem cell progenitor cells.

5. 3. The method of claim 2, wherein step (c) comprises culturing the human mesenchymal stem cell progenitor cells in the basal medium and L-glutamine supplement for at least 14 days.

6. The method of claim 2 , wherein the human pluripotent stem cells are human embryonic stem cells.

7. 3. The method of claim 2, wherein the iMSCs express one or more surface markers selected from the group consisting of CD73, CD90, CD105, CD29, CD44, and combinations thereof.

8. 3. The method of claim 2, wherein the iMSCs are capable of further differentiation into adipocytes, osteocytes, or chondrocytes.

9. 3. The method of claim 1 or 2, wherein the canonical WNT pathway agonist is a glycogen synthase kinase 3 (Gsk3) inhibitor.

10. 3. The method of claim 1 or 2, wherein the canonical WNT pathway agonist is selected from the group consisting of CHIR98014, CHIR99021, SB 216763, SB 415286, LY2090314, 3F8, A 1070722, AR-A 014418, BIO, AZD1080, WNT3A, and combinations thereof.

11. 11. The method of claim 10, wherein the canonical WNT pathway agonist is present in the culture medium at a concentration in the range of 0.25 to 0.75 μM.

12. 3. The method of claim 1 or 2, wherein the BET pathway antagonist is a triazolo-diazepine compound.

13. 3. The method of claim 1, wherein the BET pathway antagonist is selected from the group consisting of (+)-JQ1, TEN-010, OTX015, I-BET762, I-BET151, BAY1238097, ABBV-744, ABBV-075, iBET-BD1, iBET-BD2, SJ432, RVX-208, MS417, AZD5153, and combinations thereof.

14. 14. The method of claim 13, wherein the BET pathway antagonist is present in the culture medium at a concentration in the range of 25 to 75 nM.

15. The method of claim 1 or 2, wherein the PDGF pathway agonist is PDGF-BB.

16. 16. The method of claim 15, wherein PDGF-BB is present in the culture medium at a concentration in the range of 7.5 to 12.5 ng / ml.

17. 3. The method of claim 1 or 2, wherein the IGF1 pathway agonist is IGF1.

18. 18. The method of claim 17, wherein IGF1 is present in the culture medium at a concentration in the range of 15 to 25 ng / ml.

19. 3. The method of claim 1 or 2, wherein the FGF-beta pathway agonist is FGF-beta.

20. 20. The method of claim 19, wherein FGF-beta is present in the culture medium at a concentration in the range of 7.5 to 12.5 ng / ml.

21. 3. The method of claim 1 or 2, wherein the culture medium in steps (a), (b), and / or (c) comprises a basal medium containing serum.

22. 22. The method according to claim 21, wherein the serum-containing basal medium is a DMEM / F12 medium containing 10 to 15% fetal bovine serum.

23. 3. The method of claim 2, wherein the L-glutamine supplement is an L-alanine-L-glutamine dipeptide.

24. 3. The method of claim 2, further comprising isolating extracellular vesicles produced by the iMSCs from the culture.

25. A two-stage culture medium for obtaining human mesenchymal stem cell precursor cells by sequentially using a first-stage culture medium and a second-stage culture medium, the two-stage culture medium comprising: (i) a first-stage culture medium containing a canonical WNT pathway agonist and a BET pathway antagonist; and (ii) a second-stage culture medium containing a PDGF pathway agonist, an IGF1 pathway agonist, and an FGF-beta pathway agonist.

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