Culture system for directionally differentiating nerve mesoderm progenitor cells into skeletal muscle cells, induction method and application

Abstract

The invention relates to a culture system for directionally differentiating nerve mesoderm progenitor cells into skeletal muscle cells, an induction method and application. The system comprises two differential culture media, and a basic culture system comprises a liquid basic culture medium, a 0.01-5 * B27 additive, a 0.01-5 * N2 additive, 0.01-10% penicillin / streptomycin and 0.01-500 [mu] g / mL 2-phosphoric acid-vitamin C; the differential culture medium I further comprises 0.01 to 50 [mu] M of a GSK3 inhibitor and 0.01 to 500 ng / mL of FGF family growth factors; and the differential medium II also comprises 0.01 to 500 ng / mL of HGF (Hepatocyte Growth Factor) protein and 0.01 to 500 ng / mL of IGF family growth factors. The induction method comprises the following steps: differentiating nerve mesoderm progenitor cells into paraaxial mesoderm by utilizing a differential culture medium I; and inducing and differentiating the paraaxial mesoderm into skeletal muscle cells by using a differential culture medium II. The skeletal muscle cells obtained by the method can be used for preparation of cells for treating muscle diseases, muscle tissue engineering, establishment of muscle disease models, in-vitro drug screening of muscle diseases and the like.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedical engineering, and relates to a culture system, an induction method and an application for directing differentiation of neural mesodermal progenitor cells into skeletal muscle cells. Technical Background

[0002] Skeletal muscle is a low self-renewal tissue, accounting for about 40% of the body weight, and is mainly responsible for mechanical movement, maintaining body temperature and other functions. Most skeletal muscles are attached to bones through tendons, and skeletal muscles can be divided into different muscles according to the connective tissue layer (tendon, endomysium, etc.). Myofibers are the basic contractile units of skeletal muscle, which are bundles of elongated multinucleated cells. Skeletal muscle has a certain regenerative capacity, which is highly dependent on the existence of adult stem cells, especially skeletal muscle stem cells. Skeletal muscle stem cells are distributed on the periphery of muscle fibers, so they are also called satellite cells. Satellite cells are characterized by the expression of paired box gene 7 (PAX7), so PAX7 is often used as a marker for satellite cells. However, muscle degeneration affects the health of millions of people around the world, and its pathogenic factors include muscular dystrophy, sarcopenia, cachexia, metabolic disorders and chronic muscle damage. Various disease factors lead to insufficient endogenous muscle regeneration, so other treatments are needed to restore the normal function of muscles. Preclinical experiments and clinical trials have shown that cell-mediated muscle regeneration can be used to treat muscle degeneration diseases. However, both satellite cells and skeletal muscle cells have the problem of scarce sources.

[0003] For all vertebrates from fish to humans, the role of neuromesodermal progenitors (NMPs) in body axis elongation is conserved. During embryonic development, neuromesodermal progenitors are a group of stem cells with bidirectional differentiation potential, which can not only differentiate into the spinal cord, but also differentiate into paraxial mesodermal tissues (including skeletal muscle, cartilage, etc.). Therefore, neuromesodermal progenitors show great potential in cell therapy for trunk and spinal cord injuries. At present, there are literature reports on the induction of neuromesodermal progenitors to produce neurons or neuromuscular organoids, but how to achieve the directed differentiation of neuromesodermal progenitors into skeletal muscle cells remains a problem to be solved. There is still a lack of effective induction methods in this field to induce neuromesodermal progenitors into skeletal muscle cells in vitro, especially under culture conditions with clear chemical composition. Summary of the invention

[0004] The present invention is based on the above-mentioned problems and provides a directed differentiation system for directing differentiation of neural mesodermal progenitor cells into skeletal muscle cells. The system includes two differentiation culture media. Differentiation culture medium 1 induces neural mesodermal progenitor cells to become paraxial mesoderm; differentiation culture medium 2 induces the above-mentioned paraxial mesoderm to become skeletal muscle cells.

[0005] Another object of the present invention is to provide a method for directing differentiation of neural mesodermal progenitor cells into skeletal muscle cells, wherein the method can direct differentiation of neural mesodermal progenitor cells into skeletal muscle cells via the paraxial mesoderm stage, wherein the cells contain muscle fibers and satellite cells.

[0006] The third purpose of the present invention is to provide related applications of directed differentiated skeletal muscle cells, such as disease treatment, muscle tissue engineering construction, muscle disease model establishment, in vitro drug screening for muscle diseases, etc.

[0007] In order to achieve the above object, the technical solution adopted by the present invention is as follows:

[0008] In a first aspect, the present invention provides a culture system for directed differentiation of neural mesodermal progenitor cells into paraxial mesoderm, the system comprising a culture medium with a clear chemical composition, named differentiation medium 1, the differentiation medium comprising: liquid basal culture medium, 0.01-5×B27 additive, 0.01-5×N2 additive, 0.01-10% penicillin / streptomycin, 0.01-500μg / mL 2-phospho-vitamin C, 0.01-50μM GSK3 inhibitor, and 0.01-500ng / mL FGF family growth factor.

[0009] Preferably, the liquid basal culture medium is selected from one or more of MEM cell culture medium, DMEM cell culture medium, DMEM / F12 cell culture medium, IMDM cell culture medium, 199 cell culture medium, F10 cell culture medium, F12 cell culture medium, and 1640 cell culture medium.

[0010] GSK3 inhibitors include but are not limited to one or more of small molecule GSK3 inhibitors such as CHIR99021, BIO and LY2090314; FGF family growth factors include but are not limited to any one or more of FGF 1 to FGF23.

[0011] Further preferably, the differentiation medium 1 consists of the following components: the liquid basal culture medium is DMEM / F12, 1×N2, 1×B27, 70 μg / mL 2-phospho-vitamin C, and 1% penicillin / streptomycin; the GSK3 inhibitor is 10 μM CHIR99021, and the FGF family growth factor is 40 ng / mL FGF2.

[0012] In the specific embodiment of the present invention, the above components are used for experiments, but each component is not limited to the above specific components. For example, 10 μM CHIR99021 is selected for experiments among GSK3 inhibitors, but BIO and LY2090314 can also achieve corresponding functions. The same is true for the selection of components of FGF family growth factors.

[0013] In a second aspect, the present invention provides a culture system for directed differentiation of paraxial mesoderm into skeletal muscle, the system comprising a culture medium with a clear chemical composition, which is named differentiation medium 2. The differentiation medium comprises: liquid basal culture medium, 0.01-5×B27 additive, 0.01-5×N2 additive, 0.01-10% penicillin / streptomycin, 0.01-500μg / mL 2-phospho-vitamin C, 0.01-500ng / mL HGF protein, and 0.01-500ng / mL IGF family growth factor.

[0014] Preferably, the liquid basal culture medium is selected from one or more of MEM cell culture medium, DMEM cell culture medium, DMEM / F12 cell culture medium, IMDM cell culture medium, 199 cell culture medium, F10 cell culture medium, F12 cell culture medium, and 1640 cell culture medium.

[0015] The IGF family growth factors include, but are not limited to, any one or more of IGF1 and IGF2.

[0016] More preferably, the second differentiation medium consists of the following components: the liquid basal culture medium is DMEM / F12, 1×N2, 1×B27, 70 μg / mL 2-phospho-vitamin C, 1% penicillin / streptomycin; the HGF protein is 10 ng / mL, and the IGF family growth factor is 2 ng / mL IGF1.

[0017] In a specific embodiment of the present invention, the above components are used for experiments, but each component is not limited to the above specific components. For example, 2ng / mL of IGF1 is selected from the IGF family growth factors for experiments, but IGF2 can also achieve the corresponding function. The same is true for the selection of other components.

[0018] In a third aspect, the present invention provides a method for directing differentiation of neural mesodermal progenitor cells into skeletal muscle cells using the above culture system, wherein the method can direct differentiation of neural mesodermal progenitor cells into skeletal muscle cells via the paraxial mesoderm stage, comprising the following steps:

[0019] A. Using the aforementioned differentiation medium 1, the neural mesodermal progenitor cells are cultured for 1-10 days to differentiate into paraxial mesoderm;

[0020] B. Using the aforementioned differentiation medium 2, the paraxial mesoderm obtained in step A is cultured for 1-3 months to differentiate into skeletal muscle cells.

[0021] Preferably, in step A, the neural mesodermal progenitor cells are mammalian neural mesodermal progenitor cells.

[0022] Furthermore, the neural mesodermal progenitor cells are purified from early embryonic tissues or differentiated from human pluripotent stem cells, including "temporary" neural mesodermal progenitor cells and "self-renewable" neural mesodermal progenitor cells.

[0023] At present, most of the induced differentiation of embryonic stem cells still belongs to the run-through process: that is, functional, terminally differentiated cells can be obtained through the multi-step sequential differentiation process of embryonic stem cells, but the differentiation system cannot be stably maintained at a specific intermediate developmental stage (such as the tissue stem cell stage). For example, in the process of inducing human pluripotent stem cells into spinal cord neurons, they will briefly go through the neural mesodermal progenitor cell stage, so we call it "temporary" neural mesodermal progenitor cells.

[0024] It is common knowledge in the art to induce human pluripotent stem cells into "temporary" neuromesodermal progenitor cells. References include: Turner DA, Hayward PC, Baillie-Johnson P, Rué P, Broome R, Faunes F, Martinez Arias A. Wnt / β-catenin and FGF signalling direct the specification and maintenance of aneuromesodermal axial progenitor in ensembles of mousembryonic stem cells. Development. 2014 Nov; 141(22): 4243-53.

[0025] Inducing human pluripotent stem cells into "self-renewing" neural mesodermal progenitor cells, see Dr. Sun Pingxin's graduation thesis "Study on the Differentiation of Human Embryonic Stem Cells into Self-renewing Neural Mesodermal Progenitor Cells". By establishing a new culture system, the neural mesodermal progenitor cells derived from human pluripotent stem cells can maintain long-term stable self-renewal in vitro and maintain the bidirectional differentiation potential of neural and mesoderm. The method of the present invention can differentiate neural mesodermal progenitor cells into skeletal muscle cells in two stages.

[0026] The fourth aspect of the present invention provides paraxial mesoderm cells and skeletal muscle cells (NMP-SCs) obtained according to the culture method of the present invention. Molecular identification results show that when induced to 45 days, skeletal muscle cells derived from neural mesodermal progenitor cells highly express muscle fiber markers DESMIN, MF20, myoblast fusion key transcription factor MYOG, and sarcomere marker TTN. At the same time, the striated structure formed by TTN can also be observed, which indicates that skeletal muscle cells derived from neural mesodermal progenitor cells can differentiate into mature striated muscle fibers. Immunofluorescence staining shows that the proportion of satellite cells positive for transcription factor PAX7 is as high as 43.3%. It is suggested that the skeletal muscle cells obtained by the culture system and method of the present invention contain mature muscle fibers and satellite cells.

[0027] The fifth aspect of the present invention provides the application of the induced skeletal muscle cells, such as the application in the preparation of cells for the treatment of muscle diseases, muscle tissue engineering, the establishment of muscle disease models, in vitro drug screening for muscle diseases, etc.

[0028] In the specific implementation method of the present invention, a muscle injury model mouse was first constructed, and then NMP-SCs were injected. The results showed that NMP-SCs could be integrated into mouse muscle fibers, indicating that satellite cells derived from neuromesodermal progenitor cells can promote muscle regeneration in muscle injury model mice, and have important application potential and advantages in muscle injury diseases.

[0029] The beneficial protection and effects of the present invention are as follows:

[0030] The culture system with clear chemical composition of the present invention can be used to quickly obtain skeletal muscle cells through two-stage induction of neural mesodermal progenitor cells, which contain mature muscle fibers with striated structures and satellite cells positive for transcription factor PAX7. Furthermore, satellite cells derived from neural mesodermal progenitor cells can be used for cell therapy of muscle injury diseases, can be integrated into host muscles and enhance the regeneration capacity of muscles, and provide a useful exploration for the provision and application of clinical skeletal muscles (muscle fibers and satellite cells). BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1Experimental results of the differentiation of neural mesodermal progenitor cells derived from human pluripotent stem cells into skeletal muscle via the paraxial mesoderm stage: (A) Immunofluorescence staining results showed that neural mesodermal progenitor cells (NMPs) derived from human pluripotent stem cells co-expressed the neural stem cell molecular marker SOX2 and the early mesoderm molecular marker TBXT; (B) The induction scheme of neural mesodermal progenitor cells differentiating into skeletal muscle via the paraxial mesoderm stage and the molecular markers of cells at each stage; (C) qRT-PCR detection results of neural mesodermal progenitor cells differentiating into paraxial mesoderm; (D-E) Immunofluorescence staining detection of paraxial mesoderm molecular markers TBXT and TBX6 and quantitative statistical results; (F) Optical microscope photo of paraxial mesoderm cells after one month of induction in skeletal muscle differentiation medium.

[0032] Figure 2 Molecular identification results of skeletal muscle cells derived from neuromesodermal progenitor cells: (AF) Immunofluorescence staining to detect skeletal muscle cell molecular markers, including myofiber molecular markers MF20, DESMIN, MYOG, TTN and satellite cell molecular marker PAX7, as well as the statistical results of MYOG positivity (C) and PAX7 positivity (F); (GH) GO analysis results of differentially expressed genes in neuromesodermal progenitor cells and skeletal muscle cells derived from neuromesodermal progenitor cells (NMP-SM); (I) Heat map results of differentially expressed genes in neuromesodermal progenitor cells and skeletal muscle cells derived from neuromesodermal progenitor cells (NMP-SM).

[0033] Figure 3 Experimental results of promoting muscle regeneration in muscle injury model mice by satellite cells derived from neural mesodermal progenitor cells: (A) Schematic diagram of experimental design; (B-D) Photos of tibialis anterior muscles in the neural mesodermal progenitor cell-treated and control groups, and the statistical results of the wet weight and maximum diameter of the corresponding muscles; (E) Immunofluorescence staining results of tibialis anterior muscles in the neural mesodermal progenitor cell-treated and control groups, where Laminin is a molecular marker of muscle fibers, HuMitoch is a molecular marker of human mitochondria, and DAPI is a chemical dye (which can bind to DNA in the cell nucleus and color it); (F) Statistical results of mouse muscle fibers containing human mitochondria. DETAILED DESCRIPTION

[0034] The present invention is now described in detail with reference to the embodiments, but the implementation of the present invention is not limited thereto.

[0035] The reagents and raw materials used in the present invention are all commercially available or can be prepared according to literature methods. The experimental methods in the following examples without specifying specific conditions are usually carried out under conventional conditions or under conditions recommended by the manufacturer. Unless otherwise specified, percentages and parts are calculated by weight.

[0036] Example 1: Neuromesodermal progenitor cells can differentiate into skeletal muscle cells via the paraxial mesoderm pathway

[0037] Human pluripotent stem cells were induced into neural mesodermal progenitor cells. Immunofluorescence staining results showed that neural mesodermal progenitor cells co-expressed the neural stem cell marker SOX2 and the early mesoderm marker TBXT ( Figure 1 A). In vivo embryonic development studies suggest that the WNT and FGF signaling pathways need to be activated simultaneously during the elongation of the body axis. Therefore, we speculate that the process of differentiating neural mesodermal progenitor cells into paraxial mesoderm in vitro also requires the simultaneous activation of the WNT and FGF signaling pathways. To verify this hypothesis, we activated the WNT and FGF signaling pathways separately or simultaneously, and treated neural mesodermal progenitor cells for one week in three culture conditions. The three culture conditions were based on N2B27 basal medium, supplemented with 10μM CHIR99021 and / or 40ng / ml FGF2, and were recorded as C10, F40, C10F40 ( Figure 1 B). Quantitative PCR results showed that activation of the FGF signaling pathway alone could increase the expression of the neural stem cell molecular marker SOX2, while down-regulating the expression of the mesoderm molecular markers TBX6 and MSGN1; activation of the WNT signaling pathway alone had the opposite effect to the former; simultaneous activation of the WNT and FGF signaling pathways could further increase the expression of the mesoderm molecular markers TBX6 and MSGN1, and reduce the expression of the neural stem cell molecular marker SOX2 ( Figure 1 C). Therefore, we differentiated neural mesodermal progenitor cells into paraxial mesoderm by activating the WNT and FGF signaling pathways. Furthermore, immunofluorescence staining results showed that after activating the WNT and FGF signaling pathways simultaneously for one week, the positive ratios of mesoderm molecular markers TBXT and TBX6 reached 45.1% and 26.5%, respectively ( Figure 1 DE). The above results indicate that simultaneous activation of WNT and FGF signaling pathways can effectively differentiate neural mesodermal progenitor cells into paraxial mesoderm.

[0038] The above-mentioned paraxial mesoderm cells were further cultured in skeletal muscle differentiation medium ( Figure 1 B), the culture conditions are based on N2B27 basal medium, supplemented with 2ng / ml IGF1 (Gibco, PHG0071) and 10ng / ml HGF (Gibco, PHG0254). After one month of induction, a large number of muscle fibers can be seen under a light microscope ( Figure 1F). Immunofluorescence staining was used to verify the expression of skeletal muscle cell markers. At 45 days of induction, skeletal muscle cells derived from neural mesodermal progenitor cells highly expressed myofiber markers DESMIN and MF20, myoblast fusion key transcription factor MYOG, and sarcomere marker TTN ( Figure 2 AC). At the same time, the striated structure formed by TTN can be observed, which indicates that skeletal muscle cells derived from neural mesodermal progenitor cells can differentiate into mature striated muscle fibers ( Figure 2 D). It is noteworthy that these cells maintained the characteristic of continuously producing myoblasts within three months of in vitro culture, so we speculated that they contained satellite cells. Immunofluorescence staining showed that the proportion of satellite cells positive for transcription factor PAX7 was as high as 43.3% ( Figure 2 EF). We also performed transcriptome sequencing on neural mesodermal progenitor cells (NMPs) and neural mesodermal progenitor cell-derived skeletal muscle cells (NMPs-derived skeletal muscle, NMP-SM). The GO analysis results of differentially expressed genes showed that up-regulated genes were mainly enriched in terms of muscle fiber sliding, skeletal muscle contraction, and sarcomere assembly, while down-regulated genes were enriched in terms of cell differentiation, maintenance of adult stem cell populations, and stem cell proliferation ( Figure 2 GH), and the heat map results of differentially expressed genes are consistent with it ( Figure 2 I).

[0039] In summary, we have established a culture system and induction method for the differentiation of neural mesodermal progenitor cells into skeletal muscle cells via the paraxial mesoderm stage. These skeletal muscle cells contain mature myofibers with striated structures and satellite cells positive for the transcription factor PAX7.

[0040] Example 2: Satellite cells derived from neural mesodermal progenitor cells can promote muscle regeneration in muscle injury model mice

[0041] We used a cardiotoxin-induced muscle injury model to examine the muscle regeneration potential of NMPs-derived satellite cells (NMP-SCs). The left and right tibialis anterior muscles of immunodeficient NSG mice were injured by injection of cardiotoxin. Cell transplantation was performed one week after modeling. The left tibialis anterior muscle was injected with 3×10 5 NMP-SCs were injected into the right tibialis anterior muscle with an equal volume of phosphate buffered saline (PBS) as a control group ( Figure 3 A). Comparison of muscles on both sides two months after transplantation ( Figure 3BD), the tibialis anterior muscle of the NMP-SCs transplanted group had a larger wet weight (58.62mg vs.50.24mg) and diameter (4.96mm vs.3.93mm) than that of the PBS control group. Immunofluorescence staining was further used to detect whether NMP-SCs were integrated into mouse muscle fibers, among which human mitochondria (HuMitoch) was used to characterize the integration of NMP-SCs. Immunofluorescence staining results showed that HuMitoch was expressed in Laminin-positive mouse muscle fibers, and the average number of mouse muscle fibers with NMP-SCs integration was 181 ( Figure 3 EF).

[0042] In summary, satellite cells derived from neuromesodermal progenitor cells can promote muscle regeneration in muscle injury model mice and have important application potential and advantages in muscle injury diseases.

[0043] The preferred embodiments of the present invention have been specifically described above, but the present invention is not limited to the embodiments. Those skilled in the art may make various equivalent modifications or substitutions without violating the spirit of the present invention. These equivalent modifications or substitutions are all included in the scope defined by the claims of this application.

Claims

1. A culture system for directing differentiation of neural mesodermal progenitor cells into skeletal muscle cells, characterized in that: The system includes two differentiation media, Differentiation medium 1 is composed of the following components: liquid basal medium, 0.01-5×B27 supplement, 0.01-5×N2 supplement, 0.01-10% penicillin / streptomycin, 0.01-500 μg / mL 2-phospho-vitamin C, 0.01-50 μM GSK3 inhibitor, 0.01-500 ng / mL FGF family growth factor; Differentiation medium 2 is composed of the following components: liquid basal culture medium, 0.01-5×B27 additive, 0.01-5×N2 additive, 0.01-10% penicillin / streptomycin, 0.01-500 μg / mL 2-phospho-vitamin C, 0.01-500 ng / mL HGF protein, and 0.01-500 ng / mL IGF family growth factor.

2. The culture system for directed differentiation of neural mesodermal progenitor cells into skeletal muscle cells according to claim 1, characterized in that: The liquid basal culture medium is selected from a combination of MEM cell culture medium, DMEM cell culture medium, DMEM / F12 cell culture medium, IMDM cell culture medium, 199 cell culture medium, F10 cell culture medium, F12 cell culture medium, and 1640 cell culture medium; The GSK3 inhibitor is selected from one or more of CHIR99021, BIO and LY2090314; The FGF family growth factor is selected from any one or more of FGF 1 to FGF23; The IGF family growth factor is selected from any one or a combination of IGF1 and IGF2.

3. The culture system for directed differentiation of neural mesodermal progenitor cells into skeletal muscle cells according to claim 2, characterized in that: Differentiation medium 1 consisted of the following components: liquid basal medium, 1×B27 supplement, 1×N2 supplement, 1% penicillin / streptomycin, 70 μg / mL 2-phospho-vitamin C, 10 μM GSK3 inhibitor, 40 ng / mL FGF family growth factor; Differentiation medium 2 consists of the following components: liquid basal medium DMEM / F12, 1×B27 supplement, 1×N2 supplement, 1% penicillin / streptomycin, 70 μg / mL 2-phospho-vitamin C, 10 ng / mL HGF protein, and 2 ng / mL IGF family growth factor.

4. The culture system for directed differentiation of neural mesodermal progenitor cells into skeletal muscle cells according to claim 2, characterized in that: Differentiation medium 1 consisted of the following components: liquid basal medium DMEM / F12, 1×B27 supplement, 1×N2 supplement, 1% penicillin / streptomycin, 70 μg / mL 2-phospho-vitamin C, 10 μM CHIR99021, 40 ng / mL FGF2; Differentiation medium 2 consists of the following components: liquid basal medium DMEM / F12, 1×B27 supplement, 1×N2 supplement, 1% penicillin / streptomycin, 70 μg / mL 2-phospho-vitamin C, 10 ng / mL HGF protein, and 2 ng / mL IGF1.

5. A method for inducing directed differentiation of neuromesodermal progenitor cells into skeletal muscle cells using the culture system according to any one of claims 1 to 4, characterized in that: The method comprises the following steps: A. Differentiate the neuromesodermal progenitor cells into paraxial mesoderm using the aforementioned differentiation medium 1; B. Using the aforementioned differentiation medium 2, the paraxial mesoderm obtained in step A was induced to differentiate into skeletal muscle cells.

6. The induction method according to claim 5, characterized in that: The differentiation culture time in step A is 1 to 10 days, and the differentiation culture time in step B is 1 to 3 months.

7. The induction method according to claim 5, characterized in that: The neural mesodermal progenitor cells are mammalian neural mesodermal progenitor cells.

8. The induction method according to claim 5, characterized in that: The neural mesodermal progenitor cells are neural mesodermal progenitor cells purified from early embryonic tissue or obtained by in vitro induced differentiation of pluripotent stem cells.

9. The skeletal muscle cells obtained by the induction method according to any one of claims 5 to 8, characterized in that: The skeletal muscle cells include myofibers and satellite cells.

10. Use of the skeletal muscle cells according to claim 9 in preparing cells for treating muscle diseases, establishing muscle tissue engineering, establishing muscle disease models or in vitro drug screening for muscle diseases.

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