Process

Abstract

The present invention relates generally to the field of stem cell differentiation. In particular, the present invention relates to a serum-free method for differentiating stem cells into pluripotent mesenchymal stem cells (MSCs). The invention also relates to a serum-free medium for cell culture. In one aspect of the present invention, there is provided a serum-free composition for the differentiation of sub-totipotent stem cells into pluripotent MSCs, the composition comprising a Dulbecco's Modified Eagle Medium, a DMEM, a KnockOutTM serum substitute (KOSR), and a GlutaMAX, for use in the differentiation of sub-totipotent stem cells into pluripotent MSCs, the composition comprising a Dulbecco's Modified Eagle Medium, a DMEM, a KnockOutTM serum substitute.

Description

Technical Field

[0001] The present invention generally relates to the field of stem cell differentiation. In particular, the present invention relates to a serum-free method for differentiating stem cells into multipotent mesenchymal stem cells (MSCs). The present invention also relates to a serum-free medium for cell culture. Background Art

[0002] Human mesenchymal stem cells (MSCs) are currently being used in clinical trials for a variety of cellular applications. They are multipotent, safe (non-tumorigenic), and are thought to have immunomodulatory properties. However, primary human MSCs isolated from tissues are variable, heterogeneous, and have limited expansion and proliferation capacity (approximately 20 passages).

[0003] Human pluripotent stem cells (hPSCs) are renewable and can provide unlimited quantities of MSCs for cell therapy and clinical trial applications. Unfortunately, current protocols for differentiating hPSCs into MSCs often involve undefined reagents, such as fetal bovine serum (FBS) and trypsin, which do not meet clinical requirements.

[0004] Therefore, a method to generate MSCs in a sustainable and serum-free manner is urgently needed. Summary of the invention

[0005] In one aspect of the present invention, a method for differentiating sub-totipotent stem cells into multipotent MSCs is provided, the method comprising the following steps: (a) In a medium containing Dulbecco's Modified Eagle Medium (DMEM), KnockOut TM Stem cells were cultured in the first medium of serum replacement (KOSR) and GlutaMAX to form embryoid bodies (EBs); (b) replacing the first culture medium with a second culture medium comprising DMEM, KOSR and GlutaMAX, and culturing the EBs in the second culture medium to differentiate the EBs into a MSC progenitor cell population; and (c) replacing the second culture medium with a third culture medium comprising DMEM, KOSR, GlutaMAX and fibroblast growth factor 2 (FGF-2), and culturing the MSC progenitor cell population in the third culture medium to differentiate the MSC progenitor cell population into MSCs; Among them, the first culture medium, the second culture medium and the third culture medium are serum-free culture media.

[0006] As used herein, "serum-free" means not containing animal or human serum. Serum-free compositions may include compositions that do not contain fetal bovine serum or other bovine serum.

[0007] As used herein, the term "differentiating" or "differentiation" refers to the process by which a cell develops further along a developmental pathway than its immediate precursor cell. A differentiated cell is one in which a more specialized cell type is derived from a less specialized cell type during cell differentiation. A differentiated cell is a cell that is in a more stable position in a cell lineage.

[0008] The term "stem cell" as used herein refers to a cell that can self-renew and differentiate into a more specialized cell. The stem cells used herein may include embryonic stem cells or induced pluripotent stem cells. The pluripotent stem cells used herein may include, but are not limited to, human stem cells and non-human primate stem cells. Human pluripotent stem cells (hPSCs) may include human embryonic stem cells (hESCs) or human induced pluripotent stem cells (hiPSCs). In certain embodiments, the pluripotent stem cells used may be human embryonic stem cells, human induced pluripotent stem cells (hiPSCs), adult stem cells, or primate induced pluripotent stem cells. Pluripotent stem cells have the potential to differentiate into tissues from all three embryonic germ layers (i.e., endoderm, mesoderm, and ectoderm). Some tools used to characterize pluripotency include quantitative PCR and immunofluorescence staining, which can detect the upregulation of pluripotency genes (such as Oct4, Sox2, and Nanog). However, the gold standard for assessing pluripotency is the teratoma formation assay, which assesses the ability of cells to form tissues from all three germ layers in vivo in the form of encapsulated tumors called teratomas. The so-called "embryoid bodies" (EBs) are three-dimensional aggregates that include sub-totipotent stem cells.

[0009] The term "multipotent" refers to a cell type that can produce a limited number of other specific cell types. Pluripotent cells are committed to one or more embryonic cell fates, and therefore, in contrast to pluripotent cells, cannot produce each of the three embryonic cell lines and extraembryonic cells. Pluripotent cells are more differentiated than pluripotent cells, but not the most differentiated.

[0010] The term "mesenchymal stem cells" (MSC) used herein refers to multipotent cells that can differentiate into multilineage cells such as chondrocytes, osteoblasts, adipocytes, etc. The term "MSCs" used refers to cells in which 95% or more of the cells are mesenchymal stem cells, or 95% or more of the cells express MSC positive markers. MSC positive markers include CD73, CD90 and CD105. The term "MSCs" used refers to a cell population defined according to the standards proposed by the Mesenchymal and Tissue Stem Cell Committee of the International Society for Cellular Therapy (ISCT). One of the standards of ISCT is that MSCs have plastic-adherent properties under standard culture conditions. Another standard of ISCT is that ≥95% of the mesenchymal stem cell population expresses CD105, CD73 and CD90. In addition, MSCs lack the expression of CD45, CD34, CD14 or CD11b, CD79α or CD19 and HLA-DR surface molecules (positive rate ≤2%). Another criterion of ISCT is that MSCs can differentiate into osteoblasts, adipocytes and chondroblasts in vitro. The "progenitor MSC" or "MSC progenitor cell" refers to a multipotent cell that has the potential to differentiate into MSCs. The term "MSC progenitor cell population" used herein refers to a cell population in which less than 95% of the cells express MSC positive markers.

[0011] In one embodiment, DMEM is DMEM with high glucose content, also referred to as "high glucose DMEM". High glucose content in DMEM refers to a glucose content of 4.5 g / L (25 mmol / L), and low glucose content in DMEM refers to a glucose content of 1 g / L (5.55 mmol / L). In some embodiments, the first culture medium, the second culture medium, and the third culture medium contain high glucose DMEM.

[0012] Alternatively, DMEM can be DMEM / F-12 containing GlutaMAX. DMEM / F-12 containing GlutaMAX can be used for the dissociation of sub-totipotent stem cells before step (a) of the method. DMEM / F-12 containing GlutaMAX includes a 1:1 mixture of DMEM (basal medium) and Ham's F-12. The glucose content of DMEM / F-12 containing GlutaMAX is 3.151 g / L.

[0013] As used herein, the term "culture" refers to the cultivation of a cell population in a liquid or solid medium under suitable growth conditions. As used herein, the term "culture" includes subculture, also known as subculture or cell division. Subculture or subculture refers to a technique that maintains cell viability and allows cells to proliferate for a long time under culture conditions. Subculture or subculture may include removing the culture medium and transferring the cells from a previous culture to a fresh growth medium so that the cells can further proliferate. The subculture process may also include dissociating the cells before the cells are transferred to the fresh growth medium.

[0014] In one embodiment, the method further includes the step of using the reagent ReLeSR to dissociate the sub-totipotent stem cells into clumps before step (a), or using the reagent TrypLE to dissociate the sub-totipotent stem cells into single cells before step (a). It is advantageous to use ReleSR or TrypLE instead of trypsin to dissociate sub-totipotent stem cells for the following reasons. Since trypsin is of porcine origin, it is heterologous and not suitable for cell therapy applications. On the other hand, TrypLE is animal-free, while ReLeSR complies with the dynamic pharmaceutical production management specifications (cGMP standards).

[0015] Compared to trypsin, TrypLE is gentler on cells and preserves cell surface epitopes such as CD73, CD90, and CD105. Trypsin requires neutralization with serum-containing buffers / media, which poses a problem for the production of cell-based therapeutic approaches, whereas the enzymatic activity of TrypLE can be inhibited by diluting TrypLE, allowing the use of serum-free reagents during cell passaging.

[0016] The inventors have also found that ReLeSR is superior to trypsin because, depending on the length of time ReLeSR is treated, ReLeSR has different abilities to detach stem cells while keeping differentiated cells attached to the cell plate. Based on the experiments performed, the inventors found that trypsin indiscriminately detaches all (differentiated and undifferentiated) cells. Therefore, using ReLeSR in the dissociation step better ensures that the clumps formed are from sub-totipotent stem cells.

[0017] In one embodiment, before the dissociation step, sub-totipotent stem cells are cultured in MSC M0 medium or mTeSR TM 1 (Stemcell Technologies, catalog number 85850). During the dissociation of sub-totipotent stem cells into clumps using ReLeSR or into single cells using TrypLE to form embryoid bodies (EBs), sub-totipotent stem cells were cultured in hPSC M0 medium or DPBS (Cytiva, catalog number SH30028) as shown in Table 1 to dilute the dissociation reagent (i.e., ReleSR or TrypLE) and stop the reaction.

[0018] In one embodiment, sub-totipotent stem cells dissociated using ReLeSR are cultured in MSC M1 medium to form embryoid bodies (EBs). In another embodiment, sub-totipotent stem cells dissociated using TrypLE are cultured in MSC M0 medium and 5-10 μM Y-27632 for 24 hours, followed by MSC M1 medium for embryoid body (EB) formation. In one embodiment, embryoid bodies are formed after 1-2 days of culture after sub-totipotent stem cell dissociation.

[0019] In one embodiment, embryoid bodies and MSC progenitor cell populations are cultured on a plate coated with gelatin. The use of gelatin coating can provide an ideal cell attachment substrate for cell culture and delay cell senescence.

[0020] In one embodiment, the MSC progenitor population is dissociated using TrypLE prior to step (c) of the method.

[0021] In one embodiment of step (b) of the method, differentiation of embryoid bodies into MSCs begins by seeding embryoid bodies onto gelatin-coated plates and culturing them with MSC M2 medium. In one embodiment, seeding is performed on day 8. At this stage, the cells begin to have MSC-like morphology, but are classified as MSC progenitor cell populations rather than mesenchymal stem cells (MSCs) because most cells do not yet express MSC cell markers, such as CD73, CD90, and CD105. In one embodiment, the replacement of MSC M2 medium with MSC M3 medium in step (c) represents passage 1. The cells are passaged using MSC M3 medium. In the first few passages, the cells may still be MSC progenitor cell populations because most cells do not yet express MSC cell markers, such as CD73, CD90, and CD105. After the first few passages, a highly pure MSC cell population (95% or more cells express MSC positive markers and less than 2% cells express MSC negative markers (such as CD34 and CD45)) is obtained, and these cells are classified as mesenchymal stem cells (MSCs). In one embodiment, mesenchymal stem cells (MSCs) are formed after passage 5.

[0022] In one embodiment, the method further comprises passaging the MSCs obtained in step (c) in a third culture medium to maintain the multipotent state of the MSCs.

[0023] In another embodiment, after being passaged 15 times using the third culture medium, the MSCs are maintained in a multipotent state.

[0024] In another embodiment, MSCs are dissociated with TrypLE prior to each passaging using the third culture medium.

[0025] The term "maintain" refers to a process that includes maintaining cell viability. As used herein, the term "maintain" in the context of maintaining cells in a pluripotent state may include passaging of cells to maintain cells at an optimal density to continue growing and maintain the pluripotency of the cells.

[0026] In one embodiment, as shown in Table 1, the first culture medium is MSC M1 culture medium, the second culture medium is MSC M2 culture medium, and the third culture medium is MSC M3 culture medium.

[0027] MSC M3 medium can also be a MSC maintenance medium to maintain MSCs in a multipotent state. The presence of FGF-2 in MSC M3 medium may help maintain the multipotency of MSCs.

[0028] In one embodiment, subtotipotent stem cells are cultured in a first culture medium to form EBs, the EBs are cultured in a second culture medium to form a MSC progenitor cell population, the MSC progenitor cell population is cultured in a third culture medium to form MSCs, and the MSCs are maintained in a third culture medium, all at a temperature of 37° C., a carbon dioxide content of 5%, and a pH of 7.4.

[0029] In one embodiment, the sub-totipotent stem cells in step (a) of the method are human induced pluripotent stem cells (hiPSCs).

[0030] In one embodiment of the method, after step (c), at least 95% of the cells obtained are MSCs.

[0031] Another aspect of the present invention provides a serum-free composition for differentiating sub-totipotent stem cells into multipotent MSCs, the composition comprising Dulbecco's Modified Eagle Medium (DMEM), KnockOut TM Serum Replacement (KOSR) and GlutaMAX.

[0032] In one embodiment of the serum-free composition, the concentration of DMEM is about 74% to about 89% (volume fraction, v / v), the concentration of KOSR is about 10% to about 25% (volume fraction, v / v), and the concentration of GlutaMAX is about 1% (volume fraction, v / v).

[0033] In one embodiment of the serum-free composition, the concentration of DMEM is about 79% to about 84% (volume fraction, v / v), the concentration of KOSR is about 15% to about 20% (volume fraction, v / v), and the concentration of GlutaMAX is about 1% (volume fraction, v / v).

[0034] In another embodiment of the serum-free composition, the concentration of DMEM is about 79% to about 89% (volume fraction, v / v), the concentration of KOSR is about 10% to about 20% (volume fraction, v / v), and the concentration of GlutaMAX is about 1% (volume fraction, v / v).

[0035] In one embodiment, the serum-free composition further comprises Fibroblast Growth Factor 2 (FGF-2).

[0036] In another embodiment of the serum-free composition, the concentration of DMEM is about 74% to about 84% (volume fraction, v / v), the concentration of KOSR is about 15% to about 25% (volume fraction, v / v), the concentration of GlutaMAX is about 1% (volume fraction, v / v), and the concentration of FGF-2 is about 2.5 ng / mL to 5 ng / mL. In another embodiment, the concentration of FGF-2 is about 2.5 ng / mL.

[0037] In one embodiment, the sub-pluripotent stem cells are human induced pluripotent stem cells (hiPSCs).

[0038] In another aspect of the present invention, there is provided a population of mesenchymal stem cells obtained or obtainable by the method described herein.

[0039] In one embodiment, at least 95% of the cells in a population of mesenchymal stem cells express CD73, CD90 and CD105.

[0040] In one embodiment, 2% or less of the cells in the mesenchymal stem cell population express CD34 and CD45.

[0041] The listing or discussion of an apparently prior-published document in this specification is not necessarily taken as an admission that the document is part of the state of the art or is common general knowledge.

[0042] Any documents mentioned herein are incorporated by reference in their entirety.

[0043] In order that the invention may be fully understood and readily put into practical effect, preferred embodiments of the invention will be described by way of non-limiting examples, the description of which will refer to the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] In the attached figure: Figure 1 is a schematic diagram of a clinically acceptable serum-free protocol for generating hPSC-derived MSCs from a starting hPSC culture; Figure 2 Representative bright field images of hPSC-derived MSCs generated by different methods are shown; (A) shows hPSC-derived MSCs generated with FBS and trypsin; (B) shows hPSC-derived MSCs generated with serum-free defined substitutes (KOSR and TrypLE); scale bar represents 10 μm; Figure 3Figure 2 shows flow cytometric analysis of MSC populations generated using different methods; (A) shows hPSC-derived MSCs generated using FBS and trypsin; (B) shows hPSC-derived MSCs generated using serum-free defined replacements (KOSR and TrypLE); the MSC populations used were cultured for the same period of time; Figure 4 Representative bright field images of EBs generated by different methods after 7 days in culture are shown; EBs were generated from: (A) serum-free defined replacement (KOSR), and (B) FBS; scale bar represents 10 μm; Figure 5 Flow cytometry analysis of MSC populations generated by different methods is shown; hiPSCs were dissociated with TrypLE or ReLeSR, and then hiPSC-derived MSCs were generated with FBS or serum-free defined substitute KOSR; the MSC populations used were cultured for the same period of time; wherein, (A) shows the expression of CD73, (B) shows the expression of CD90, (C) shows the expression of CD105, (D) shows the expression of CD34, and (E) shows the expression of CD45; Figure 6 Doubling rates of MSCs generated after prolonged passaging are shown; hiPSCs were dissociated with TrypLE or ReLeSR and then hiPSC-derived MSCs were generated with FBS or the serum-free defined replacement KOSR; the MSC populations used were cultured for the same time period; Figure 7 Representative autopsy images of nonobese diabetic severe combined immunodeficient (NOD SCID) mice injected with (a) undifferentiated hiPSCs (positive control) and (b) hPSC-derived MSCs generated using a serum-free protocol according to an embodiment of the present invention for teratoma detection are shown; arrows indicate injection sites; Figure 8 Histological analysis of the sites of injection of (a, b, c) hPSCs, (d) hPSC-derived MSCs generated using a serum-containing regimen, and (e) hPSC-derived MSCs generated using a serum-free regimen are shown; the scale bar represents 10 μm, and arrows indicate the respective germ layers. DETAILED DESCRIPTION

[0045] hPSCs (e.g., hiPSCs) are differentiated into MSCs using serum-free media. An example of a serum-free differentiation protocol is shown in FIG1 . The media indicated by the arrows in FIG1 are used to generate cells in the next process. Thus, MSC M1 (or MSC M0 and MSC M1 for cells dissociated with TrypLE) are used to generate EBs from sub-totipotent stem cells. Briefly, sub-totipotent stem cells are first cultured in MSC M0 medium. After dissociation, cell clumps (generated using ReLeSR dissociation) are cultured in MSC M1 for 8 days; or, if single cells (generated using TrypLE dissociation), in MSC M0 for 1 day; and then in MSCM1 for 7 days to form and maintain EBs. EBs are usually formed 1-2 days after dissociation of sub-totipotent stem cells.

[0046] hiPSCs are first dissociated into clumps or single cells suitable for embryoid body formation. The dissociation step can be performed using ReLeSR or TrypLE, both of which are clinically compatible reagents. When using ReLeSR, hiPSCs are dissociated into clumps. When using TrypLE, hiPSCs are dissociated into single cells. Subsequent sub-totipotent embryoid bodies are plated and directed to differentiate into plastic-adherent MSCs. Various academic protocols were compared and modified (e.g., use of FBS, trypsin) so that the reagents used to generate hPSC-derived MSCs are serum-free (e.g., use of KnockOut Serum Replacement (KOSR), TrypLE). These hPSC-derived MSCs were characterized for MSC marker expression and found to be comparable to primary MSCs.

[0047] Existing serum-free donor-derived MSC culture protocols generally require the use of serum-containing reagents during initial culture and early passages to maximize cell viability. Unlike traditional serum-free protocols, the method of the present invention can use serum-free reagents throughout the entire MSC culture process and throughout all MSC passages while maintaining the viability of MSCs.

[0048] The ability to differentiate hiPSCs into MSCs allows for unlimited MSCs to be generated from hiPSCs, whereas primary MSCs from human tissues, in contrast, have limited expansion potential. By creating a serum-free differentiation protocol, the generation of clinical-grade hiPSC-derived MSCs could be targeted as the ultimate goal for cell therapy and clinical applications in humans.

[0049] Existing FBS-containing protocols are often unreliable and vary widely due to batch-to-batch differences, FBS sources, and quality. In the present invention, existing FBS-containing differentiation protocols have been modified to be serum-free. This requires careful identification of serum-containing components, swapping them with serum-free reagents, and verifying that they are equally effective (if not better). FBS-containing protocols were compared to the serum-free protocols of the present invention, and cells were characterized ( Figures 2 to 6 Advantageously, the scheme of the present invention meets clinical requirements, has highly consistent results, and is suitable for use in biomanufacturing. In particular, the scheme of the present invention can be used to facilitate large-scale cell manufacturing processes.

[0050] hiPSC-derived MSCs produced by the method of the present invention can be used in various cell applications, including chronic wounds, diabetic wounds, cardiovascular diseases, lung diseases (such as COVID-19), etc.

[0051] Serum-free differentiation protocol Medium formulation Table 1

[0052] In one embodiment, MSC M1 medium consists of 74-89% DMEM, 10-25% KOSR, and 1% GlutaMAX. In one embodiment, MSC M2 medium consists of 79-89% DMEM, 10-20% KOSR, and 1% GlutaMAX. In one embodiment, MSC M3 medium consists of 74-84% DMEM, 15-25% KOSR, 1% GlutaMAX, and 2.5 ng / mL FGF-2.

[0053] Detailed steps of serum-free differentiation protocol Day 1 Dissociation of hPSCs with ReLeSR or TrypLE (a) For ReLeSR dissociation, ReLeSR was added to hPSCs at room temperature and removed within 30 seconds. hPSCs were incubated at 37˚C for 2.5-3.5 minutes to dissociate clumps. The resulting hPSC aggregates were collected with hPSC M0 medium and resuspended as necessary to obtain hPSC clumps of 50-200µm in size. hPSC clumps from confluent 10 cm plates were seeded onto non-tissue culture treated six-well plates with 3 mL of MSC M1 medium and 10 μM Rho-associated, coiled coil containing protein kinase (ROCK) inhibitor Y-27632 per well.

[0054] (b) For TrypLE dissociation, add TrypLE to hPSCs and incubate at 37˚C for 3 minutes. Afterwards, aspirate TrypLE and collect hPSCs with hPSC M0 medium. Resuspend hPSCs thoroughly and filter through a 40 µM cell strainer to ensure a single-cell suspension. Seed cells at 3-4 million cells per well in a six-well plate that has not been treated with tissue culture, and add 3 mL of MSC M0 medium and 10 μM ROCK inhibitor Y-27632 to prevent apoptosis.

[0055] Place the cell suspension on a shaker at 80-85 rpm to allow embryoid bodies (EBs) to form.

[0056] Day 2 24 hours after seeding the cells, the spent medium was replaced with MSC M1 medium.

[0057] EBs should begin to form.

[0058] Day 4-7 Every 2-3 days, replace the spent culture medium with MSC M1 culture medium.

[0059] Day 8 Re-seed EBs with MSC M2 medium on gelatin-coated six-well plates, with approximately 20 EBs per well.

[0060] Day 10-15 (1) Replace the culture medium with MSC M2 medium every 2-3 days.

[0061] (2) When the wells are confluent (approximately 5-7 days from reseeding), dissociate the cells with TrypLE and plate them onto gelatin-coated 10 cm plates using MSC M3 medium. This is passage 1.

[0062] From passage 1 (1) Replace the culture medium with MSC M3 medium every 2-3 days.

[0063] (2) Cells may take longer to become confluent in passage 1 and require two weeks of growth before the next passage can be started.

[0064] (3) Starting from passage 2, cells can be passaged every 7 days.

[0065] (4) Cells can be stored on gelatin-coated plates or on plates treated with normal tissue culture.

[0066] (5) MSCs can be maintained for more than 15 passages using the MSC M3 medium formula, with more than 90% of the cell population expressing MSC-positive markers (CD73, CD90, and CD105).

[0067] Serum-free reagent components In one embodiment, different reagents are used in the serum-free formulation.

[0068] (1) High glucose DMEM (Cytiva, catalog number SH30317) The components of the high glucose DMEM are shown in Table 2.

[0069] Table 2

[0070] (2) KOSR (Cytiva and Thermo Fisher, catalog number 10828028) It is a defined, serum-free preparation. Unlike FBS, the composition of KOSR is constant from batch to batch, so there is no need to test each batch before use.

[0071] KOSR is composed of amino acids (glycine, L-histidine, L-isoleucine, L-methionine, L-phenylalanine, L-proline, L-hydroxyproline, L-serine, L-threonine, L-tryptophan, L-tyrosine, L-valine), vitamins / antioxidants (thiamine, reduced glutathione, ascorbic acid 2-PO4), trace elements (Ag + , Al 3+ , Ba2+ , Cd 2+ , Co 2+ , Cr 3+ , Ge 4+ ,Se 4+ , Br − , I − , F − , Mn 2+ , Si 4+ , V 5+ , Mo 6+ , Ni 2+ , Rb + , Sn 2+ , Zr 4+ ) and proteins (transferrin (iron saturated), insulin, lipid-rich albumin (AlbuMAX)).

[0072] (3) GlutaMAX supplement (Gibco or Thermo Fisher, catalog number 35050079) GlutaMAX supplements are made from 200mM L-alanyl-L-glutamine dipeptide and 0.85% sodium chloride (NaCl).

[0073] (4) FGF-2 (Miltenyi Biotec, catalog number 130-093-842) The composition of the FGF-2 reagent is 100 ng / mL FGF-2 in sterile aqueous solution.

[0074] (5) TryPLE (Thermo Fisher, catalog number 12605010) The components of TrypLE are shown in Table 3.

[0075] Table 3

[0076] (6) ReseLR (Stemcell Technologies, catalog number 05872) This is a chemically defined, enzyme-free, animal component-free, cGMP reagent for the dissociation and passaging of hPSCs without the need for manual selection or scraping. The drug master file is available from Stemcell Technologies.

[0077] (7) DMEM / F-12 (Thermo Fisher, catalog number 10565018) This is a 1:1 mixture of DMEM and Ham's F-12.

[0078] (8) MEM Non-Essential Amino Acids (Thermo Fisher, catalog number 11140050) Contains non-essential amino acids such as glycine, L-alanine, L-asparagine, L-aspartic acid, L-glutamic acid, L-proline and L-serine.

[0079] (9) Penicillin / Streptomycin (Thermo Fisher, catalog number 15140122) The solution contained 10,000 units / mL of penicillin and 10,000 μg / mL of streptomycin.

[0080] (10) Essential 8 (Stemcell Technologies, catalog number 05990) TeSR TM -E8 TM It is a feeder-free, animal-free culture medium for human embryonic stem cells and human induced pluripotent stem cells.

[0081] Example 1 Human sub-totipotent stem cells (hPSCs) such as hiPSCs are differentiated into MSCs using serum-free media. Various academic protocols were compared and modified to be serum-free. For example, FBS was replaced by KOSR and trypsin was replaced by TrypLE. These hiPSC-derived MSCs were characterized for MSC marker expression, and Figures 3 to 6 show that these marker expression characterizations are comparable to primary MSCs. The use of serum-free reagents to differentiate hiPSCs into MSCs makes these cells clinically compatible and ultimately usable for downstream clinical applications.

[0082] Example 2 Compared with the use of FBS, the method of the present invention can generate more EBs on day 7 using a serum-free regimen. Figure 4As shown, the serum-free protocol in one embodiment of the present invention produces more EBs in greater numbers and more consistent sizes than EBs produced using FBS-containing medium. This difference can be observed as early as day 3 of the setup. The increase in the number of EBs and the consistency of size may be critical for initial scale-up work, as less starting material is needed to generate large numbers of MSCs.

[0083] Example 3 The cells cultured using the serum-free method of the present invention that meets clinical requirements have sustained MSC characteristics during long-term in vitro culture.

[0084] According to the criteria established by the International Society for Cellular Therapy (ISCT), ≥95% of the MSC population must express CD73, CD90, and CD105, and ≤2% of the population may express negative MSC markers such as CD45, CD34, CD14 or CD11b, CD79a or CD19, and HLA class II.

[0085] Here, the expression of MSC positive markers (CD73, CD90, CD105) and negative markers by MSCs generated by a serum-free differentiation protocol were compared with those generated using fetal bovine serum (FBS).

[0086] Figure 5 A to E show that hiPSC-derived MSCs generated by the present invention always have ≥95% of the population expressing positive MSC markers (CD73, CD90, CD105), and ≤2% of the population expressing negative MSC markers (CD34 and CD45). In contrast, in hiPSC-derived MSCs generated using FBS, the number expressing CD90 decreased significantly starting from passage 6. In hiPSC-derived MSCs generated using FBS, the fluctuation of the percentage of the population expressing negative MSC markers was also greater.

[0087] Example 4 Cells generated using serum-free medium exhibited more stable doubling rates and could be passaged for longer periods without losing the ability to become confluent within the 7-day passaging cycle.

[0088] like Figure 6 As shown in Figure 2, hiPSC-derived MSCs generated using FBS have inconsistent doubling rates, which may lead to unstable passage times due to fluctuations in growth rate. In contrast, hiPSC-derived MSCs generated using the serum-free protocol of the present invention have consistent doubling rates (approximately 1 population doubling rate per passage), which prevents fluctuations in growth rate between batches.

[0089] Example 5 In order to determine whether there are residual sub-totipotent stem cells in the hPSC-derived MSCs produced according to the serum-free protocol of the present invention, the hPSC-derived MSCs were injected into the gastrocnemius muscle of immunodeficient NOD SCID mice, and the formation of teratoma was observed ( Figure 7 In the positive control injected with undifferentiated hPSCs ( Figure 7 a), teratoma formation was observed as early as eight weeks after injection. However, after injection of hPSC-derived MSCs in NOD SCID mice, no obvious teratoma was observed at the injection site ( Figure 7 b).

[0090] In vivo teratoma assays demonstrated that the positive control undifferentiated hiPSC line i70b is sub-totipotent and can generate derivatives of the three germ layers corresponding to ectoderm (neuronal rosette), mesoderm (cartilage), and finally endoderm (gut-like epithelium). Figure 8 a, b, c). However, gastrocnemius tissue injected with hiPSC-derived MSCs only showed normal striated muscle morphology ( Figure 8 d and e), which indicates that there are no residual sub-totipotent stem cells in the hiPSC-derived MSCs. Therefore, the serum-free differentiation protocol according to the embodiment of the present invention can effectively generate MSCs without residual tumorigenicity and undifferentiated hPSCs.

[0091] i70b was used as a starting hiPSC cell source for directed differentiation into MSCs. In vivo teratoma assays showed that i70b was sub-totipotent and could produce teratomas within eight weeks after injection. In contrast, no obvious tumors were seen in NOD SCID mice transplanted with hiPSC-derived MSCs produced using a serum-free regimen according to an embodiment of the present invention. NOD SCID mice transplanted with hiPSC-derived MSCs remained tumor-free for 20 weeks and were ultimately euthanized according to the IACUC protocol after 20 weeks. Subsequently, histological analysis of tissue sites injected with the respective cell lines also confirmed that only undifferentiated i70b cells were sub-totipotent. Even 20 weeks after injection, the gastrocnemius tissue injected with hiPSC-derived MSCs contained only normal mouse striated muscle cells, with no obvious signs of teratoma cells. This shows that hPSC-derived MSCs generated using a serum-free regimen according to an embodiment of the present invention can be safely transplanted.

[0092] Furthermore, according to Ahfeldt, T. et al., Programming human pluripotent stem cells into white and brown adipocytes. Nat Cell Biol, 2012. 14(2): p. 209-19. Experiments have also been conducted to direct differentiation of i70b into MSCs using a conventional serum-containing protocol. Histological analysis showed that hiPSC-derived MSCs produced by a serum-free protocol according to an embodiment of the present invention were comparable to MSCs produced by a serum-containing protocol, with no residual sub-totipotent cells detected by in vivo teratoma assays.

[0093] In vivo teratoma detection method Collect 1×10 6 i70b cells or i70b-derived MSCs were used for in vivo teratoma assays. At the time of collection, i70b-derived MSCs were cultured for 6 passages. The cell pellets were resuspended in 100 to 150 μl of Matrigel. Then, the respective prepared cell lines were injected intramuscularly into the gastrocnemius muscle of 6-week-old NOD SCID mice. Mice injected with i70b developed teratomas within 8 weeks after injection and were euthanized. Meanwhile, mice injected with i70b-derived MSCs showed no tumors by palpation or autopsy. These mice were euthanized only after 20 weeks. To further verify teratoma formation, gastrocnemius muscles of all mice were excised, fixed, paraffin-sectioned, and stained with hematoxylin and eosin (H&E) for subsequent analysis. All animal experiments were performed in accordance with relevant ethical regulations and were approved by A*STAR (approval number: IACUC 211660).

[0094] Although the embodiments of the present invention have been particularly shown and described with reference to specific embodiments, it should be understood by those skilled in the art that various changes may be made to the form and details of the present invention without departing from the scope of the present invention as defined by the appended claims. Therefore, the scope of the present invention is indicated by the appended claims, and all changes within the meaning and equivalent range of the claims should be included therein.

Claims

1. A method for differentiating subtotipotent stem cells into multipotent mesenchymal stem cells (MSCs), the method comprising the following steps: Step (a) was performed in a medium containing Dulbecco's modified Eagle's medium (DMEM), KnockOut TM Subtotipotent stem cells were cultured in the first medium of serum replacement (KOSR) and GlutaMAX to form embryoid bodies (EBs); Step (b), replacing the first culture medium with a second culture medium comprising DMEM, KOSR and GlutaMAX, and culturing EBs in the second culture medium to differentiate the EBs into a MSC progenitor cell population; as well as Step (c), replacing the second culture medium with a third culture medium comprising DMEM, KOSR, GlutaMAX and fibroblast growth factor 2 (FGF-2), and culturing the MSC progenitor cell population in the third culture medium to differentiate the MSC progenitor cell population into MSCs; Among them, the first culture medium, the second culture medium and the third culture medium are serum-free culture media.

2. The method according to claim 1, further comprising the step of dissociating the sub-totipotent stem cells into clumps using the reagent ReLeSR before step (a), or dissociating the sub-totipotent stem cells into single cells using the reagent TrypLE before step (a).

3. The method according to claim 1 or 2, wherein the EBs and MSC progenitor cell populations are cultured on a plate coated with gelatin. 4 . The method according to claim 1 , wherein the MSC progenitor cell population is dissociated using TrypLE prior to step (c). 5 . The method according to claim 1 , further comprising passaging the MSCs obtained in step (c) in a third culture medium to maintain the multipotent state of the MSCs. 6 . The method according to claim 5 , wherein after being passaged 15 times using the third culture medium, MSCs are maintained in a multipotent state.

7. The method according to claim 5 or 6, wherein MSCs are dissociated with TrypLE before each passaging using the third culture medium.

8. The method according to any one of claims 1 to 7, wherein the sub-totipotent stem cells are human induced sub-totipotent stem cells (hiPSCs).

9. The method according to any one of claims 1 to 8, wherein: After step (c), at least 95% of the obtained cell population are MSCs.

10. A serum-free composition for differentiating subtotipotent stem cells into multipotent mesenchymal stem cells (MSCs), the composition comprising Dulbecco's modified Eagle's medium (DMEM), KnockOut TM Serum Replacement (KOSR) and GlutaMAX. The serum-free composition according to claim 10 , further comprising fibroblast growth factor 2 (FGF-2).

12. The serum-free composition according to claim 10, wherein the concentration of DMEM is about 74% to about 89% (v / v), the concentration of KOSR is about 10% to about 25% (v / v), and the concentration of GlutaMAX is about 1% (v / v).

13. The serum-free composition according to claim 10, wherein the concentration of DMEM is about 79% to about 84% (v / v), the concentration of KOSR is about 15% to about 20% (v / v), and the concentration of GlutaMAX is about 1% (v / v).

14. The serum-free composition of claim 10, wherein the concentration of DMEM is about 79% to about 89% (v / v), the concentration of KOSR is about 10% to about 20% (v / v), and the concentration of GlutaMAX is about 1% (v / v).

15. The serum-free composition of claim 11, wherein the concentration of DMEM is about 74% to about 84% (v / v), the concentration of KOSR is about 15% to about 25% (v / v), the concentration of GlutaMAX is about 1% (v / v), and the concentration of FGF-2 is about 2.5 ng / mL to 5 ng / mL.

16. The serum-free composition according to claim 15, wherein The concentration of FGF-2 was about 2.5 ng / mL. 17 . The serum-free composition according to claim 10 , wherein the sub-totipotent stem cells are human induced pluripotent stem cells (hiPSCs).

18. A mesenchymal stem cell population, obtainable by the method according to any one of claims 1 to 9.

19. The mesenchymal stem cell population of claim 18, wherein at least 95% of the cells of the mesenchymal stem cell population express CD73, CD90 and CD105. 20 . The mesenchymal stem cell population according to claim 18 or 19 , wherein 2% or less of the cells of the mesenchymal stem cell population express CD34 and CD45.

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