Differential culture medium composition and method for preparing mesenchymal stem cells by inducing stem cells to differentiate in body segment direction
Through the combination of differentiation culture media and step-by-step induction methods, the problem of unstable differentiation efficiency of pluripotent stem cells into mesenchymal stem cells was solved, and efficient and high-purity mesenchymal stem cell preparation was achieved, which was applied in fields such as regenerative medicine and immunotherapy.
Patent Information
- Application Number
- CN202510954407.6
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-07-11
- Publication Date
- 2025-09-30
AI Technical Summary
In the existing technology, the efficiency of inducing pluripotent stem cells to differentiate into mesenchymal stem cells is unstable, and the differentiation process is prone to bias towards other types of cells, making it difficult to achieve efficient and precise differentiation control.
Using a specifically formulated differentiation medium combination, including mediums A, B, C, and D, pluripotent stem cells are gradually induced to differentiate into paraxial mesoderm and somites, ultimately yielding highly purified mesenchymal stem cells. The medium combination contains small molecule compounds such as Activin A, CHIR-99021, FGF2, and SB431542. IMDM and F12 are used as basal media, supplemented with chemically defined lipids and insulin-transferrin-selenium.
It achieves stable and efficient differentiation from pluripotent stem cells to mesenchymal stem cells, provides a high-purity cell source, improves the safety and effectiveness of clinical applications, and is suitable for fields such as regenerative medicine, tissue engineering, and immunotherapy.
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Figure CN120718845A_ABST
Abstract
Description
Technical Field
[0001] The present disclosure relates to the biological field, and in particular to a differentiation culture medium combination and a method for inducing stem cells to differentiate in the somite direction to prepare mesenchymal stem cells. Background Art
[0002] Mesenchymal stem cells (MSCs) are multipotent stem cells that possess all the common properties of stem cells, namely, self-renewal and multidirectional differentiation. MSCs can differentiate into different cell types, such as osteocytes, chondrocytes, and adipocytes, and therefore have broad application prospects in regenerative medicine, tissue repair, immunomodulation, and anti-inflammatory therapy. MSCs not only directly promote the repair of damaged tissues through differentiation but also modulate the microenvironment through the secretion of cytokines and exosomes, further accelerating tissue regeneration and functional recovery. Therefore, MSCs are considered to have great potential in clinical treatment, particularly in the treatment of degenerative diseases, wound repair, and immune-related disorders.
[0003] Induced pluripotent stem cells (iPSCs) are stem cells that are genetically reprogrammed to become pluripotent. iPSCs possess pluripotency similar to that of embryonic stem cells (ESCs). Differentiation is the result of selective gene expression and does not alter the genetic material, while reprogramming is, in a sense, a reversal of differentiation. Unlike classic embryonic stem cell technology and somatic cell nuclear transfer, iPSC technology does not use embryonic cells or oocytes, thus raising no ethical concerns.
[0004] Although iPSC-derived MSCs can theoretically address the heterogeneity problem of traditional MSCs, the practical differentiation of iPSCs into MSCs still faces several challenges. First, the differentiation efficiency is unstable, and different culture conditions and differentiation methods may lead to different results. Second, although iPSCs are pluripotent, their differentiation process may favor other cell types, such as osteoblasts or chondrocytes. Therefore, how to precisely regulate the differentiation route to ensure efficient differentiation of iPSCs into MSCs remains a difficult problem in technology development.
[0005] Public content To address the problems of the prior art, the present disclosure provides a differentiation medium combination and a method for inducing stem cells to differentiate along somite lines to produce mesenchymal stem cells. The technical solution is as follows: In one aspect, the present disclosure provides a differentiation medium combination, comprising: medium A, medium B, medium C, and medium D.
[0006] The culture medium A comprises: a basal culture medium and a first small molecule compound combination, wherein the first small molecule compound combination comprises: Activin A, CHIR-99021, FGF2 and PIK90; The culture medium B comprises: the basal culture medium and a second small molecule compound combination, wherein the second small molecule compound combination comprises: SB431542, CHIR-99021, FGF2 and Dorsomorphin; The culture medium C comprises: the basal culture medium and a third small molecule compound combination, wherein the third small molecule compound combination comprises: SB431542, Dorsomorphin, Wnt-C59 and PD173074; The culture medium D includes: the basal culture medium and a fourth small molecule compound combination, wherein the fourth small molecule compound combination includes SB590885.
[0007] Specifically, the basic culture medium includes: IMDM, F12, chemically defined lipid, insulin-transferrin-selenium and 1-thioglycerol.
[0008] Specifically, in the culture medium A, the volume ratio of the IMDM and the F12 is 3:(1~9), the chemically defined lipid and the insulin-transferrin-selenium each account for 0.5%~2% of the total volume of the basal culture medium, the volume ratio of the IMDM and the insulin-transferrin-selenium is 49:1, the final concentration of the 1-thioglycerol is 225μM~600μM, the final concentration of the Activin A is 10ng / mL~50ng / mL, the final concentration of the CHIR-99021 is 1μM~6μM, the final concentration of the FGF2 is 10ng / mL~50ng / mL, and the final concentration of the PIK90 is 50μM~200μM.
[0009] Specifically, in the culture medium B, the final concentration of the SB431542 is 1 μM to 6 μM, the final concentration of the CHIR-99021 is 1 μM to 6 μM, the final concentration of the FGF2 is 10 ng / mL to 50 ng / mL, and the final concentration of the Dorsomorphin is 1 μM to 6 μM.
[0010] Specifically, in the culture medium C, the final concentration of the SB431542 is 1 μM to 6 μM, the final concentration of the Dorsomorphin is 0.5 μM to 6 μM, the final concentration of the Wnt-C59 is 0.5 μM to 4 μM, and the final concentration of the PD173074 is 50 nM to 200 nM.
[0011] Specifically, the final concentration of SB590885 is 1 μM to 10 μM.
[0012] In another aspect, the present disclosure provides a method for preparing mesenchymal stem cells by inducing stem cells to differentiate in the somite direction using the above-mentioned differentiation medium combination, the method comprising: Inducing differentiation of pluripotent stem cells or embryonic stem cells using the culture medium A to obtain a first product; Using the culture medium B to induce differentiation of the first product to obtain paraxial mesoderm; Using the culture medium C to induce differentiation of the paraxial mesoderm to obtain somites; The culture medium D is used to induce differentiation of the somites, and then amplified and cultured to obtain mesenchymal stem cells.
[0013] The beneficial effects of the technical solutions provided by the embodiments of the present disclosure are as follows: the present invention provides a differentiation medium combination and a method for inducing stem cell differentiation to prepare mesenchymal stem cells. The provided differentiation medium combination and induction method can prepare mesenchymal stem cells from pluripotent stem cells or embryonic stem cells, which can be widely used in the fields of regenerative medicine, tissue engineering, immunotherapy, etc. High-purity mesenchymal stem cells can be used in a variety of clinical applications such as tissue repair, osteoarthritis treatment, immunomodulation, and anti-aging. In particular, the technology of inducing the differentiation of mesenchymal stem cells from somites can provide a more stable and standardized cell source for clinical practice, thereby improving the safety and effectiveness of clinical treatment. BRIEF DESCRIPTION OF THE DRAWINGS
[0014] In order to more clearly illustrate the technical solutions in the embodiments of the present disclosure, the following briefly introduces the drawings required for use in the description of the embodiments. Obviously, the drawings described below are only some embodiments of the present disclosure. For ordinary technicians in this field, other drawings can be obtained based on these drawings without any creative work.
[0015] Figure 1 This is a cell morphology diagram at differentiation D3 provided in Example 1 of the present disclosure; Figure 2 This is a morphological diagram of the P2 generation iMSC provided in Example 1 of the present disclosure; Figure 3 This is a morphological diagram of the P4 generation iMSC provided in Example 1 of the present disclosure; Figure 4 This is a flow cytometric graph of CD73 expression in iMSCs provided in Example 1 of the present disclosure; Figure 5 This is a flow cytometric graph of CD90 expression by iMSCs provided in Example 1 of the present disclosure; Figure 6This is a flow cytometric graph of CD105 expression in iMSCs provided in Example 1 of the present disclosure. DETAILED DESCRIPTION
[0016] In order to make the objectives, technical solutions and advantages of the present disclosure more clear, the embodiments of the present disclosure will be further described in detail below with reference to the accompanying drawings.
[0017] Example 1 The present disclosure provides a differentiation medium combination, which includes: medium A, medium B, medium C and medium D, wherein medium A includes: a basal medium and a first small molecule compound combination, the basal medium includes: IMDM, F12, chemically defined lipid (lipid concentrate), insulin-transferrin-selenium, and 1-thioglycerol, the volume ratio of IMDM to F12 is 1:1, the chemically defined lipid and insulin-transferrin-selenium each account for 1% of the total volume of the basal medium, the volume ratio of IMDM to insulin-transferrin-selenium is 49:1, the final concentration of 1-thioglycerol is 450 μM, and the first small molecule compound combination includes: Activin A (purchased from PeproTech), CHIR-99021 (purchased from MCE), FGF2 (purchased from ORIGENE), and PIK90 (purchased from MCE), Activin The final concentration of medium A was 30 ng / mL, the final concentration of CHIR-99021 was 4 μM, the final concentration of FGF2 was 20 ng / mL, and the final concentration of PIK90 was 100 μM. The formula of the basal medium of medium A is shown in Table 1.
[0018] Table 1 is the formula of the basal culture medium
[0019] Specifically, culture medium B includes: the same basal culture medium as culture medium A provided in this embodiment and a second small molecule compound combination, the second small molecule compound combination includes: SB431542, CHIR-99021, FGF2 and Dorsomorphin, the final concentration of SB431542 is 2 μM, the final concentration of CHIR-99021 is 3 μM, the final concentration of FGF2 is 20 ng / mL, and the final concentration of Dorsomorphin is 4 μM.
[0020] Specifically, culture medium C includes: the same basal culture medium as culture medium A provided in this embodiment and a third small molecule compound combination, the third small molecule compound combination includes: SB431542, Dorsomorphin, Wnt-C59 and PD173074, the final concentration of SB431542 is 2 μM, the final concentration of Dorsomorphin is 4 μM, the final concentration of Wnt-C59 is 1 μM, and the final concentration of PD173074 is 100 nM.
[0021] Specifically, culture medium D includes: a basal culture medium identical to culture medium A provided in this embodiment and a fourth small molecule compound combination, wherein the fourth small molecule compound combination includes SB590885, and the final concentration of SB590885 is 2 μM.
[0022] The present disclosure also provides a method for preparing mesenchymal stem cells by inducing stem cells to differentiate in the somite direction using a combination of differentiation mediums, the method comprising: Induced pluripotent stem cells (iPSCs) were differentiated using medium A to obtain a first product with primitive streak characteristics; The first product was induced to differentiate using medium B to obtain the paraxial mesoderm; Medium C was used to induce paraxial mesoderm differentiation and obtain somites; Culture medium D was used to induce somite differentiation and expansion culture, and then expansion culture was performed to obtain mesenchymal stem cells.
[0023] D0: Gently aspirate the iPSC culture medium E8 (purchased from Gibco) in the culture wells of the culture plate, gently wash the culture wells once with 1 mL of calcium- and magnesium-free DPBS (Dulbecco's Phosphate-Buffered Saline) per well, then add 2 mL of culture medium A to each well. Return the culture plate to the incubator for induction culture for 1 day to differentiate into the first product with primitive streak characteristics.
[0024] D1: Gently aspirate the culture medium in the culture wells, gently wash each well once with 1 mL of DPBS without calcium and magnesium, add 2 mL of culture medium B to each well, return the culture plate to the incubator and induce culture for 1 day to differentiate into paraxial mesoderm.
[0025] D2: Gently aspirate the culture medium in the culture wells, gently wash each well once with 1 mL of DPBS without calcium and magnesium, add 2 mL of culture medium C to each well, and return the culture plate to the incubator for induction culture for 1 day to differentiate into somites.
[0026] D3~D14: Gently aspirate the culture medium in the culture wells, gently wash each well with 1 mL of DPBS without calcium and magnesium, add 2 mL of culture medium D to each well, return the culture plate to the incubator for induction culture, change the medium every day, and continue the culture from D3 to D14 for a total of 11 days, and pass the cells to P0 on D14. The cell morphology at D3 of differentiation is shown in the figure below. Figure 1 shown.
[0027] iMSC passaging stage Prepare several centrifuge tubes, label them with the cell names, and wash each well of a 6-well plate with 1 mL of DPBS. Add 1 mL of Accutase digestion buffer and incubate at 37°C for 2–3 minutes.
[0028] Observe cells under a microscope to ensure complete digestion. Once the cell edges appear shiny and some cells have detached, add 1 mL of commercially available MSC culture medium to each well to terminate digestion. Gently pipette to mix thoroughly. Transfer the cell suspension to a centrifuge tube and centrifuge at 300g for 5 minutes. Discard the supernatant, retain the pellet, and resuspend the cells. Gently pipette to obtain a single-cell suspension for counting. Based on the number of viable cells, seed the cells into a culture vessel of appropriate size and culture using commercially available MSC culture medium supplemented with Y27632 at a final concentration of 10 μM. The next day, switch to the appropriate MSC culture medium without Y27632. Change the medium every 1–3 days depending on the specific situation.
[0029] When the P0 cell fusion reaches 80%~90%, the digested cells are continued to be subcultured: add an appropriate amount of PBS to each bottle to wash once, add TrypLE digestion solution (purchased from Gibco), and incubate at room temperature or incubator for digestion for 2~5 minutes. Under the microscope, the cells are transparent and more than 80% of the cells in the bottle fall off the bottle wall. The digestion is terminated and the cell suspension is transferred to a centrifuge tube of appropriate specifications. After centrifugation at 300g for 5 minutes, the supernatant is discarded, the precipitate is retained, and it is resuspended and mixed with MSC culture medium. The cells are counted, and then a cell culture flask of appropriate specifications is selected for expansion and subculture. Cells are collected at P2 and P4 generations. The morphology of iMSC at P2 is shown in the figure below. Figure 2 As shown in Figure 4, the morphology of iMSCs at P4 is shown in Figure 4. Figure 3 As shown, the collected P2 cells were subjected to flow cytometry detection as shown in Figures 4 to 6 As shown by Figures 4 to 6 The iMSCs obtained showed a CD73 expression of 98.12%, a CD90 expression of 98.22%, and a CD105 expression of 98.85%. This indicates that the iMSCs differentiated from iPSCs provided by the present invention have consistent surface markers with adult-derived MSCs.
[0030] Example 2 The present disclosure provides a differentiation medium combination, which includes: medium A, medium B, medium C and medium D. Medium A includes: a basal medium and a first small molecule compound combination. The basal medium includes: IMDM (purchased from Gibco), F12 (purchased from Gibco), chemically defined lipid (lipid concentrate) (purchased from Gibco), insulin-transferrin-selenium (purchased from MCE), and 1-thioglycerol (purchased from Sigma). The small molecule compound combination includes: Activin A (purchased from PeproTech), CHIR-99021 (purchased from MCE), FGF2 (purchased from ORIGENE), and PIK90 (purchased from MCE). The volume ratio of IMDM to F12 is 3:1, the chemically defined lipid and insulin-transferrin-selenium each account for 0.5% of the total volume of the culture medium, the final concentration of 1-thioglycerol is 225 μM, and the Activin A is 0.5% of the total volume of the culture medium. The final concentration of A was 10 ng / mL, the final concentration of CHIR-99021 was 1 μM, the final concentration of FGF2 was 10 ng / mL, and the final concentration of PIK90 was 50 μM. The formula of the basal medium of medium A provided in this example is shown in Table 2.
[0031] Table 2 is the formula of the basal culture medium
[0032] Culture medium B includes: the same basal culture medium as culture medium A provided in this example and a second small molecule compound combination, the second small molecule compound combination includes: SB431542 (purchased from MCE), CHIR-99021 (purchased from MCE), FGF2 (purchased from MCE) and Dorsomorphin (purchased from MCE), the final concentration of SB431542 is 1 μM, the final concentration of CHIR-99021 is 1 μM, the final concentration of FGF2 is 10 ng / mL, and the final concentration of Dorsomorphin is 1 μM.
[0033] Culture medium C includes: the same basal culture medium as culture medium A provided in this example and a third small molecule compound combination, the third small molecule compound combination includes: SB431542 (purchased from MCE), Dorsomorphin (purchased from MCE), Wnt-C59 (purchased from MCE) and PD173074 (purchased from MCE), the final concentration of SB431542 is 1 μM, the final concentration of Dorsomorphin is 0.5 μM, the final concentration of Wnt-C59 is 0.5 μM, and the final concentration of PD173074 is 50 nM.
[0034] Culture medium D includes: the same basal culture medium as culture medium A provided in this example and a fourth small molecule compound combination, wherein the fourth small molecule compound combination includes SB590885 (purchased from MCE Company), and the final concentration of SB590885 is 0.5 μM.
[0035] Example 3 The present disclosure provides a differentiation medium combination, which includes: medium A, medium B, medium C and medium D, wherein medium A includes: a basal medium and a first small molecule compound combination, the basal medium includes: IMDM (purchased from Gibco), F12 (purchased from Gibco), chemically defined lipid (lipid concentrate) (purchased from Gibco), insulin-transferrin-selenium (purchased from MCE), and 1-thioglycerol (purchased from Sigma), and the small molecule compound combination includes: Activin A (purchased from PeproTech), CHIR-99021 (purchased from MCE), FGF2 (purchased from ORIGENE), and PIK90 (purchased from MCE), wherein the volume ratio of IMDM to F12 is 1:3, the chemically defined lipid and insulin-transferrin-selenium each account for 2% of the total volume of the culture medium, the volume ratio of IMDM to insulin-transferrin-selenium is 49:1, the final concentration of 1-thioglycerol is 600 μM, Activin The final concentration of A was 50 ng / mL, the final concentration of CHIR-99021 was 6 μM, the final concentration of FGF2 was 50 ng / mL, and the final concentration of PIK90 was 200 μM. The formula of the basal medium of medium A provided in this example is shown in Table 3.
[0036] Table 3 is the formula of the basal culture medium
[0037] Culture medium B includes: the same basal culture medium as culture medium A provided in this example and a second small molecule compound combination, the second small molecule compound combination includes: SB431542 (purchased from MCE), CHIR-99021 (purchased from MCE), FGF2 (purchased from MCE) and Dorsomorphin (purchased from MCE), the final concentration of SB431542 is 6 μM, the final concentration of CHIR-99021 is 6 μM, the final concentration of FGF2 is 50 ng / mL, and the final concentration of Dorsomorphin is 6 μM.
[0038] Culture medium C includes: the same basal culture medium as culture medium A provided in this example and a third small molecule compound combination, the third small molecule compound combination includes: SB431542 (purchased from MCE), Dorsomorphin (purchased from MCE), Wnt-C59 (purchased from MCE) and PD173074 (purchased from MCE), the final concentration of SB431542 is 6 μM, the final concentration of Dorsomorphin is 6 μM, the final concentration of Wnt-C59 is 4 μM, and the final concentration of PD173074 is 200 nM.
[0039] Culture medium D includes: the same basal culture medium as culture medium A provided in this example and a fourth small molecule compound combination, wherein the fourth small molecule compound combination includes SB590885 (purchased from MCE Company), and the final concentration of SB590885 is 5 μM.
[0040] The above description is merely an optional embodiment of the present disclosure and is not intended to limit the present disclosure. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present disclosure shall be included in the scope of protection of the present disclosure.
Claims
1. A differentiation medium composition, characterized in that: The differentiation medium combination includes: medium A, medium B, medium C and medium D, The culture medium A comprises: a basal culture medium and a first small molecule compound combination, wherein the first small molecule compound combination comprises: Activin A, CHIR-99021, FGF2 and PIK90; The culture medium B comprises: the basal culture medium and a second small molecule compound combination, wherein the second small molecule compound combination comprises: SB431542, CHIR-99021, FGF2 and Dorsomorphin; The culture medium C comprises: the basal culture medium and a third small molecule compound combination, wherein the third small molecule compound combination comprises: SB431542, Dorsomorphin, Wnt-C59 and PD173074; The culture medium D includes: the basal culture medium and a fourth small molecule compound combination, wherein the fourth small molecule compound combination includes SB590885.
2. The differentiation medium combination according to claim 1, characterized in that The basic culture medium comprises: IMDM, F12, chemically defined lipid, insulin-transferrin-selenium and 1-thioglycerol.
3. The differentiation medium combination according to claim 2, characterized in that In the culture medium A, the volume ratio of the IMDM and the F12 is 3:(1-9), the chemically defined lipid and the insulin-transferrin-selenium each account for 0.5%-2% of the total volume of the basal culture medium, the volume ratio of the IMDM and the insulin-transferrin-selenium is 49:1, the final concentration of the 1-thioglycerol is 225 μM-600 μM, the final concentration of the Activin A is 10 ng / mL-50 ng / mL, the final concentration of the CHIR-99021 is 1 μM-6 μM, the final concentration of the FGF2 is 10 ng / mL-50 ng / mL, and the final concentration of the PIK90 is 50 μM-200 μM.
4. The differentiation medium combination according to claim 1, characterized in that In the culture medium B, the final concentration of the SB431542 is 1 μM to 6 μM, the final concentration of the CHIR-99021 is 1 μM to 6 μM, the final concentration of the FGF2 is 10 ng / mL to 50 ng / mL, and the final concentration of the Dorsomorphin is 1 μM to 6 μM.
5. The differentiation medium combination according to claim 1, characterized in that In the culture medium C, the final concentration of the SB431542 is 1 μM to 6 μM, the final concentration of the Dorsomorphin is 0.5 μM to 6 μM, the final concentration of the Wnt-C59 is 0.5 μM to 4 μM, and the final concentration of the PD173074 is 50 nM to 200 nM.
6. The differentiation medium combination according to claim 1, characterized in that The final concentration of SB590885 is 1 μM to 10 μM.
7. A method for preparing mesenchymal stem cells by inducing stem cells to differentiate in the somite direction using the differentiation medium combination according to any one of claims 1 to 6, characterized in that: The method comprises: Inducing differentiation of pluripotent stem cells or embryonic stem cells using the culture medium A to obtain a first product; Using the culture medium B to induce differentiation of the first product to obtain paraxial mesoderm; Using the culture medium C to induce differentiation of the paraxial mesoderm to obtain somites; The culture medium D is used to induce differentiation of the somites, and then amplified and cultured to obtain mesenchymal stem cells.