Inducing method for oriented myogenic differentiation of sheep iPSCs (induced pluripotent stem cells) and application thereof

Abstract

The invention discloses an induction method for oriented myogenic differentiation of sheep iPSCs (induced pluripotent stem cells) and application of the induction method. After sheep induced pluripotent stem cells are subcultured for one day, four-stage induced differentiation is performed by respectively using a differential medium I, a differential medium II, a differential medium III and a differential medium IV; and finally, directionally inducing and differentiating into myoblasts and fusing into myotubes. According to the invention, by applying micromolecules and / or growth factors of a plurality of key signal channels, single-layer adherent sheep iPSCs are successfully gradually and directionally induced and differentiated into myoblasts and fused into myotubes; the method is of great significance in exploring the mechanism of formation of muscle-derived cells in the early stage of sheep embryonic development, exploring molecular breeding markers valuable for muscle development, carrying out in-vitro muscle tissue engineering, researching and developing cultured meat and the like.

Description

Technical Field

[0001] The present invention belongs to the technical field of stem cells, and more specifically, to a method for inducing directed myogenic differentiation of sheep induced pluripotent stem cells (iPSCs) and its application. Background Art

[0002] Skeletal muscle is the most abundant tissue in vertebrates, and its growth and development are important indicators of the production performance of meat-producing livestock and poultry. With the vigorous development of animal husbandry and the improvement of living standards, the muscle yield and quality of livestock and poultry have received more and more attention. Embryonic myoblast differentiation is the key to determining the quality of muscle development, and the formation of early myogenic cells is the premise and basis for ensuring good myogenic differentiation. Myogenic cell formation is an extremely complex and highly coordinated biological process that strictly follows the process of embryonic development and differentiation and is regulated by a variety of key signaling pathways and transcription factors. At present, the mechanism of early development of myogenic cells is not fully understood. Due to the difficulty of establishing livestock embryonic stem cells (ESCs) and the problems of acquisition sources and ethics, the development of in vitro stem cell differentiation technology and muscle tissue engineering has been hindered.

[0003] Induced pluripotent stem cells (iPSCs) have biological characteristics very similar to those of ESCs, with good self-renewal and multidirectional differentiation capabilities. They overcome the problems of ESCs in terms of source and ethics, and provide a powerful means for studying the mechanism of myogenic cell formation in early embryonic development. Although studies have induced the differentiation of iPSCs from species such as humans, mice, and pigs into myogenic progenitor cells or myoblasts by overexpressing myogenic transcription factors, this differentiation protocol bypasses the formation process of myogenic cells and is severely limited in the study of skeletal muscle development mechanisms. The stepwise induction of differentiation using small molecules and / or growth factors of key signaling pathways can simulate the development process of myogenic cells, but the induction differentiation stage is more complicated, and the interaction mechanism of the main signaling pathways involved in each stage and their key activators / inhibitors has not yet been systematically elucidated. At present, the myogenic differentiation induction protocol of iPSCs using only small molecules and / or growth factors without involving exogenous myogenic transcription factors has only been successfully established in humans and mice, but not in other species. However, the differentiation induction protocols for human and mouse iPSCs are different. This is because there are certain differences in the developmental processes and mechanisms between different species, and different signaling pathways are required to work together during the differentiation induction process. Therefore, there are still major challenges for the directed induction of myogenic differentiation of iPSCs in other species, especially livestock and poultry.

[0004] As a major livestock, sheep have important value in animal husbandry and biomedical fields. Therefore, the combined use of activators / inhibitors of multiple key signaling pathways and cytokines to establish a stable, safe and efficient induction scheme for directed myogenic differentiation of sheep iPSCs can not only lay the foundation for studying the formation mechanism of myogenic cells in early embryonic development, but also provide an ideal cell source for in vitro muscle tissue engineering of sheep and the development of cultured meat. Summary of the invention

[0005] Based on this, the purpose of the present invention is to provide an induction method for directed myogenic differentiation of sheep iPSCs and its application. The induction method of the present invention can stably, efficiently and safely induce sheep induced pluripotent stem cells to differentiate into myoblasts.

[0006] The specific technical solutions for achieving the above-mentioned invention objectives include the following.

[0007] The first aspect of the present invention provides a method for inducing directed myogenic differentiation of sheep iPSCs, comprising the following steps:

[0008] (1) Inducing differentiation of sheep iPSCs for 1 day using differentiation medium I to obtain mesoderm cells; the differentiation medium I comprises: 85% to 87% DMEM basal medium, 8% to 12% FBS, 0.8% to 1.2% GultaMAX, 0.8% to 1.2% NEAA, 0.8% to 1.2% PS, 0.08mM to 0.12mM β-ME, and 2μM to 5μM CHIR99021;

[0009] (2) replacing the culture medium with differentiation medium II, inducing differentiation for 2 days to obtain somite cells; the differentiation medium II comprises: 85% to 87% DMEM basal medium, 8% to 12% FBS, 0.8% to 1.2% GultaMAX, 0.8% to 1.2% NEAA, 0.8% to 1.2% PS, 0.08mM to 0.12mM β-ME, 180nM to 220nM LDN193189, and 8μM to 12μM SB431542;

[0010] (3) replacing the culture medium with differentiation medium III, inducing differentiation for 1 day to obtain myoblasts; the differentiation medium III comprises: 85% to 87% DMEM basal medium, 8% to 12% FBS, 0.8% to 1.2% GultaMAX, 0.8% to 1.2% NEAA, 0.8% to 1.2% PS, 0.08mM to 0.12mM β-ME, 2μM to 5μM CHIR99021, and 15ng / mL to 25ng / mL FGF2;

[0011] (4) The culture medium was replaced with differentiation medium IV, and differentiation was induced for 3 days to obtain multinucleated myotubes; the differentiation medium IV included: 95% to 97% DMEM basal medium, 1.8% to 2.2% HS, 0.8% to 1.2% PS, and 1.8 ng / mL to 2.2 ng / mL IGF-1.

[0012] The second aspect of the present invention provides the use of the above-mentioned method for inducing directed myogenic differentiation of sheep iPSCs in sheep in vitro muscle tissue engineering.

[0013] In the present invention, based on the current research on the formation and differentiation mechanism of myogenic cells, combined with and compared the currently established human and mouse iPSCs directed induction differentiation schemes, the inventors found that in the directed myogenic differentiation process of sheep iPSCs, certain signaling pathways (including WNT signaling pathway, BMP signaling pathway, TGF-β signaling pathway and FGF signaling pathway) at different developmental stages such as pluripotent stem cells-gastrula stage-paraxial mesoderm-somite-myosome-myogenic progenitor cells-mononuclear myoblasts-multinuclear myotubes) played a key role in the directed myogenic differentiation process of sheep iPSCs, and We further systematically explored the combination, action time, concentration, etc. of small molecules and / or cytokines of these key signaling pathways (including WNT signaling pathway activator CHIR99021, BMP signaling pathway inhibitor LDN193189, TGF-β signaling pathway inhibitor SB431542, FGF signaling pathway activator FGF2, etc.), and finally successfully induced the monolayer adherent sheep iPSCs to differentiate into myoblasts and fuse into myotubes, thus establishing a stable, safe and efficient induction method for directed myogenic differentiation of sheep iPSCs.

[0014] The induction method for directed myogenic differentiation of sheep iPSCs of the present invention adopts a plurality of key signaling pathway small molecules and / or cytokines with clear chemical compositions, does not involve gene editing in the differentiation induction process, avoids the risk of exogenous gene integration, and is of great significance for exploring the mechanism of myogenic cell formation in the early stage of sheep embryonic development, discovering valuable molecular breeding markers for muscle development, and carrying out in vitro muscle tissue engineering.

[0015] In addition, the present invention is a directed myogenic differentiation induction scheme for iPSCs of other species established by simply using small molecules and / or cytokines instead of myogenic transcription factors after humans and mice. It is also of great significance for studying the differences in the developmental mechanisms of myogenic cells between different species and establishing and optimizing directed myogenic differentiation induction schemes for other species. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1The cell morphology and pluripotency detection of sheep iPSCs cultured without feeder layer in Example 1 of the present invention are shown in Figure 1. A is the cell morphology of sheep iPSCs on gelatin; B is the cell morphology of sheep iPSCs on matrix gel; C is continuous passage on matrix gel to P50; D is alkaline phosphatase staining; E is the mRNA expression level of endogenous core pluripotency genes.

[0017] Figure 2 This is a flow chart of inducing directed myogenic differentiation of sheep iPSCs in Example 2 of the present invention.

[0018] Figure 3 The cell morphology and pluripotency marker gene detection of induced sheep iPSCs differentiation in Example 2 of the present invention are shown in Figure 2. A is the cell morphology on days 0 to 3 of differentiation; B is the mRNA expression level of pluripotency genes; C is alkaline phosphatase staining; and D is the immunofluorescence staining result of pluripotency proteins.

[0019] Figure 4 The cell morphology and marker gene detection of sheep iPSCs differentiated into primitive streak cells in Example 2 of the present invention. A is the mRNA expression level of primitive streak genes on days 0 to 3 of differentiation; B is the cell morphology induced by different concentrations of CHIR99021 for 1 day; C is the mRNA expression level of primitive streak genes induced by different concentrations of CHIR99021 for 1 day; D is the cell morphology induced by CHIR99021 combined with other signaling pathway small molecules for 1 day; E is the mRNA expression level of primitive streak genes induced by CHIR99021 combined with other signaling pathway small molecules for 1 day.

[0020] Figure 5 The cell morphology and marker gene detection of sheep iPSCs differentiated into mesoderm cells in Example 2 of the present invention. A is the mRNA expression level of mesoderm genes on days 0 to 3 of differentiation; B is the cell morphology of differentiation induced by small molecules of other signaling pathways alone or in combination on day 2; C is the mRNA expression level of mesoderm genes induced by small molecules of other signaling pathways alone or in combination on day 2.

[0021] Figure 6The cell morphology diagram and marker gene detection of sheep mesoderm cells differentiated into somite cells in Example 2 of the present invention. A is the cell morphology on the 3rd and 4th days of differentiation; B is the mRNA expression level of somite genes; C is the cell morphology induced by different concentrations of LDN193189 on the 3rd day of differentiation; D is the mRNA expression level of somite genes induced by different concentrations of LDN193189 on the 3rd day of differentiation; E is the cell morphology induced by LDN193189 combined with other signaling pathway small molecules on the 3rd day of differentiation; F is the mRNA expression level of primitive streak genes induced by LDN193189 combined with other signaling pathway small molecules on the 3rd day of differentiation.

[0022] Figure 7 The cell morphology diagram and marker gene detection of sheep somite cells differentiating into myogenic cells in Example 2 of the present invention. A is the cell morphology induced by CHIR99021 on the 4th and 5th days of differentiation; B is the cell morphology induced by CHIR99021 combined with FGF2 on the 4th and 5th days of differentiation; C is the mRNA expression level of myotome, myogenic progenitor cell and myoblast marker genes; D is the immunofluorescence staining result of MYOD1 on the 4th day of differentiation.

[0023] Figure 8 The cell morphology and marker gene detection of sheep myoblasts differentiated into fused myotubes in Example 2 of the present invention. A is the cell morphology on days 5 to 7 of differentiation; B is the mRNA expression level of the MyHC2 gene; and C is the immunofluorescence staining result of MyHC2 on day 7.

[0024] Fig. 9 The cell morphology diagrams are of the myogenic differentiation of sheep iPSCs induced by the embryoid body method in Experimental Example 1 of the present invention. A is the cell morphology of differentiation induced by 5% HS; B is the cell morphology of differentiation induced by 2% HS (scale bar: 200 μm).

[0025] Fig.10 The cell morphology diagram of the induction of myogenic differentiation of sheep iPSCs in Experimental Example 2 of the present invention. A is a flowchart of the induction of differentiation; B is the cell morphology during the induction of differentiation; C is the cell morphology during the induction of differentiation by removing SB431542 in the differentiation medium I (scale bar: 200 μm).

[0026] Fig.11The cell morphology diagram of the myogenic differentiation process of sheep iPSCs induced by different serum types and concentrations in Experimental Example 3 of the present invention. A is a flow chart of induced differentiation; B is serum-free induced differentiation; C is 2% FBS induced differentiation; D is 2% HS induced differentiation; E is 5% FBS induced differentiation; F is 5% HS induced differentiation; G is 10% FBS induced differentiation; H is 10% FBS combined with 2% HS induced differentiation; I is 10% FBS combined with 5% HS induced differentiation (scale bar: 200 μm).

[0027] Fig.12 The cell morphology of the sheep iPSCs in Experimental Example 4 of the present invention after one day of subculture is shown in the process of differentiation induction. A is a flowchart of differentiation induction; B is a flowchart of 3×10 4 / cm 2 One day after cell culture, differentiation was induced; C is 10×10 4 / cm 2 Differentiation was induced one day after density passaging (scale bar: 200 μm).

[0028] Fig.13 This is a cell morphology diagram of induced differentiation of sheep iPSCs after one day of passage and two days of natural differentiation in Experimental Example 4 of the present invention. A is a flowchart of induced differentiation; B is the cell morphology during the induced differentiation process (scale bar: 200 μm). DETAILED DESCRIPTION

[0029] In order to facilitate the understanding of the present invention, the present invention will be described more fully below. The present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosure of the present invention more thorough and comprehensive.

[0030] Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as those commonly understood by those skilled in the art to which the present invention belongs. The terms used in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention. The term "and / or" used in the present invention includes any and all combinations of one or more of the related listed items.

[0031] The experimental methods in the following examples where specific conditions are not specified are generally performed according to conventional conditions, such as the conditions described in Green and Sambrook et al., Molecular Cloning: A Laboratory Manual (2013), or according to the conditions recommended by the manufacturer.

[0032] In some embodiments of the present invention, a method for inducing directed myogenic differentiation of sheep iPSCs is disclosed, comprising the following steps:

[0033] (1) Inducing differentiation of sheep iPSCs for 1 day using differentiation medium I to obtain mesoderm cells; the differentiation medium I comprises: 85% to 87% DMEM basal medium, 8% to 12% FBS, 0.8% to 1.2% GultaMAX, 0.8% to 1.2% NEAA, 0.8% to 1.2% PS, 0.08mM to 0.12mM β-ME, and 2μM to 5μM CHIR99021;

[0034] (2) replacing the culture medium with differentiation medium II, inducing differentiation for 2 days to obtain somite cells; the differentiation medium II comprises: 85% to 87% DMEM basal medium, 8% to 12% FBS, 0.8% to 1.2% GultaMAX, 0.8% to 1.2% NEAA, 0.8% to 1.2% PS, 0.08mM to 0.12mM β-ME, 180nM to 220nM LDN193189, and 8μM to 12μM SB431542;

[0035] (3) replacing the culture medium with differentiation medium III, inducing differentiation for 1 day, and obtaining myoblasts (expressing MYF5, MYF6, MYOD1, MYOG genes and MYOD1 protein); the differentiation medium III comprises: 85% to 87% DMEM basal medium, 8% to 12% FBS, 0.8% to 1.2% GultaMAX, 0.8% to 1.2% NEAA, 0.8% to 1.2% PS, 0.08mM to 0.12mM β-ME, 2μM to 5μM CHIR99021, and 15ng / mL to 25ng / mL FGF2;

[0036] (4) The culture medium was replaced with differentiation medium IV, and differentiation was induced for 3 days to obtain multinucleated myotubes (expressing MyHC2 marker gene and protein); the differentiation medium IV comprised: 95% to 97% DMEM basal medium, 1.8% to 2.2% HS, 0.8% to 1.2% PS, and 1.8 ng / mL to 2.2 ng / mL IGF-1.

[0037] In some of the embodiments, the method further includes the step of passage of the sheep iPSCs to a cell culture dish pre-treated with matrix gel and cultured for 1 day using M15 growth medium containing Y-27632.

[0038] In some embodiments, the mass concentration of the matrix gel is 0.8% to 1.2%, preferably 1%.

[0039] In some embodiments, the concentration of Y-27632 in the M15 growth medium is 8 μM to 12 μM, preferably 10 μM.

[0040] In some embodiments, the density of the ovine iPSCs is 4×10 4 / cm 2 ~6×10 4 / cm 2 , preferably 4×10 4 / cm 2 .

[0041] In some embodiments, the concentration of CHIR99021 in the differentiation medium I in step (1) is 2 μM to 4 μM.

[0042] In some of the embodiments, the concentration of LDN193189 in the differentiation medium II in step (2) is 195nM to 205nM, and the concentration of SB431542 is 9.5μM to 10.5μM.

[0043] In some of the embodiments, the concentration of CHIR99021 in the differentiation medium III in step (3) is 2 μM to 4 μM, and the concentration of FGF2 is 19 ng / mL to 21 ng / mL.

[0044] In some embodiments, in step (2) and step (4), the differentiation medium is replaced every day.

[0045] In some embodiments, the method for inducing directed myogenic differentiation of sheep iPSCs comprises the following steps:

[0046] (1) The density is 4×10 4 / cm 2 ~6×10 4 / cm 2 The sheep iPSCs were passaged into cell culture dishes pretreated with 0.8% to 1.2% Matrigel and cultured for 1 day using M15 growth medium containing Y-27632;

[0047] (2) Inducing differentiation of sheep iPSCs for 1 day using differentiation medium I to obtain mesoderm cells; the differentiation medium I comprises: 85% to 87% DMEM basal medium, 8% to 12% FBS, 0.8% to 1.2% GultaMAX, 0.8% to 1.2% NEAA, 0.8% to 1.2% PS, 0.08mM to 0.12mM β-ME, and 2μM to 4μM CHIR99021;

[0048] (3) replacing the culture medium with differentiation medium II, inducing differentiation for 2 days to obtain somite cells; the differentiation medium II comprises: 85% to 87% DMEM basal medium, 8% to 12% FBS, 0.8% to 1.2% GultaMAX, 0.8% to 1.2% NEAA, 0.8% to 1.2% PS, 0.08mM to 0.12mM β-ME, 195nM to 205nM LDN193189, and 9.5μM to 10.5μM SB431542;

[0049] (4) replacing the culture medium with differentiation medium III, inducing differentiation for 1 day to obtain myoblasts; the differentiation medium III comprises: 85% to 87% DMEM basal medium, 8% to 12% FBS, 0.8% to 1.2% GultaMAX, 0.8% to 1.2% NEAA, 0.8% to 1.2% PS, 0.08mM to 0.12mM β-ME, 2μM to 4μM CHIR99021, and 19ng / mL to 21ng / mL FGF2;

[0050] (5) The culture medium was replaced with differentiation medium IV, and differentiation was induced for 3 days to obtain multinucleated myotubes; the differentiation medium IV included: 95% to 97% DMEM basal medium, 1.8% to 2.2% HS, 0.8% to 1.2% PS, and 1.8 ng / mL to 2.2 ng / mL IGF-1.

[0051] In other embodiments of the present invention, the application of the above-mentioned method for inducing directed myogenic differentiation of sheep iPSCs in in vitro sheep muscle tissue engineering is disclosed.

[0052] In the following examples, the sheep induced pluripotent stem cells used were prepared according to the applicant's prior patent CN116064660B (Sheep induced pluripotent stem cells and preparation method thereof). The main experimental reagents used in the present invention are shown in Table 1.

[0053] Table 1 Main experimental reagents

[0054]

[0055]

[0056] The present invention is described in detail below with reference to the accompanying drawings and specific embodiments.

[0057] Example 1 Exploration of feeder-free culture conditions for sheep iPSCs

[0058] The directed induction differentiation of pluripotent stem cells (ESCs and iPSCs) depends on the culture environment without feeder layer. However, the feeder-free culture system of sheep iPSCs is not yet mature. It is very easy to differentiate under feeder-free conditions and cannot be stably passaged. For this reason, before the directed myogenic differentiation of sheep iPSCs, this embodiment uses iPSCs cell clones with high pluripotency to explore the culture system of sheep iPSCs under feeder-free conditions. In this embodiment, AP staining and qRT-PCR detection methods are performed with reference to the applicant's prior patent CN116064660B, and the primer sequence information involved is shown in Table 2.

[0059] Table 2 qRT-PCR primer sequence information

[0060]

[0061]

[0062] The main steps are as follows:

[0063] Observe the confluence of sheep iPSCs cells. When it reaches 85% to 90%, discard the original cell culture medium and wash twice with DPBS. Add 0.25% trypsin to digest for 3min to 4min, and observe the digestion during the period. When the cell bulges become round, add an equal volume of M15 culture medium to terminate the digestion, collect the cells into a 15mL centrifuge tube, and centrifuge at 1000r / min for 5min. After centrifugation, discard the supernatant, add an appropriate amount of embryoid body culture medium to the cell pellet, mix it by blowing, and evenly inoculate the cell suspension into a cell culture dish pretreated with 0.1% gelatin. After shaking, place it in a 37°C, 5% CO2 cell culture incubator and culture it for 30min to 40min to remove the feeder layer cells. Collect the non-adherent cells into a 15mL centrifuge tube and centrifuge it at 1000r / min for 5min. After centrifugation, discard the supernatant, add an appropriate amount of M15 culture medium to the cell pellet, mix it by blowing, and count the cells. The cell suspension was evenly inoculated into cell culture dishes pretreated with 0.1% gelatin and 1% matrigel, and the cells were cultured in M15 medium and Y-27632 was added. The medium was changed one day after subculturing, and the M15 medium was changed every other day thereafter.

[0064] On the 3rd to 4th day of culture, the ovine iPSCs clones on the gelatin-pretreated culture dish were small and dome-shaped, and some cells showed obvious differentiation ( Figure 1 A in the figure); cells can maintain good cell morphology on the culture dish pretreated with matrix gel, showing flat round or oval shape, and cells in the colony are tightly aggregated ( Figure 1 B). After continuous passage for more than 40 generations under the condition of Matrigel, sheep iPSCs can maintain good cell morphology ( Figure 1C), AP staining was stained blue-black and positive ( Figure 1 D), the expression levels of endogenous core pluripotency genes OCT4 and SOX2 are still at a high level ( Figure 1 E).

[0065] The results of this example show that the sheep iPSCs cell clones can maintain an undifferentiated state and good pluripotency under the condition of 1% matrix gel, which lays a foundation for the subsequent induction of directed myogenic differentiation of sheep iPSCs.

[0066] Example 2 Direct method to gradually induce the myogenic differentiation of sheep iPSCs

[0067] The activators / inhibitors of multiple key signaling pathways and growth factors were used to directly induce the monolayer adherent sheep iPSCs in stages. In this example, AP staining, qRT-PCR detection and cell immunochemical detection were all performed with reference to the applicant's prior patent CN116064660B, and the primer sequence information involved is shown in Table 3.

[0068] Table 3 qRT-PCR primer sequence information

[0069]

[0070]

[0071]

[0072] For specific differentiation induction methods, please refer to Figure 2 , including the following steps:

[0073] 1. The first stage of induction differentiation: differentiating sheep iPSCs into mesoderm cells

[0074] Observe the growth confluence of sheep iPSCs cells. When it reaches 85% to 90%, discard the original cell culture medium and wash three times with PBS. Add 0.25% trypsin to digest for 3min to 4min, and observe the digestion during the period. When the cell bulges become round, add an equal volume of M15 culture medium to terminate the digestion, collect the cells into a 15mL centrifuge tube, and centrifuge at 1000r / min for 5min. After centrifugation, discard the supernatant, add an appropriate amount of M15 culture medium to the cell pellet, mix it by blowing, and then mix the cell suspension at 4×10 4 / cm 2 The cells were uniformly inoculated in a 1% matrix gel pre-treated cell culture dish at a density of 100 μg / mL. The cells were cultured in M15 medium and Y-27632 was added. After shaking, they were placed in a 37°C, 5% CO2 cell culture incubator. After one day of culture, the differentiation of sheep iPSCs was induced, which was marked as differentiation day 0 (D0) ( Figure 3A), the M15 medium was replaced with the initially prepared differentiation medium I (86% DMEM basal medium, 10% FBS, 1% GultaMAX, 1% NEAA, 1% PS, 0.1 mM β-ME, 3 μM CHIR99021), and Y-27632 was removed.

[0075] The results showed that on the first day of differentiation (D1), the cells were in small clumps and evenly distributed; on the second day (D2), most of the cells began to adhere to the wall and differentiate, with irregular shapes; on the third day (D3), the cells gradually differentiated to the surrounding areas and became round or short spindle-shaped ( Figure 3 qRT-PCR results showed that the expression level of pluripotency marker gene OCT4 continued to decrease on D1 and D2; the expression level of SOX2 decreased on D1 and then increased on D2 ( Figure 3 D1 cells have a light or only partially stained AP ( Figure 3 C), the expression levels of pluripotency proteins OCT4 and SOX2 decreased ( Figure 3 D).

[0076] The expression level of the marker gene T (Brachyury) of the primitive streak stage increased rapidly on D1 and continued to decrease on D2 and D3; the expression level of MIXL1 increased on D1 and D2 and decreased on D3 ( Figure 4 A in Figure 1). The combined expression trends of T and MIXL1 genes indicate that CHIR99021 (CHIR) can differentiate iPSCs into primitive streak cells after one day of induction.

[0077] The differentiation effects of ovine iPSCs induced by different concentrations of CHIR (1.5, 3, 6, 10 μM) in differentiation medium I were compared for one day. The results showed that the density and morphology of differentiated cells induced by 6 μM and 10 μM CHIR were similar to those induced by 3 μM CHIR ( Figure 4 There was no significant difference in the relative expression of T and MIXL1 genes ( Figure 4 C); however, the cell density induced by 1.5 μM CHIR was higher, and some cells adhered to the wall and showed a flat morphology ( Figure 4 B), the relative expression levels of T and MIXL1 genes were low ( Figure 4 C).

[0078] Based on the use of 3μM CHIR, we further explored the effects of adding BMP signaling pathway activators BMP2 and BMP4, and FGF signaling pathway activator FGF2 on the effect and efficiency of enhancing the differentiation of sheep iPSCs into primitive streak cells. The results showed that compared with the use of CHIR alone to induce differentiation, the cell morphology of the combined 25ng / mL BMP2-induced differentiation was more aggregated ( Figure 4D), the relative expression levels of T and MIXL1 genes were decreased ( Figure 4 E); The cell morphology induced by the combination of 25ng / mL BMP4 and BMP2 was similar ( Figure 4 D), the relative expression of T gene increased, while the relative expression of MIXL1 gene decreased significantly ( Figure 4 E); The cell morphology induced by the combination of 20ng / mL FGF2 is flatter ( Figure 4 The relative expression levels of T and MIXL1 genes were also decreased ( Figure 4 E).

[0079] The primitive streak stage is a transient stage in which pluripotent stem cells differentiate into mesoderm, and the increase in gene expression indicates the formation of mesoderm. The inventors also detected the expression trend of mesoderm marker genes, and the results showed that the expression levels of TBX6 and MSGN1 increased rapidly on D1 and continued to decrease on D2 and D3 ( Figure 5 A), indicating that 3μM CHIR induction for one day can differentiate iPSCs into mesoderm cells, and continued induction may differentiate mesoderm cells into other types of cells. Therefore, based on the use of 3μM CHIR to induce differentiation for one day, the effects of continuing to add BMP signaling pathway inhibitor LDN193189 (LDN) and TGF-β signaling pathway inhibitor SB431542 (SB) alone or in combination on improving the effect and efficiency of sheep iPSCs differentiation into mesoderm cells were further explored. The results showed that the cell density induced by the combination of 3μM CHIR and 200nM LDN (CL) was higher, and some cells began to adhere to the wall for differentiation ( Figure 5 B); cells induced to differentiate using 200 nM LDN alone have already attached to the wall and gradually differentiated to the surrounding areas, showing round or short spindle shapes ( Figure 5 B), the relative expression of TBX6 gene increased ( Figure 5 C); The cell morphology induced by the combination of 200 nM LDN and 10 μM SB (LS) was similar to that induced by LDN alone, but the cell density was higher ( Figure 5 B).

[0080] The results showed that after one day of induction of differentiation using a WNT signaling pathway activator (3 μM CHIR), further induction for one day alone or in combination with a BMP signaling pathway inhibitor and a TGF-β signaling pathway inhibitor failed to increase the expression levels of the mesoderm marker genes TBX6 and MSGN1 ( Figure 5C). Therefore, the WNT signaling pathway activator (3 μM CHIR) was used alone as the key small molecule of differentiation medium I, that is, the initially formulated differentiation medium I was used as the final differentiation medium I, and the sheep iPSCs were differentiated into mesoderm cells by inducing differentiation for one day, and then the second stage of induced differentiation was performed.

[0081] 2. The second stage is to induce differentiation - differentiating mesoderm cells into somite cells

[0082] After the sheep iPSCs were differentiated into mesoderm cells on D1, CHIR was removed and the differentiation was continued using the preliminary proposed differentiation medium II (86% DMEM basal medium, 10% FBS, 1% GultaMAX, 1% NEAA, 1% PS, 0.1mM β-ME, 200nM LDN193189) containing 200nM LDN. The results showed that some cells adhered to the wall on D2, showing round or short spindle shapes, and gradually differentiated to the surrounding areas ( Figure 5 In the figure (B), on D3, the cells have already attached to the wall, the cell density has increased, and there is no obvious change in cell morphology. On the fourth day (D4), the cell density is more uniform, and long spindle cells can be seen ( Figure 6 A in the figure). qRT-PCR results showed that the expression level of the somite marker gene MEOX1 continued to increase on D2 and D3, but showed a downward trend on D4, indicating that LDN induction can differentiate mesoderm cells into somite cells within two days, and continued induction may further differentiate somite cells into other types of cells ( Figure 6 B).

[0083] The inventors compared the differentiation effects of different concentrations of LDN (100nM, 200nM, 400nM, 800nM) on sheep mesoderm cells for two days. The results showed that the cell morphology induced by different concentrations of LDN was similar, but the cell density was lower at 100nM, 400nM and 800nM ( Figure 6 A and C in Figure ), there was no significant difference in the relative expression of MEOX1 gene ( Figure 6 D).

[0084] Based on the use of 200nM LDN, we further explored the effects of adding RA signaling pathway activator Retinoic acid (RA), FGF signaling pathway inhibitor PD173074 (PD) and TGF-β signaling pathway inhibitor SB on the effect and efficiency of enhancing the differentiation of mesoderm cells into somites. The results showed that the cells induced by the combination of 1μM RA were round or short spindle-shaped, and the cells induced by the combination of 100nM PD and 10μM SB were mostly spindle-shaped, among which the cell density of the combination of SB was more uniform ( Figure 6E in Figure ). qRT-PCR results showed that compared with the use of LDN alone to induce differentiation, the combination of SB and LDN significantly increased the expression level of the MEOX1 gene ( Figure 6 F).

[0085] Therefore, a BMP signaling pathway inhibitor (200 nM LDN) and a TGF-β signaling pathway inhibitor (10 μM SB) were used in combination as key small molecules of differentiation medium II. That is, 10 μM SB was supplemented on the basis of the initially prepared differentiation medium II as the final differentiation medium II. The mesoderm cells were differentiated into somite cells for two days after induction of differentiation, and then the third stage of induced differentiation was carried out.

[0086] 3. The third stage is to induce differentiation - differentiate somites into myoblasts

[0087] After the sheep mesoderm cells were differentiated into somite cells on D3, LDN and SB were removed, and the differentiation was continued using the preliminary proposed differentiation medium III (86% DMEM basal medium, 10% FBS, 1% GultaMAX, 1% NEAA, 1% PS, 0.1mMβ-ME, 3μM CHIR99021) containing 3μM CHIR. On D4, the cells showed various morphologies, some were spindle-shaped, some were round or polygonal and aggregated, and more dead cells were seen. On the 5th day (D5), there was no obvious change in cell morphology, but the cell viability decreased ( Figure 7 A).

[0088] On the basis of using 3μM CHIR, the inventors added FGF2, an activator of the FGF signaling pathway, to improve the efficiency of sheep somite cells differentiating into myoblasts. The results showed that D4 cells were spindle-shaped, with fewer cells of other morphologies and dead cells, and a uniform distribution density. D5 cells gradually elongated, with a small amount of cell fusion ( Figure 7 B). Compared with the use of 3μM CHIR alone to induce differentiation, the combination of 20ng / mL FGF2 not only maintained the viability of cells at a higher density, but also promoted the differentiation of somite cells. The expression of myotome marker gene EN1 increased significantly on D4 of differentiation and decreased on D5; the expression of myoblast progenitor cell marker genes PAX3 and PAX7 increased slowly in the first three days of differentiation, increased significantly on D4, and decreased on D5; the expression of myoblast marker genes MYF5, MYF6, MYOD1, and MYOG increased significantly on D4 and decreased on D5 ( Figure 7 C). Cell immunochemistry results showed that D4 cells expressed MYOD1 protein ( Figure 7 The above results show that the characteristics of angel myogenic cells are most obvious when CHIR and FGF2 are combined to induce differentiation on the first day, but the subsequent addition is not conducive to myogenic differentiation.

[0089] Therefore, WNT signaling pathway activator (3 μM CHIR) and FGF signaling pathway activator (20 ng / mL FGF2) were used in combination as the key small molecules of differentiation medium III, that is, 20 ng / mL FGF2 was supplemented on the basis of the initially prepared differentiation medium III as the final differentiation medium III, and the somites cells were differentiated into myoblasts for one day of induction differentiation, and then the fourth stage of induced differentiation was carried out.

[0090] 4. The fourth stage is to induce differentiation - differentiate myoblasts into multinucleated myotubes

[0091] After the sheep somites were differentiated into myoblasts on D4, CHIR and FGF2 were removed and the differentiation medium IV (96% DMEM basal medium, 2% HS, 1% GultaMAX, 1% PS, 2ng / mL IGF-1) was replaced to continue inducing differentiation. The results showed that the cells were spindle-shaped on D5, and gradually elongated on the 6th day (D6). The adjacent cells arranged in a consistent manner, and cell fusion was observed. On the 7th day (D7), myotubes were clearly formed ( Figure 8 A in the figure), expressing MyHC2 marker genes and proteins ( Figure 8 B and C in ).

[0092] Experimental Example 1: Inducing Myogenic Differentiation of Sheep iPSCs by Embryoid Body Method

[0093] Embryoid body formation is one of the main methods for differentiating pluripotent stem cells into adult cells. This method simulates the physiological process of early embryonic development. This test example refers to the currently reported protocol for inducing myogenic differentiation of human and mouse pluripotent stem cells (ESCs and iPSCs) through embryoid body method, and myogenic differentiation of sheep iPSCs was performed.

[0094] The preparation method of embryoid bodies is as follows: observe the growth confluence of sheep iPSCs cells. When it reaches 85% to 90%, discard the original cell culture medium and wash three times with PBS. Add 0.25% trypsin to digest for 3min to 4min, and observe the digestion during the period. When the cell bulges and becomes round, add an equal volume of M15 culture medium to terminate the digestion, collect the cells into a 15mL centrifuge tube, and centrifuge at 1000r / min for 5min. After centrifugation, discard the supernatant, add an appropriate amount of embryoid body growth medium (86% DMEM basal medium, 10% FBS, 1% GlutaMAX, 1% NEAA, 1% PS, 0.1mMβ-ME) to the cell pellet, blow and mix, and evenly inoculate the cell suspension into a 0.1% gelatin pretreated cell culture dish, shake well and place it in a 37°C, 5% CO2 cell culture incubator, and culture for 30min to 40min to remove the feeder layer cells (if the sheep iPSCs cell density is high, the waiting time can be appropriately extended). Collect the non-adherent cells into a 15 mL centrifuge tube and centrifuge at 1000 r / min for 5 min. After centrifugation, discard the supernatant, add an appropriate amount of embryoid body culture medium to the cell pellet, count the cells, and dilute the cell suspension to 5×10 5 Prepare a cell culture dish, open the lid so that the inner surface faces upward, add an appropriate amount of PBS to the dish, and carefully drip the diluted cell suspension onto the lid, 20 μL per drop. After dripping the cell suspension at appropriate intervals, carefully turn the lid upside down onto the dish and place it in a 37°C, 5% CO2 cell culture incubator for hanging drop culture. After three days of hanging drop culture of sheep iPSCs, obvious spheroids can be observed under the hanging drop liquid, which are embryoid bodies.

[0095] During the myogenic differentiation of sheep iPSCs, embryoid body differentiation medium (81% DMEM basal medium, 10% FBS, 5% HS, 1% GultaMAX, 1% NEAA, 1% PS, 0.1mM β-ME) was used to induce differentiation during the hanging drop-suspension-adherence process. After three days of hanging drop culture, the embryoid bodies were carefully aspirated and transferred to a 12-well plate to which embryoid body differentiation medium had been added, with 8 to 10 cells per well, and placed in a 37°C, 5% CO2 cell culture incubator for suspension culture. During the suspension culture, be careful to prevent the embryoid bodies from adhering to the wall. If there are embryoid bodies adhering to the wall, they can be gently blown with a pipette to suspend them. After three days of cell suspension culture, the embryoid bodies were carefully aspirated, and the embryoid bodies were evenly inoculated in a 12-well plate with 1% matrix gel and embryoid body culture medium. After shaking well, they were placed in a 37°C, 5% CO2 cell culture incubator to allow them to adhere to the wall and differentiate. After three days of culture, i.e., differentiation day 9 (D9), embryoid bodies were observed to grow on the wall. The cells in the center were denser and round or short spindle-shaped; the surrounding cells continued to differentiate outward and became spindle-shaped. After three more days of differentiation, i.e., differentiation day 12 (D12), the cell density was more uniform, most of them were spindle-shaped, and other morphologies were also observed ( Fig. 9 A), indicating that embryoid bodies may differentiate into multiple cell types.

[0096] At the same time, the inventors tested the effects of different concentrations of HS on embryoid body differentiation. When the concentration of HS in the embryoid body differentiation medium was reduced from 5% to 2%, there was no significant difference in the differentiation process and differentiation effect ( Fig. 9 B). Although the induction method of embryoid body formation simulates the physiological process of embryonic development, it also has obvious disadvantages: first, the cells at the periphery of the embryoid body are sparse and can better contact the differentiation medium, while the cells in the center are denser and may not be completely differentiated. Because the cell distribution density at the periphery and center of the embryoid body is uneven, the differentiation process of cells may be inconsistent, resulting in low induced differentiation efficiency; second, the embryoid body induction cycle is long, and most embryoid body induction methods require flow cytometry sorting.

[0097] The results of this experiment indicate that the currently reported induction method for embryoid body formation is not suitable for myogenic differentiation of ovine iPSCs.

[0098] Experimental Example 2: Direct induction of myogenic differentiation of sheep iPSCs by monolayer adherent cells

[0099] The differentiation of pluripotent stem cells into myogenic cells requires multiple developmental stages, and different key signaling pathways and transcription factors play a role in different developmental stages. Currently, the protocols for inducing myogenic differentiation of human and mouse pluripotent stem cells through direct methods are different, including the type of basal culture medium, the type and concentration of serum, the combination and concentration of key signaling pathway small molecules, etc.

[0100] 1. Refer to the currently more mature and efficient human pluripotent stem cell myogenic differentiation induction protocol to differentiate sheep iPSCs into myogenic cells. Follow the flow chart ( Fig.10 A) in the experiment was used to induce differentiation. In this test example, differentiation medium I included: 93% DMEM basal medium, 5% HS, 1% GultaMAX, 1% PS, 3μM CHIR, 2μM SB, 200μM Vc, 10ng / mL EGF, 10mg / mL insulin, 0.4mg / mL dexamethasone; differentiation medium II included: 93% DMEM basal medium, 5% HS, 1% GultaMAX, 1% PS, 0.5μM LDN, 2μM SB, 200μM Vc, 10ng / mL EGF, 20ng / mL HGF, 20ng / mL FGF2, 10mg / mL insulin, 10ng / mL IGF-1; differentiation medium III included: 84% DMEM basal medium, 15% KSR, 10ng / mL IGF-1. The results showed that D2 cells showed various morphologies; D4 cells had a reduced survival rate and were long spindle-shaped ( Fig.10 B). Discard the original cell culture medium, wash three times with PBS, add 0.25% trypsin to digest for 3min~4min, and observe the digestion during the process. When the cell bulges become round, add an equal volume of differentiation medium I to stop digestion, collect the cells into a 15mL centrifuge tube, and centrifuge at 1000r / min for 5min. After centrifugation, discard the supernatant, add an appropriate amount of differentiation medium II (containing ROCK inhibitor) to the cell pellet, mix well by pipetting, and then add the cell suspension at 1×10 4 / cm 2 The cells were uniformly inoculated in the cell culture dish pretreated with matrix gel and induced to differentiate. The D6 cells were uniformly inoculated in various forms ( Fig.10 B); then continue to differentiate, and a large number of cells die. The results show that sheep iPSCs cannot be induced to differentiate into myogenic cells by referring to the current more mature and efficient human pluripotent stem cell myogenic differentiation induction protocol, indicating that there are certain differences in the developmental mechanisms between sheep and humans.

[0101] 2. The inventors tried to change the induction differentiation conditions based on the scheme of 1 to observe whether sheep iPSCs could be induced to differentiate into myogenic cells. Many studies have shown that the use of WNT signaling pathway activator CHIR alone can induce pluripotent stem cells to differentiate into mesoderm cells. Therefore, SB in differentiation medium I was removed, and different culture media were replaced in stages according to the above flowchart and steps to induce differentiation. The results showed that the cell morphology of D2 was similar to that of the combined use of CHIR and SB, but the cell density was higher; D4 cells were short spindle-shaped and evenly distributed; D6 cells were obviously paving stone-shaped; D8 cell density increased, and more dead cells were visible; D10 cell morphology changed from round to long spindle; D12 cells were further elongated, and adjacent cells had the same arrangement trend; D13 most cells were slender, cell fusion was visible, and cells of other morphologies were also visible ( Fig.10 C); after that, the differentiation continued, and a large number of cells died. By removing SB in differentiation medium I, the induced differentiation of sheep iPSCs was maintained for a longer time and the induced differentiation efficiency was higher, but the differentiation process of sheep iPSCs was inconsistent with that of humans in this scheme, the induction efficiency was still low, and no obvious fusion myotubes were observed.

[0102] The results showed that the removal of a small molecule in the first four days of differentiation significantly changed the cell activity and differentiation efficiency of sheep iPSCs, indicating that small molecules in the signaling pathway play an important role in the differentiation induction process, and the combination, action time and concentration of small molecules are very likely to be the key to inducing myogenic differentiation of sheep iPSCs. In addition, since the growth medium components of sheep iPSCs are different from those of humans and mice, differentiation cannot be carried out according to the differentiation medium of humans and mice. On the basis of the growth medium, key conditions such as the composition of the medium suitable for the differentiation of sheep iPSCs, the combination of small molecules, action time and concentration should be systematically explored.

[0103] Experimental Example 3 Exploration of serum types and concentrations during the myogenic differentiation stage of sheep iPSCs

[0104] According to the flow chart ( Fig.11A) in the experiment was used to induce differentiation, and new basal differentiation medium, small molecules and cytokines were replaced every day. This experimental example focused on testing the type and concentration of serum in the stage of differentiation of pluripotent stem cells into myogenic cells (i.e., the stage of basal differentiation medium I combined with small molecule induction). Basic differentiation medium I includes: DMEM basal medium (the proportion of the culture medium added needs to be adjusted according to the proportion of FBS or / and HS), FBS or / and HS, 1% GultaMAX, 1% NEAA, 1% PS, 0.1mM β-ME; basic differentiation medium II includes: 83% DMEM basal medium, 15% FBS, 1% GultaMAX, 1% PS; basic differentiation medium III includes: 96% DMEM basal medium, 2% HS, 1% GultaMAX, 1% PS.

[0105] First, ovine iPSCs were cultured at 3 × 10 4 / cm 2 After three days of subculture at a density of 100 μg / cm2, the cells were flat, round or oval in shape. At this time, the sheep iPSCs were induced to differentiate and marked as D0. The types and concentrations of serum in the basic differentiation medium I were tested, including serum-free, 2% FBS, 2% HS, 5% FBS, 5% HS, and 10% FBS. The results showed that when serum was not added, D2 cells differentiated from round clones to irregular paving stones, and the cells maintained a high survival rate. A large number of D3 cells died ( Fig.11 B); When 2% FBS or 2% HS was added, D2 cells were able to maintain a higher survival rate, D4 ​​cells had a decreased survival rate and were round or short spindle-shaped, and D6 cells had a further decreased survival rate, with most cells being long spindle-shaped, and cells of other morphologies were also seen ( Fig.11 C and D in the figure); When 5% FBS or 5% HS was added, the morphological changes of cells at each stage of differentiation (D2, D4 and D6) were similar to those when 2% FBS or 2% HS was added, but the cell survival rate was higher. At D8, the long spindle-shaped cells gradually elongated, a small number of cells fused, and the overall cell activity was poor. There was no obvious change in the morphology of cells that continued to differentiate ( Fig.11 E and F in Figure 3); When 10% FBS was added, the morphological changes of cells at each differentiation stage (D2, D4, D6 and D8) were similar to those when 5% FBS or 5% HS was added, but the cell survival rate was higher. At D10, the cells were further elongated, and the adjacent cells arranged in a consistent manner. The activity of cells decreased after continued differentiation until D12 ( Fig.11 The above results show that the cell survival rate and differentiation efficiency are the best when 10% FBS is used, however, the overall differentiation and fusion efficiency of cells is still low, and cells of other morphologies can be seen during the differentiation process, indicating that heterogeneous differentiation may be accompanied by induction of myogenic differentiation.

[0106] Further tests were conducted on the basis of 10% FBS combined with 2% HS and 5% HS. The results showed that the morphological changes and cell survival rates of cells at each stage of differentiation were similar to those of adding only 10% FBS, and the differentiation efficiency was not significantly improved ( Fig.11 H, I in.

[0107] Therefore, 10% FBS was used in the basal differentiation medium I to further explore and optimize the conditions for the directed myogenic differentiation of sheep iPSCs to improve the efficiency of myogenic differentiation.

[0108] Experimental Example 4: Exploration of the initial induction differentiation time of sheep iPSCs

[0109] Considering that direct differentiation of sheep iPSCs cell clones may cause uneven cell differentiation and heterogeneous differentiation, heterogeneous cells will affect directional myogenic differentiation and cell fusion, thereby reducing differentiation efficiency. In order to make the cells differentiate evenly, this experimental example attempts to induce differentiation before sheep iPSCs form cell clones, and tests the induced differentiation effect of sheep iPSCs after one day of subculture and one day of subculture + two days of natural differentiation. Basic differentiation medium I includes: 86% DMEM basic medium, 10% FBS, 1% GultaMAX, 1% NEAA, 1% PS, 0.1mM β-ME; basic differentiation medium II includes: 83% DMEM basic medium, 15% FBS, 1% GultaMAX, 1% PS; basic differentiation medium III includes: 96% DMEM basic medium, 2% HS, 1% GultaMAX, 1% PS.

[0110] 1. Induced differentiation of sheep iPSCs after one day of subculture

[0111] Sheep iPSCs were cultured at 3 × 10 4 / cm 2 After one day of subculturing, the cells have not yet grown into a clone morphology and are evenly distributed in the culture dish. At this time, differentiation induction begins, marked as D0, according to the flow chart ( Fig.12 A) in the above was used to induce differentiation, and the corresponding basic differentiation medium was replaced every day. Small molecules and cytokines were added to the above basic differentiation medium I, II, and III. The results showed that D1 cells were in small clumps with a sparse density; the survival rate of D2 cells further decreased ( Fig.12 To this end, we explored the initial cell density and the results showed that after increasing the initial cell density (4×10 4 / cm 2 ~10×10 4 / cm 2 ) effectively improved cell survival rate and differentiation efficiency, among which 7×10 4 / cm2 ~10×10 4 / cm 2 The differentiation effect is similar across the density range. 4 / cm 2 After 1 day of subculture at a density of 10, the cells were induced to differentiate. The cell density was high on D1; the cells began to adhere to the wall and differentiate on D2; the cells grew evenly and were short spindle-shaped on D4; the cells gradually elongated on D6 and were long spindle-shaped, with few cells of other morphologies; the cells continued to differentiate on D8, and the adjacent cells arranged in the same trend; the cells on D10 were long spindle-shaped or long strips, with clear boundaries between cells and obvious cell fusion; and the cell activity gradually decreased as the differentiation continued. Therefore, the initial cell density was increased to 7×10 4 / cm 2 ~10×10 4 / cm 2 After that, the efficiency of myogenic differentiation of sheep iPSCs was effectively improved. However, oxidative damage of cells was observed at D8, and too high cell density was not conducive to myoblast fusion in the later stage ( Fig.12 Therefore, we further add 4×10 4 / cm 2 ~6×10 4 / cm 2 The density range is determined by 4×10 4 / cm 2 As the final initial cell density (see Example 2).

[0112] 2. Sheep iPSCs were cultured for one day and then differentiated naturally for two days before induction of differentiation

[0113] Sheep iPSCs were cultured at 10 × 10 4 / cm 2 After one day of subculturing at a density of 100 μg / cm2, the basal differentiation medium I was replaced to allow the cells to differentiate naturally for two days. The cells began to differentiate from the clumps and adhered to the wall. The cell density was high and the distribution was uniform. At this time, differentiation was induced, marked as D0, and the flow chart ( Fig.13 A) in the above was used to induce differentiation, and the corresponding basic differentiation medium was replaced every day. Small molecules and cytokines were added to the above basic differentiation medium I, II, and III. The results showed that the cell density was relatively uniform at D1, and the cells were round or short spindle-shaped; the cells gradually elongated at D4; the cells were long spindle-shaped at D6, and the adjacent cells arranged in the same trend; the arrangement trend was more obvious at D8, and more cell fusion was seen; after continued differentiation, the cell activity gradually decreased ( Fig.13 B).

[0114] Compared with directly inducing differentiation of sheep iPSCs cell clones, inducing differentiation before forming cell clones is more efficient and produces fewer heterogeneous differentiated cells. Among them, more cell fusion can be seen in the cells induced and differentiated after one day of subculture, and the adjacent cells have a consistent arrangement trend, and a large number of slender myotubes are formed. Therefore, one day after subculture is used as the initial induction differentiation time.

[0115] The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0116] The above-mentioned embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. A method for inducing directed myogenic differentiation of sheep iPSCs, characterized in that: The following steps are involved: (1) Inducing differentiation of sheep iPSCs for 1 day using differentiation medium I to obtain mesoderm cells; the differentiation medium I comprises: 85% to 87% DMEM basal medium, 8% to 12% FBS, 0.8% to 1.2% GultaMAX, 0.8% to 1.2% NEAA, 0.8% to 1.2% PS, 0.08mM to 0.12mM β-ME, and 2μM to 5μM CHIR99021; (2) replacing the culture medium with differentiation medium II, inducing differentiation for 2 days to obtain somite cells; the differentiation medium II comprises: 85% to 87% DMEM basal medium, 8% to 12% FBS, 0.8% to 1.2% GultaMAX, 0.8% to 1.2% NEAA, 0.8% to 1.2% PS, 0.08mM to 0.12mM β-ME, 180nM to 220nM LDN193189, and 8μM to 12μM SB431542; (3) replacing the culture medium with differentiation medium III, inducing differentiation for 1 day to obtain myoblasts; the differentiation medium III comprises: 85% to 87% DMEM basal medium, 8% to 12% FBS, 0.8% to 1.2% GultaMAX, 0.8% to 1.2% NEAA, 0.8% to 1.2% PS, 0.08mM to 0.12mM β-ME, 2μM to 5μM CHIR99021, and 15ng / mL to 25ng / mL FGF2; (4) The culture medium was replaced with differentiation medium IV, and differentiation was induced for 3 days to obtain multinucleated myotubes; the differentiation medium IV included: 95% to 97% DMEM basal medium, 1.8% to 2.2% HS, 0.8% to 1.2% PS, and 1.8 ng / mL to 2.2 ng / mL IGF-1.

2. The method for inducing directed myogenic differentiation of sheep iPSCs according to claim 1, characterized in that: The method also includes the following steps before step (1): subculturing the sheep iPSCs into a cell culture dish pretreated with matrix gel, and culturing them for 1 day using M15 growth medium containing Y-27632.

3. The method for inducing directed myogenic differentiation of sheep iPSCs according to claim 2, characterized in that: The mass concentration of the matrix gel is 0.8% to 1.2%, preferably 1%.

4. The method for inducing directed myogenic differentiation of sheep iPSCs according to claim 2, characterized in that: The concentration of Y-27632 in the M15 growth medium is 8 μM to 12 μM, preferably 10 μM.

5. The method for inducing directed myogenic differentiation of sheep iPSCs according to claim 2, characterized in that: The density of the ovine iPSCs was 4×10 4 / cm 2 ~6×10 4 / cm 2 , preferably 4×10 4 / cm 2 .

6. The method for inducing directed myogenic differentiation of sheep iPSCs according to any one of claims 1 to 5, characterized in that: The concentration of CHIR99021 in the differentiation medium I in step (1) is 2 μM to 4 μM.

7. The method for inducing directed myogenic differentiation of sheep iPSCs according to any one of claims 1 to 5, characterized in that: In the differentiation medium II in step (2), the concentration of LDN193189 is 195nM to 205nM, and the concentration of SB431542 is 9.5μM to 10.5μM.

8. The method for inducing directed myogenic differentiation of sheep iPSCs according to any one of claims 1 to 5, characterized in that: In step (3), the concentration of CHIR99021 in the differentiation medium III is 2 μM to 4 μM, and the concentration of FGF2 is 19 ng / mL to 21 ng / mL.

9. The method for inducing directed myogenic differentiation of sheep iPSCs according to claim 1, characterized in that: The following steps are involved: (1) The density is 4×10 4 / cm 2 ~6×10 4 / cm 2 The sheep iPSCs were passaged into cell culture dishes pretreated with 0.8% to 1.2% Matrigel and cultured for 1 day using M15 growth medium containing Y-27632; (2) Inducing differentiation of sheep iPSCs for 1 day using differentiation medium I to obtain mesoderm cells; the differentiation medium I comprises: 85% to 87% DMEM basal medium, 8% to 12% FBS, 0.8% to 1.2% GultaMAX, 0.8% to 1.2% NEAA, 0.8% to 1.2% PS, 0.08mM to 0.12mM β-ME, and 2μM to 4μM CHIR99021; (3) replacing the culture medium with differentiation medium II, inducing differentiation for 2 days to obtain somite cells; the differentiation medium II comprises: 85% to 87% DMEM basal medium, 8% to 12% FBS, 0.8% to 1.2% GultaMAX, 0.8% to 1.2% NEAA, 0.8% to 1.2% PS, 0.08 mM to 0.12 mM β-ME, 195 nM to 205 nM LDN193189, and 9.5 μM to 10.5 μM SB431542; (4) replacing the culture medium with differentiation medium III, inducing differentiation for 1 day to obtain myoblasts; the differentiation medium III comprises: 85% to 87% DMEM basal medium, 8% to 12% FBS, 0.8% to 1.2% GultaMAX, 0.8% to 1.2% NEAA, 0.8% to 1.2% PS, 0.08mM to 0.12mM β-ME, 2μM to 4μM CHIR99021, and 19ng / mL to 21ng / mL FGF2; (5) The culture medium was replaced with differentiation medium IV, and differentiation was induced for 3 days to obtain multinucleated myotubes; the differentiation medium IV included: 95% to 97% DMEM basal medium, 1.8% to 2.2% HS, 0.8% to 1.2% PS, and 1.8 ng / mL to 2.2 ng / mL IGF-1.

10. Use of the method for inducing directed myogenic differentiation of sheep iPSCs according to any one of claims 1 to 9 in sheep in vitro muscle tissue engineering.

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