A method for differentiating human pluripotent stem cells into mesenchymal stem cells

Abstract

The present invention discloses a method for differentiating human pluripotent stem cells into mesenchymal stem cells. The method can significantly shorten the culture period of obtaining MSCs by using an induction method of SIK and its pathway-related inhibitors, and the obtained MSCs are phenotypically mature and of high quality.

Description

Technical Field

[0001] The present invention relates to the technical field of stem cell induction differentiation, and in particular to a method for differentiating human pluripotent stem cells into mesenchymal stem cells. Background Art

[0002] Mesenchymal stem cells (MSC) are a type of specialized stem cells discovered in the bone marrow in 1976. Studies have shown that mesenchymal stem cells originate from the mesoderm and ectoderm in the early stages of embryonic development and are distributed in the umbilical cord, placenta, fat, bone marrow and other tissues in the human or animal body. They have the potential to differentiate into three lines of fat, bone and cartilage, and exert immune regulation through cell-to-cell interactions and the production of cytokines; trophoblast cells may exist in all tissues and support the survival and growth of a variety of cells; secrete related anti-inflammatory molecules and anti-apoptotic molecules to promote tissue repair. Although mesenchymal stem cells have shown unique advantages in injury repair, immune regulation, anti-aging, etc., MSCs derived from human tissues are difficult to mass produce, have large differences in quality between production batches, and are difficult to ensure the effectiveness and quality of MSCs from different individuals. With the advent of in vitro reprogramming technology for human pluripotent stem cells (hPSCs), the use of human induced pluripotent stem cells to differentiate into MSCs has gradually become the preferred solution for large-scale production of MSCs.

[0003] hPSCs include induced pluripotent stem cells (iPSCs) produced by reprogramming human terminally differentiated cells, and embryonic stem cells (ESCs) derived from early human embryos. hPSCs have stable self-renewal ability, can maintain normal karyotype and developmental pluripotency, and have strong proliferation ability. Under specific conditions, they can be directed to differentiate into MSCs. Due to the stability of their proliferation and expansion, they can greatly reduce the batch-to-batch differences in MSC production and provide a stable and sustainable cell source.

[0004] The classic differentiation route of PSC-derived MSCs is to convert PSCs into mesoderm cells by inhibiting the TGF-β signaling pathway and then induce them into MSCs. However, this route takes a long time to obtain MSCs, and they also need to be cultured after flow sorting through specific phenotypes, and the process is relatively complicated. In addition, the inhibition of a single pathway has certain defects. Since the sequence of lineage branch points and the role of induction and other inhibitory signals at each developmental stage are still not fully understood, traditional mesoderm differentiation protocols take weeks to months and may produce heterogeneous cells that include cell types of the desired lineage subsets and other contaminating lineages. The differentiation process is long and not precise enough.

[0005] SIK (Salt-inducible kinases) belongs to the AMP-activated protein kinase (AMPK) family, and its functions mainly involve regulating physiological processes related to energy responses, such as gluconeogenesis and lipid metabolism. Summary of the invention

[0006] The present invention aims to solve at least one of the above-mentioned technical problems existing in the prior art. To this end, the object of the present invention is to provide a method for promoting the differentiation of human pluripotent stem cells (hPSC) into mesenchymal stem cells, by using SIK inhibitors to promote the differentiation of hPSC into mesoderm, and then induce differentiation into MSCs. The method shortens the culture period for obtaining MSCs by using SIK inhibitors, and the obtained MSCs are phenotypically mature, high in quality, easy to operate throughout the process, and no flow screening is required. Compared with the original differentiation method, it is faster and can obtain qualified MSC-P0 generation cells in a shorter culture period.

[0007] In a first aspect of the present invention, a method for differentiating human pluripotent stem cells into mesenchymal stem cells is provided, the method comprising the following steps:

[0008] Mesenchymal stem cells are obtained by differentiating and culturing human pluripotent stem cells using differentiation medium;

[0009] Wherein, the differentiation culture medium contains SIK and its pathway-related inhibitors.

[0010] In some embodiments of the present invention, a mesenchymal stem cell maintenance medium is further used for maintenance culture after cell differentiation culture.

[0011] In some embodiments of the present invention, the differentiation medium is a human pluripotent stem cell differentiation medium.

[0012] In some embodiments of the present invention, the human pluripotent stem cell differentiation medium includes but is not limited to Essential 6 medium, Essential 7 medium, differentiation basal medium containing N2B27, hPLWⅨ-BM1 medium, hPLWⅨ-plus-BM1 medium, hPLWⅩ-BM1 medium, hPLWXI-BM1 medium and BioCISO-BM1 medium.

[0013] In some embodiments of the present invention, the mesenchymal stem cell maintenance medium includes but is not limited to MEM medium, a-MEM medium, DMEM medium, DMEM / F12 medium and other basic maintenance medium.

[0014] In some embodiments of the present invention, mesenchymal stem cell maintenance medium may be further added during the differentiation culture process.

[0015] In some embodiments of the present invention, the SIK and its pathway-related inhibitors include at least one of YKL-06-062, MRT199665, HG-9-91-01, YKL-06-061, WH-4-025, SIK2-IN-1, GLPG3970, MRIA9, YKL-05-099, ARN-3236, SIKs-IN-1, Pterosin B or MR22.

[0016] In some embodiments of the present invention, the SIK and its pathway-related inhibitor is one of YKL-06-062, MRT199665, HG-9-91-01, YKL-06-061, WH-4-025, SIK2-IN-1, GLPG3970, MRIA9, YKL-05-099, ARN-3236, SIKs-IN-1, Pterosin B or MR22.

[0017] In some embodiments of the present invention, the working concentration of YKL-06-062 is 0.01-20 μM.

[0018] In some embodiments of the present invention, the working concentration of MRT199665 is 0.01-10 μM.

[0019] In some embodiments of the present invention, the working concentration of HG-9-91-01 is 0.01-10 μM.

[0020] In some embodiments of the present invention, the working concentration of YKL-06-061 is 0.1-20 μM.

[0021] In some embodiments of the present invention, the working concentration of WH-4-025 is 0.1-20 μM.

[0022] In some embodiments of the present invention, the working concentration of SIK2-IN-1 is 0.01-10 μM.

[0023] In some embodiments of the present invention, the working concentration of GLPG3970 is 0.1-10 μM.

[0024] In some embodiments of the present invention, the working concentration of MRIA9 is 0.1-10 μM.

[0025] In some embodiments of the present invention, the working concentration of YKL-05-099 is 0.1-10 μM.

[0026] In some embodiments of the present invention, the working concentration of ARN-3236 is 0.01-10 μM.

[0027] In some embodiments of the present invention, the working concentration of SIKs-IN-1 is 0.01-10 μM.

[0028] In some embodiments of the present invention, the working concentration of Pterosin B is 0.01-10 μM.

[0029] In some embodiments of the present invention, the working concentration of MR22 is 0.1-10 μM.

[0030] In some embodiments of the present invention, the working concentration of YKL-06-062 is 0.1 μM.

[0031] In some embodiments of the present invention, the working concentration of MRT199665 is 0.01 μM.

[0032] In some embodiments of the present invention, the working concentration of HG-9-91-01 is 0.08 μM.

[0033] In some embodiments of the present invention, the working concentration of YKL-06-061 is 0.2 μM.

[0034] In some embodiments of the present invention, the working concentration of WH-4-025 is 2 μM.

[0035] In some embodiments of the present invention, the working concentration of SIK2-IN-1 is 5 μM.

[0036] In some embodiments of the present invention, the working concentration of GLPG3970 is 0.5 μM.

[0037] In some embodiments of the present invention, the working concentration of MRIA9 is 0.8 μM.

[0038] In some embodiments of the present invention, the working concentration of YKL-05-099 is 3 μM.

[0039] In some embodiments of the present invention, the working concentration of ARN-3236 is 0.05 μM.

[0040] In some embodiments of the present invention, the working concentration of SIKs-IN-1 is 0.08 μM.

[0041] In some embodiments of the present invention, the working concentration of Pterosin B is 0.4 μM.

[0042] In some embodiments of the present invention, the working concentration of MR22 is 0.8 μM.

[0043] In some embodiments of the present invention, the culture period of the differentiation culture is 3-30 days.

[0044] In some embodiments of the present invention, the culture period of the maintenance culture is 2-6 days.

[0045] In a second aspect of the present invention, there is provided use of SIK and its pathway-related inhibitors in inducing differentiation of human pluripotent stem cells to generate mesenchymal stem cells.

[0046] The beneficial effects of the present invention are:

[0047] The present invention provides a method for inducing human pluripotent stem cells to produce mesenchymal stem cells using a culture medium containing SIK and its pathway-related inhibitors. Compared with the classical differentiation route of inducing human pluripotent stem cells to produce mesenchymal stem cells by inhibiting the TGF-β signaling pathway, the induction method using SIK and its pathway-related inhibitors in the present invention can significantly shorten the culture period for obtaining MSCs, and the obtained MSCs are phenotypically mature and of high quality. According to the results of Example 1 and Comparative Example 1 of the present invention, the culture period for obtaining MSCs with qualified phenotypes using SIK and its pathway-related inhibitors is 5-36 days, while the culture period for obtaining MSCs with qualified phenotypes using TGF-β inhibitors is 40-50 days. It can be seen that the use of SIK and its pathway-related inhibitors can significantly shorten the culture period for obtaining MSCs. In addition, the full process of the present invention is simple to operate, and qualified MSCs can be obtained without flow screening. It is faster than the original differentiation method using TGF-β inhibitors, and qualified MSC-P0 generation cells can be obtained under a shorter culture period. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] Figure 1 For undifferentiated hPSCs.

[0049] Figure 2 The cells are induced using a differentiation medium containing SIK and its pathway-related inhibitors.

[0050] Figure 3 The MSC-P0 cells were obtained by inducing with a differentiation medium including SIK and its pathway-related inhibitors and then culturing with a mesenchymal stem cell maintenance medium (experimental groups 1-13), and by inducing with a differentiation medium including the TGF-β inhibitor SB431542 (control group).

[0051] Figure 4 These are the karyotype detection results of MSC-P0 cells obtained in Example 1 and Comparative Example 1. DETAILED DESCRIPTION

[0052] The present invention is further described in detail below by specific examples. Unless otherwise specified, the raw materials, reagents or devices used in the examples and comparative examples can be obtained from conventional commercial sources or can be obtained by prior art methods. Unless otherwise specified, the experiments or test methods are conventional methods in the art.

[0053] Example 1: Using SIK inhibitors to obtain phenotypically qualified MSCs

[0054] hPSCs (future Homo sapiens) were seeded on P100 dishes (Thermo, 150466) at a density of 10,000-30,000 cells / cm 2 , undifferentiated hPSCs such as Figure 1 After the cells adhered to the wall, the original culture medium was discarded and 10 mL of differentiation medium was added. The differentiation medium was a human pluripotent stem cell culture medium (Essential6 medium (A1516401)) containing SIK and its pathway-related inhibitors. The types and working concentrations of SIK and its pathway-related inhibitors used are shown in Table 1. The medium was changed every other day and cultured for 3-30 days depending on the growth status of the cells. Figure 1 As shown, cells obtained by culturing with differentiation medium are as follows Figure 2 shown.

[0055] Table 1: Types and working concentrations of SIK and its pathway-related inhibitors

[0056]

[0057]

[0058] The cells were digested to prepare a single cell suspension, which was inoculated into a new P100 culture dish and cultured for 2-6 days using a mesenchymal stem cell maintenance medium (DMEM / F12 medium). The medium was changed every day. When the cells grew to 90%, MSC-P0 cells were obtained. Figure 3 shown.

[0059] In this example, the culture period of MSCs with qualified phenotypes obtained by using SIK and its pathway-related inhibitors is 5-36 days.

[0060] Comparative Example 1: Obtaining Phenotypically Qualified MSCs Using TGF-β Inhibitors

[0061] hPSCs were seeded on P100 dishes (Thermo, 150466) coated with extracellular matrix at a seeding density of 10,000-30,000 cells / cm2 After the cells adhered to the wall, the original culture medium was discarded and 10 mL of mTeSR1 culture medium (stemcell, 85850) containing 12 μM SB431542 was added. The medium was changed every other day and the cells were subcultured according to their growth conditions and cultured for 35-40 days.

[0062] When the cells grow to 85-90%, digest the cells to make a single cell suspension, inoculate it into a new extracellular matrix-coated P100 culture dish, and continue to culture for 5-10 days using mesenchymal stem cell maintenance medium (DMEM / F12 medium), and change the medium every other day. When the cells grow to 90%, MSC-P0 cells are obtained. Figure 3 (control group) as shown.

[0063] In this comparative example, the culture period of MSCs with qualified phenotypes obtained by using TGF-β inhibitors was 40-50 days.

[0064] Example 2: Identification results of MSCs

[0065] Karyotype analysis

[0066] The MSC-P0 cells obtained in Example 1 and Comparative Example 1 (control group) were added to fresh culture medium containing colchicine and continued to be cultured at 37°C for 2 hours. The cells were then digested, centrifuged, resuspended, and gently blown evenly. 3 mL of hypotonic solution (McLean, P816348) was added dropwise, the cells were evenly distributed in the suspension, and then 5-7 mL of hypotonic solution was added dropwise, placed in a 37°C water bath for 15 minutes, and then about 200 μL of freshly prepared fixative (methanol: glacial acetic acid (v / v) = 5:3) was added, pre-fixed for 2-3 minutes; centrifuged at 800 rpm for 8 minutes. The supernatant was removed, and the fixative was added to repeat the fixation of the cells 2-3 times, and the cells were gently blown away with a gun.

[0067] Pipette the cell suspension onto the pre-cooled slide, bake it in an oven at 70℃ for 2 hours, then place the slide in trypsin working solution, immediately place the digested slide in saline and swing it back and forth several times, add Giemsa working solution, cover it, and stain it for 5 minutes; rinse with tap water to terminate the staining, and place it in a 35℃ oven to dry. Observe and take pictures using a microscope, and the results are as follows: Figure 4 The results of the karyotype test showed that the cell chromosomes had not mutated and the bands were normal.

[0068] 2.2. Flow cytometry

[0069] The MSC-P0 cells obtained in Example 1 and Comparative Example 1 were digested and collected in a centrifuge tube for centrifugation and horizontally transferred at 300g for 5min. The supernatant was discarded and resuspended with PBS, horizontally transferred at 300g, and centrifuged for 5min. The supernatant was discarded, resuspended with PBS containing 1% FBS, and dispensed into EP tubes, 150μL per tube. In a light-proof environment, 3μL positive-cocktail and 3μL negative-cocktail (BD, human mesenchymal stem cell analysis and detection kit, 562245) were added to each tube in the experimental group, and 3μL positive-iso and 3μL negative-iso (BD, human mesenchymal stem cell analysis and detection kit, 562245) were added to the negative control tube, incubated at room temperature for 30-45min, washed twice with PBS, and horizontally transferred at 300g for centrifugation for 5min. 200μL PBS was resuspended and analyzed by flow cytometry.

[0070] The obtained cells were subjected to flow cytometry, and the results showed that the experimental group cells highly expressed CD90, CD73 and CD105 molecules, but basically did not express CD34, CD45, CD11b, CD19 and HLA-DR molecules, judging that the experimental group cells successfully differentiated into MSCs. The molecular expressions of the cells in each experimental group and control group are shown in Table 2 below:

[0071] Table 2: Expression of positive and negative molecules in induced MSCs

[0072]

[0073]

[0074] As shown in Table 2 above, compared with MSCs induced by TGF-β inhibitors, MSCs induced by SIK and its pathway-related inhibitors have a higher expression rate of positive molecules, especially CD105 and CD90, indicating that SIK and its pathway-related inhibitors have a better induction effect on MSCs.

[0075] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A method for differentiating human pluripotent stem cells into mesenchymal stem cells, the method comprising the following steps: Mesenchymal stem cells are obtained by differentiating and culturing human pluripotent stem cells using differentiation medium; in, The differentiation medium contains SIK and its pathway-related inhibitors; After differentiation culture, mesenchymal stem cell maintenance medium is also used for maintenance culture; The differentiation medium is a human pluripotent stem cell differentiation medium; The SIK and its pathway-related inhibitor is at least one of YKL-06-062, MRT199665, HG-9-91-01, YKL-06-061, WH-4-025, SIK2-IN-1, GLPG3970, MRIA9, YKL-05-099, ARN-3236, SIKs-IN-1, Pterosin B or MR22.

2. The method according to claim 1, characterized in that The human pluripotent stem cell differentiation medium includes Essential 6 medium, Essential 7 medium, differentiation basal medium containing N2B27, hPLWⅨ-BM1 medium, hPLWⅨ-plus-BM1 medium, hPLWⅩ-BM1 medium, hPLWXI-BM1 medium and BioCISO-BM1 medium.

3. The method according to claim 1, characterized in that The mesenchymal stem cell maintenance medium includes MEM medium, a-MEM medium, DMEM medium and DMEM / F12 medium.

4. The method according to claim 1, characterized in that The working concentration of YKL-06-062 is 0.01-20 μM; the working concentration of MRT199665 is 0.01-10 μM; the working concentration of HG-9-91-01 is 0.01-10 μM; the working concentration of YKL-06-061 is 0.1-20 μM; the working concentration of WH-4-025 is 0.1-20 μM; the working concentration of SIK2-IN-1 is 0.01-10 μM; the working concentration of GLPG3970 is 0.1-10 μM; the working concentration of MRIA9 is 0.1-10 μM; the working concentration of YKL-05-099 is 0.1-10 μM; the working concentration of ARN-3236 is 0.01-10 μM; the working concentration of SIKs-IN-1 is 0.01-10 μM; the working concentration of Pterosin The working concentration of B is 0.01-10 μM; or the working concentration of MR22 is 0.1-10 μM.

5. The method according to claim 1, characterized in that The culture period of the differentiation culture is 3-30 days.

6. The method according to claim 1, characterized in that The culture period of the maintenance culture is 2-6 days.

7. Use of SIK and its pathway-related inhibitors in inducing differentiation of human pluripotent stem cells to produce mesenchymal stem cells; the SIK and its pathway-related inhibitors are at least one of YKL-06-062, MRT199665, HG-9-91-01, YKL-06-061, WH-4-025, SIK2-IN-1, GLPG3970, MRIA9, YKL-05-099, ARN-3236, SIKs-IN-1, Pterosin B or MR22.

Images