Stem cell-derived function-enhanced mesenchymal stromal cell as well as preparation method and application thereof

Abstract

The invention discloses a stem cell-derived function-enhanced mesenchymal stromal cell as well as a preparation method and application thereof. The preparation method comprises the following steps: inserting an IL-10 gene coding sequence and a CD47 gene coding sequence into a human pluripotent stem cell; a neural crest cell differentiation culture medium containing a Wnt pathway agonist and an ALK5 inhibitor is used for carrying out induced differentiation culture on the CD47 positive human pluripotent stem cells to obtain the neural crest cells. The neural crest cells are subjected to induced differentiation culture by adopting a transition culture medium containing an epidermal growth factor, a fibroblast growth factor 2 and a cell apoptosis inhibitor and a mesenchymal stromal cell differentiation culture medium containing the epidermal growth factor, the fibroblast growth factor 2 and a platelet growth factor in sequence; according to the present invention, with the combination of the specific embodiments, the obtained mesenchymal stromal cell highly expresses CD47 and IL-10, the expression of the CD47 can help the transplanted cell escape from the attack of the immune system and prolong the cell survival time, and the expression of the IL-10 can be used for anti-inflammation.

Description

Technical Field

[0001] The present invention relates to the field of cell therapy, and in particular to a stem cell-derived function-enhanced mesenchymal stromal cell and a preparation method and application thereof. Background Art

[0002] There are many methods for skin injury repair, including traditional wound suturing, flap transplantation, skin substitutes, cell therapy, etc. However, each method has its limitations. 1) Wound suturing: For large, complex or deep wounds, suturing may not be effective enough. In addition, it may cause scar formation. 2) Flap transplantation: It requires surgical operation and may cause scars after surgery. Scars may also be left in the donor area, and there is a risk of local flap ischemia and necrosis. 3) Skin substitutes: The cost is high and the effect of repairing deep wounds may be limited. Some artificial substitutes may cause rejection. 4) Cell therapy: It is still in the research stage in clinical application, and its safety and effectiveness need more verification. The cost may be high. 5) Bioengineered skin: It is expensive and may cause rejection. The production and application process is relatively complicated.

[0003] Similarly, there are many limitations to the current treatments for osteoarthritis: 1) Most treatments for osteoarthritis are mainly symptomatic rather than targeting the root cause of the disease. These methods include relieving pain, reducing inflammation, and improving joint function, but they cannot completely cure the disease; 2) Side effects of drug treatment: Drugs commonly used to treat osteoarthritis, such as nonsteroidal anti-inflammatory drugs (NSAIDs), differential kinase inhibitors, and analgesics, may cause a range of side effects, including gastrointestinal problems, cardiovascular risks, and kidney problems; 3) Limitations of joint replacement surgery: For some patients with more severe osteoarthritis, joint replacement surgery may be an effective treatment. However, surgery is not suitable for all patients and has some potential risks and limitations, such as a longer recovery period after surgery, possible infection risks, and limited prosthesis life.

[0004] The successful isolation and culture of human embryonic stem cells (hESCs) and the establishment of human pluripotent stem cells (hPSCs) technology have provided a large number of cell resources for scientific research and clinical applications. In recent years, methods for obtaining cells of specific tissues with normal physiological functions by directed differentiation of stem cells in vitro have been continuously established. These stem cell-derived tissue cells have broad application prospects in the construction of disease models, drug screening, and in vivo transplantation to treat diseases.

[0005] Mesenchymal stromal cells (MSCs) are a type of multipotent adult stem cells that have the potential to self-renew and differentiate into a variety of cell types. Therefore, they have received widespread attention and have important applications in clinical and research fields: 1) Multipotential differentiation: MSCs can differentiate into a variety of cell types, including osteoblasts, chondrocytes, adipocytes, etc. This multipotency makes them have a wide range of application potential in tissue engineering and regenerative medicine. 2) Self-renewal: MSCs have a certain degree of self-renewal ability and can maintain their number in vivo or in vitro, which is crucial for long-term treatment and application. 3) Immunomodulation: MSCs exhibit immunomodulatory properties, which can inhibit the activity of immune cells and reduce inflammatory responses. This property makes MSCs have therapeutic potential in autoimmune diseases and transplant rejection. 4) Secreted factors: MSCs can secrete a variety of bioactive factors, which regulate cell proliferation, differentiation, apoptosis and other processes. The role of these secreted factors is of great significance in maintaining tissue homeostasis and promoting the healing process. 5) Clinical application: Due to their multipotency, immunomodulatory and secretory properties, MSCs have been widely studied and applied clinically, including in fracture healing, cartilage repair, cardiovascular disease, autoimmune diseases, organ transplantation and other fields.

[0006] The properties of MSCs make them a strong candidate for promoting healing, reducing inflammation, and repairing tissues. Mesenchymal stromal cells can promote wound healing in the following ways: 1) Promote healing and tissue repair: MSCs have the potential to differentiate into different cell types, including fibroblasts, epidermal cells, etc., so they can participate in and promote the healing and repair process of the skin. 2) Secrete growth factors: MSCs can secrete a variety of growth factors, such as epidermal growth factor (EGF), vascular endothelial growth factor (VEGF), etc., which help stimulate cell proliferation, angiogenesis, and tissue regeneration. 3) Anti-inflammatory and immunomodulatory: MSCs exhibit anti-inflammatory and immunomodulatory properties, which help reduce inflammatory responses and improve the tissue healing environment, thereby promoting the recovery of skin injuries. 4) Fight scar formation: Some studies have shown that the application of MSCs can help slow down or reduce scar formation and promote better tissue repair. 5) Improve blood supply: The presence of MSCs and the growth factors they secrete help improve local blood circulation, provide more oxygen and nutrients, and thus facilitate the healing of the injured area.

[0007] MSCs can treat osteoarthritis through the following pathways: 1) Anti-inflammatory effect: MSCs are believed to have anti-inflammatory and immunomodulatory abilities. They can release a series of cytokines and chemical signals to inhibit inflammatory responses and reduce inflammatory symptoms caused by arthritis. This includes regulating the production of inflammatory mediators and cell-to-cell interactions, which helps maintain tissue homeostasis. 2) Promoting repair and regeneration: MSCs are able to differentiate into a variety of cell types, including bone cells, chondrocytes, and adipocytes. Therefore, they may help promote the repair and regeneration of damaged tissues in the treatment of osteoarthritis. This includes helping to rebuild joint cartilage, repair ligaments, and relieve joint pain. 3) Anti-apoptotic effect: MSCs may protect cells in damaged joints by reducing apoptosis (cell death). This helps maintain tissue integrity and function. 4) Stimulating angiogenesis: MSCs are also believed to have the ability to promote the formation of new blood vessels (angiogenesis). By increasing blood flow, MSCs can provide more oxygen and nutrients to the damaged area, promoting the healing process. 5) Immunomodulation: The regulatory effect of MSCs on the immune system helps balance inflammatory responses, inhibit autoimmune responses, and reduce symptoms caused by arthritis.

[0008] At present, the preparation of mesenchymal stromal cells (MSCs) mostly adopts the embryoid body generation method, which induces human pluripotent stem cells (hPSCs) to differentiate into mesenchymal stromal cells (MSCs) through small molecules and growth factors. Although embryoid bodies may be one of the methods to generate MSCs, it also has some disadvantages and challenges: 1) Since embryoid bodies are formed by spontaneous or induced differentiation, the generated MSCs may have different cell types and functions. This may lead to differences between batches, making the produced MSCs less consistent. 2) Complexity and uncertainty: The formation and differentiation of embryoid bodies is a complex process that involves multiple cell types and signaling pathways. This complexity increases the uncertainty of preparing MSCs, making it more difficult to control and optimize the production process. 3) Cost and efficiency: The production of embryoid bodies and the acquisition of MSCs from them can be an expensive and complicated process. 4) Safety: When using embryoid bodies to generate MSCs, careful research is needed to ensure that the generated MSCs do not have adverse immunogenicity or other safety issues. Summary of the invention

[0009] Based on this, it is necessary to provide a method for preparing stem cell-derived function-enhanced mesenchymal stromal cells that can solve the above problems.

[0010] In addition, it is also necessary to provide the stem cell-derived function-enhanced mesenchymal stromal cells prepared by the preparation method of the stem cell-derived function-enhanced mesenchymal stromal cells and the application of the stem cell-derived function-enhanced mesenchymal stromal cells.

[0011] A method for preparing stem cell-derived function-enhanced mesenchymal stromal cells comprises the following steps: Providing human pluripotent stem cells; Inserting an IL-10 gene coding sequence and a CD47 gene coding sequence into the human pluripotent stem cells, and obtaining CD47-positive human pluripotent stem cells after screening, wherein the CD47-positive human pluripotent stem cells can also express IL-10; Using a neural crest cell differentiation medium to induce differentiation and culture the CD47-positive human pluripotent stem cells to obtain neural crest cells, wherein the neural crest cell differentiation medium includes a Wnt pathway agonist and a TGF-β receptor kinase inhibitor; The neural crest cells are induced to differentiate and cultured using a transition medium and a mesenchymal stromal cell differentiation medium in sequence to obtain the desired stem cell-derived function-enhanced mesenchymal stromal cells, wherein the transition medium includes epidermal growth factor, fibroblast growth factor 2 and a cell apoptosis inhibitor, and the mesenchymal stromal cell differentiation medium includes epidermal growth factor, fibroblast growth factor 2 and platelet growth factor.

[0012] In one embodiment, the neural crest cell differentiation medium, the transition medium, and the mesenchymal stromal cell differentiation medium do not contain animal-derived components.

[0013] In one embodiment, the operation of inserting the IL-10 gene coding sequence and the CD47 gene coding sequence into the human pluripotent stem cells is: electroporating a main plasmid including the IL-10 gene coding sequence and the CD47 gene coding sequence and an auxiliary plasmid for locating the insertion position into the human pluripotent stem cells.

[0014] In one embodiment, the master plasmid is prepared by the following operation: using CRISPR Cas9 to insert the IL-10 gene coding sequence and the CD47 gene coding sequence into the CLYBL site of the master plasmid.

[0015] In one embodiment, the main plasmid is a donor plasmid, and the auxiliary plasmid is a gRNA plasmid.

[0016] In one embodiment, the operation of inducing differentiation culture of the CD47-positive human pluripotent stem cells using a neural crest cell differentiation medium is as follows: after subculturing the CD47-positive human pluripotent stem cells for 24 hours, the medium is replaced with the neural crest cell differentiation medium, the medium is replaced every 24 hours, and the culture is continued for 7 days.

[0017] In one embodiment, the operation of sequentially using a transition medium and a mesenchymal stromal cell differentiation medium to induce differentiation of the neural crest cells is as follows: resuspending the neural crest cells in the transition medium to a cell density of 1×10 5 Pieces / cm 2 ~2.5×10 5 Pieces / cm 2 Then, the neural crest cells were cultured in a cell culture incubator at 37° C. and 5% CO 2 , with the medium changed every 24 hours for 1 day, and then the culture medium was replaced with the mesenchymal stromal cell differentiation medium, with the medium changed every 24 hours for 14 days.

[0018] In one embodiment, the Wnt pathway agonist is CHIR99021, the TGF-β receptor kinase inhibitor is SB431542, and the apoptosis inhibitor is Y27632; The formula of the neural crest cell differentiation medium is as follows:

[0019] The formula of the transition medium is as follows:

[0020] The formula of the mesenchymal stromal cell differentiation medium is as follows: .

[0021] A stem cell-derived function-enhanced mesenchymal stromal cell is prepared by the above-mentioned method for preparing the stem cell-derived function-enhanced mesenchymal stromal cell.

[0022] A use of stem cell-derived function-enhanced mesenchymal stromal cells for preparing therapeutic devices and / or drugs for skin damage and / or osteoarthritis.

[0023] The method for preparing stem cell-derived function-enhanced mesenchymal stromal cells of the present invention comprises the following steps: inserting an IL-10 gene coding sequence and a CD47 gene coding sequence into the human pluripotent stem cells, inducing differentiation and culturing the CD47-positive human pluripotent stem cells using a neural crest cell differentiation medium comprising a Wnt pathway agonist and a TGF-β receptor kinase inhibitor, thereby obtaining neural crest cells; sequentially inducing differentiation and culturing the neural crest cells using a transition medium comprising epidermal growth factor, fibroblast growth factor 2 and a cell apoptosis inhibitor and a mesenchymal stromal cell differentiation medium comprising epidermal growth factor, fibroblast growth factor 2 and platelet growth factor, thereby obtaining the desired stem cell-derived function-enhanced mesenchymal stromal cells; in combination with a specific embodiment, the obtained stem cell-derived function-enhanced mesenchymal stromal cells highly express CD47 and IL-10, and the expression of CD47 can help transplanted cells escape the attack of the immune system and prolong the cell survival time, and the expression of IL-10 can be used for anti-inflammatory purposes.

[0024] Among them, Wnt is an acronym in the field of genetics, which stands for "Wingless / Integrated". The Wnt signaling pathway is a complex protein action network, whose functions are most commonly seen in embryonic development and cancer, but also involved in the normal physiological processes of adult animals. The Wnt signaling pathway is a signal transduction pathway of a group of multiple downstream channels stimulated by the binding of the ligand protein Wnt and the membrane protein receptor. Through this pathway, the extracellular signal is transmitted to the cell through the activation process of the intracellular segment of the cell surface receptor.

[0025] Among them, transforming growth factor-β (TGF-β) belongs to a newly discovered TGF-β superfamily that regulates cell growth and differentiation.

[0026] Among them, CD47 is a cell surface protein that is widely expressed in various cell types. When it binds to its ligand SIRPα (signal regulatory protein α), it can send a "don't eat me" signal and inhibit the phagocytosis of macrophages. In immune rejection reactions (such as transplant rejection), the role of CD47 antibodies is mainly to regulate immune responses by blocking the interaction between CD47 and SIRPα, thereby affecting transplant rejection reactions.

[0027] Among them, IL-10 (interleukin-10) is an anti-inflammatory cytokine that is widely involved in regulating immune and inflammatory responses. IL-10 inhibits inflammatory responses and promotes immune tolerance through multiple mechanisms, and is an important regulator of the balance of the immune system.

[0028] In combination with specific embodiments, the mesenchymal stromal cells with high expression of IL-10 and CD47 prepared by the method for preparing functionally enhanced mesenchymal stromal cells derived from stem cells of the present invention can effectively alleviate the immune rejection reaction during cell therapy and effectively treat osteoarthritis.

[0029] Preferably, in the method for preparing stem cell-derived function-enhanced mesenchymal stromal cells of the present invention, the neural crest cell differentiation medium, the transition medium and the mesenchymal stromal cell differentiation medium do not contain animal-derived components (for example, FBS, BSA), and the entire cell culture process is in a serum-free culture medium and matrix. The differences between batches are small, and the obtained cell components are clear, thereby achieving the purpose of serum-free differentiation and reducing differentiation costs, thereby providing an effective method for the treatment of skin injuries.

[0030] The preparation method of the stem cell-derived functionally enhanced mesenchymal stromal cells of the present invention is based on a non-animal-derived and chemically determined culture medium, and the obtained MSC has a strong proliferation ability and can effectively treat skin damage and osteoarthritis. The differentiation scheme does not use animal-derived components such as FBS and BSA, excludes allergens and pathogens that may be brought by animal components, and has higher safety. At the same time, the chemical composition of each culture medium is determined, and a more stable differentiation efficiency can be maintained between batches, providing a more high-quality MSCs resource for cell therapy. At the same time, the present invention can produce mesenchymal mechanism cells with high expression of IL-10 and CD47, which can effectively alleviate the immune rejection reaction during cell therapy, and effectively treat osteoarthritis. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0032] in: Figure 1 The present invention is a flowchart of a method for preparing stem cell-derived mesenchymal stromal cells with enhanced function according to one embodiment.

[0033] Figure 2 This is the plasmid map of the main plasmid donor plasmid.

[0034] Figure 3 This is the plasmid map of the auxiliary plasmid gRNA plasmid.

[0035] Figure 4 This is a flow cytometry diagram of CD47-positive human pluripotent stem cells in Example 2.

[0036] Figure 5 This is a diagram showing the IL-10 ELISA test results of the CD47-positive human pluripotent stem cells sorted in Example 2.

[0037] Figure 6 Flow cytometry analysis of MSCs differentiation efficiency under four differentiation strategies in Example 3, wherein the red line is the negative control.

[0038] Figure 7 This is a graph showing the marker gene detection results of NCCs in test example 1.

[0039] Figure 8 This is a graph showing the marker expression detection results of NCCs in test example 2.

[0040] Fig. 9 This is a flow cytometry analysis diagram of the differentiation efficiency of MSCs in Test Example 3, wherein the red line is the negative control.

[0041] Fig.10 These are the staining photos of MSCs in Test Example 4 after they differentiated into chondrocytes, adipocytes and osteoblasts, respectively.

[0042] Fig.11 This is a graph showing the results of MSCs passaging and cell counting in Test Example 4.

[0043] Fig.12 This is a graph showing the results of flow cytometry detection of GFP positive ratio after co-culture and passaging of GPF-MSCs and UCSMCs in Test Example 4.

[0044] Fig.13 for Fig.12 Statistical chart of .

[0045] Fig.14 This is a graph showing the results of treating skin damage with MSCs and PBS in Test Example 4.

[0046] Fig.15 This is a graph showing the results of test example 4 for the survival of MSCs in the skin injury site of NSG mice.

[0047] Fig.16 This is a graph showing the survival of MSCs in the joint cavity of nude mice in Test Example 4.

[0048] Fig.17 The results of CT imaging of rats with osteoarthritis treated with MSCs, eMSCs and PBS groups in Test Example 4 are shown.

[0049] Fig.18 For Fig.17The results of quantitative analysis are shown in the figure, and the measured data include bone mineral density (BMD), trabecular bone volume per total volume (BV / TV) and trabecular bone pattern factor (Tb. Pf).

[0050] Fig.19 This is a graph showing the IL-10 ELISA test results of MSC and eMSC in Test Example 4. DETAILED DESCRIPTION

[0051] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0052] Combination Figure 1 The present invention discloses a method for preparing stem cell-derived function-enhanced mesenchymal stromal cells according to an embodiment of the present invention, comprising the following steps: S10. Provide human pluripotent stem cells.

[0053] Preferably, in the method for preparing stem cell-derived function-enhanced mesenchymal stromal cells of this embodiment, all culture media used do not contain animal-derived components, including various animal sera.

[0054] More preferably, the animal-derived ingredients also include animal protein.

[0055] That is, in the method for preparing stem cell-derived mesenchymal stromal cells with enhanced function according to the present embodiment, the added polypeptides and proteins are all human or recombinant products.

[0056] In this embodiment, the human pluripotent stem cells are purchased hPSC cell line H9. In other embodiments, human induced pluripotent stem cells can also be obtained by artificial induction.

[0057] Generally, the commercially available hPSC cell line H9 is stored in liquid nitrogen.

[0058] The specific steps of providing human pluripotent stem cells are as follows: performing operations of reviving, replacing fluid and subculturing the human pluripotent stem cells.

[0059] S20. Inserting the IL-10 gene coding sequence and the CD47 gene coding sequence into the human pluripotent stem cells obtained in S10, and obtaining CD47-positive human pluripotent stem cells after screening.

[0060] In conjunction with the specific embodiment, the screened CD47-positive human pluripotent stem cells can also express IL-10.

[0061] Immune rejection is a major challenge in cell therapy, especially in allogeneic (i.e., donor-derived) cell transplants.

[0062] The method for preparing stem cell-derived functionally enhanced mesenchymal stromal cells of this embodiment inserts the IL-10 gene coding sequence and the CD47 gene coding sequence into human pluripotent stem cells through gene editing, so that the ultimately obtained stem cell-derived functionally enhanced mesenchymal stromal cells can express CD47 and IL-10. CD47 can help transplanted cells escape the attack of the immune system and prolong cell survival time, and IL-10 has an anti-inflammatory effect.

[0063] CD47 is a cell surface protein that is widely expressed in various cell types. When it binds to its ligand SIRPα (signal regulatory protein α), it can send a "don't eat me" signal and inhibit the phagocytosis of macrophages. In immune rejection reactions (such as transplant rejection), the role of CD47 antibodies is mainly to regulate immune responses by blocking the interaction between CD47 and SIRPα, thereby affecting transplant rejection reactions.

[0064] Specifically, the mechanism of CD47 anti-rejection is as follows: Inhibit the phagocytosis of macrophages: CD4-expressing cells bind to SIRPα on macrophages, sending a "don't eat me" signal to prevent macrophages from engulfing these cells. By using anti-CD4 antibodies, this signal can be blocked, allowing macrophages to recognize and eliminate these cells. This can reduce the host immune system's rejection of the transplant during the transplant process.

[0065] Modulating immune response: The interaction between CD47 and SIRPα not only affects the activity of macrophages, but may also regulate the function of other immune cells. By blocking CD47, the immune response can be modulated, thereby reducing transplant rejection.

[0066] Promoting graft survival: By using anti-CD4 antibodies, the immune attack on the graft can be reduced, thereby promoting graft survival. This is of great significance for organ transplantation and stem cell transplantation.

[0067] IL-10 (interleukin-10) is an anti-inflammatory cytokine that is widely involved in regulating immune and inflammatory responses. IL-10 inhibits inflammatory responses and promotes immune tolerance through multiple mechanisms, and is an important regulator of the balance of the immune system.

[0068] The anti-inflammatory mechanism of IL-10: 1) Inhibit the production of pro-inflammatory cytokines: IL-10 can inhibit the production of a variety of pro-inflammatory cytokines such as TNF-α, IL-6 and IL-12 by antigen-presenting cells such as macrophages and dendritic cells, thereby reducing the intensity of the inflammatory response. 2) Inhibit the expression of antigen presentation and co-stimulatory molecules: IL-10 can reduce the expression of MHC class II molecules and co-stimulatory molecules (such as CD80 and CD86) on the surface of macrophages and dendritic cells, thereby inhibiting the activation of T cells and further reducing the immune response. 3) Promote the function of regulatory T cells: IL-10 can enhance the generation and function of regulatory T cells (Tregs). Tregs are a type of cell that can inhibit immune responses and maintain immune tolerance. IL-10 regulates immune responses by supporting the activity of Tregs to prevent excessive inflammation and autoimmune responses. 4) Promote tissue repair: IL-10 also has the function of promoting tissue repair. By reducing the inflammatory response, IL-10 can reduce tissue damage and promote the regenerative process.

[0069] IL-10 anti-inflammatory application in treatment: IL-10 has important application potential in the treatment of chronic inflammatory diseases, autoimmune diseases and tissue damage. 1) Treatment of inflammatory diseases: In diseases such as inflammatory bowel disease (such as Crohn's disease and ulcerative colitis), rheumatoid arthritis and psoriasis, IL-10 can be used to reduce inflammatory responses and relieve disease symptoms. 2) Control of transplant immune rejection: By using IL-10, the host immune system's attack on the transplant after transplantation can be inhibited, reducing the occurrence of transplant rejection. 3) Tissue engineering and regenerative medicine: In the process of tissue repair and regeneration, the use of matrix materials containing IL-10 can reduce the inflammatory response at the transplant site and promote tissue healing and regeneration.

[0070] Preferably, in this embodiment, the operation of inserting the IL-10 gene coding sequence and the CD47 gene coding sequence into human pluripotent stem cells is: electroporating a main plasmid including the IL-10 gene coding sequence and the CD47 gene coding sequence and an auxiliary plasmid for locating the insertion position into human pluripotent stem cells.

[0071] More preferably, in this embodiment, the master plasmid is prepared by the following operation: using CRISPR Cas9 to insert the IL-10 gene coding sequence and the CD47 gene coding sequence into the CLYBL site of the master plasmid.

[0072] Specifically, in this embodiment, the main plasmid is a donor plasmid, and the auxiliary plasmid is a gRNA plasmid.

[0073] Specifically, in this embodiment, combined with Figure 2 , the main plasmid is pTLL98.

[0074] The functions of the key components of pTLL98 are as follows: CLYBL homology arms: where in the genome to insert the sequence to be overexpressed; CAG: drives the expression of IL-10, CD47, and HygR; IL-10: coding sequence of human IL-10; CD47: coding sequence of human CD47; IRES: internal ribosome entry site; HygroR: hygromycin resistance gene coding sequence.

[0075] Specifically, in this embodiment, combined with Figure 3 , the helper plasmid is pTLL56.

[0076] The functions of the key elements of the pTLL56 plasmid are as follows: Cas9: cuts genomic DNA; sgRNA: guide RNA (gRNA), which determines where the Cas9 protein cuts the genome.

[0077] Among them, the sequence of sgRNA is shown as SEQ ID No.1.

[0078] The specific principle is as follows: pTLL56 uses Cas9 protein to cut at the sgRNA site in the human genome, pTLL98 provides a gene repair template, and a part of the cells will homologously recombine the left and right homologous arms of pTLL98 and the sequence between the left and right homologous arms (including CAG promoter, overexpressed human IL-10 and CD47 sequences, and the hygromycin resistance gene HygroR used for hygromycin screening) into the genome during the DNA self-repair process. These cells are selected through screening to obtain a cell line overexpressing IL-10 and CD47.

[0079] S30. Inducing differentiation and culturing the CD47-positive human pluripotent stem cells obtained in S20 using a neural crest cell differentiation medium to obtain neural crest cells.

[0080] The neural crest cell differentiation medium includes a Wnt pathway agonist and a TGF-β receptor kinase inhibitor.

[0081] Wnt is an acronym in the field of genetics, standing for "Wingless / Integrated". The Wnt signaling pathway is a complex network of protein interactions, whose functions are most commonly seen in embryonic development and cancer, but also involved in normal physiological processes in adult animals. The Wnt signaling pathway is a signal transduction pathway of a group of multiple downstream channels stimulated by the binding of the ligand protein Wnt and the membrane protein receptor. Through this pathway, extracellular signals are transmitted to the cell through the activation process of the intracellular segment of the cell surface receptor.

[0082] Transforming growth factor-β (TGF-β) belongs to a recently discovered group of TGF-β superfamily that regulates cell growth and differentiation.

[0083] Preferably, in this embodiment, the neural crest cell differentiation medium does not contain animal-derived components, and the specific description is referred to above.

[0084] More preferably, in this embodiment, the Wnt pathway agonist and the TGF-β receptor kinase inhibitor are both human or recombinant products.

[0085] Specifically, in this embodiment, the operation of inducing differentiation culture of CD47-positive human pluripotent stem cells using neural crest cell differentiation medium is as follows: after subculturing CD47-positive human pluripotent stem cells for 24 hours, the culture medium is replaced with neural crest cell differentiation medium, and the medium is replaced every 24 hours for 7 days.

[0086] Specifically, in this embodiment, the Wnt pathway agonist is CHIR99021, the TGF-β receptor kinase inhibitor is SB431542, and the cell apoptosis inhibitor is Y27632.

[0087] Specifically, in this embodiment, the formula of the neural crest cell differentiation medium is shown in Table 1 below.

[0088] Table 1: Neural crest cell differentiation medium

[0089] Among them, GlutaMAX is L-alanyl-L-glutamine solution, Pen / Strep is penicillin-streptomycin dual antibody, NEAA is non-essential amino acids, N2 is N-2 additive, and 2-mercaptoethanol is β-mercaptoethanol.

[0090] S40, sequentially using a transition culture medium and a mesenchymal stromal cell differentiation culture medium to induce differentiation of the neural crest cells obtained in S30, to obtain the desired stem cell-derived function-enhanced mesenchymal stromal cells.

[0091] Among them, the transition culture medium includes epidermal growth factor (EGF), fibroblast growth factor 2 (FGF2) and cell apoptosis inhibitor, and the mesenchymal stromal cell differentiation medium includes epidermal growth factor (EGF), fibroblast growth factor 2 (FGF2) and platelet-derived growth factor (PDGFBB).

[0092] Preferably, in this embodiment, neither the transition culture medium nor the mesenchymal stromal cell differentiation culture medium contains animal-derived components, and the specific description is referred to above.

[0093] More preferably, in this embodiment, epidermal growth factor, fibroblast growth factor 2, cell apoptosis inhibitor and platelet-derived growth factor are all human or recombinant products.

[0094] Specifically, in this embodiment, the operation of sequentially using the transition medium and the mesenchymal stromal cell differentiation medium to induce differentiation of neural crest cells is as follows: resuspending the neural crest cells in the transition medium to a cell density of 1×10 5 Pieces / cm 2 ~2.5×10 5 Pieces / cm 2 Then, the neural crest cells were cultured in a cell culture incubator at 37°C and 5% CO2, with the medium changed every 24 hours for 1 day. Then, the culture medium was replaced with mesenchymal stromal cell differentiation medium, with the medium changed every 24 hours for 14 days.

[0095] Specifically, in this embodiment, the Wnt pathway agonist is CHIR99021, the TGF-β receptor kinase inhibitor is SB431542, and the cell apoptosis inhibitor is Y27632.

[0096] Specifically, in this embodiment, the formula of the transition culture medium is shown in Table 2 below.

[0097] Table 2: Transition culture medium

[0098] Specifically, in this embodiment, the formula of the mesenchymal stromal cell differentiation medium is shown in Table 3 below.

[0099] Table 3: Mesenchymal stromal cell differentiation medium

[0100] Among them, Vc is vitamin C.

[0101] Glutamine is an essential component for maintaining cell protein synthesis and proliferation in in vitro cell culture. However, since glutamine is prone to spontaneous decomposition, a more stable alanine-glutamic acid (alanine-glutamic acid dipeptide, trade name GlutaMAX) is currently often used as a substitute for glutamine. Therefore, 1×GlutaMAX should be included in the basal culture medium.

[0102] Vitamin C (Vitamin C, Vc) has antioxidant and apoptosis-inhibiting effects in in vitro cell culture. During the cell differentiation process, since cells are stimulated by strong exogenous signals, most current differentiation strategies include Vc as a component of the basal culture medium, but its concentration should be optimized for different systems.

[0103] The method for preparing stem cell-derived function-enhanced mesenchymal stromal cells of the present invention comprises the following steps: inserting an IL-10 gene coding sequence and a CD47 gene coding sequence into human pluripotent stem cells, inducing differentiation and culturing CD47-positive human pluripotent stem cells using a neural crest cell differentiation medium comprising a Wnt pathway agonist and a TGF-β receptor kinase inhibitor, thereby obtaining neural crest cells; and sequentially inducing differentiation and culturing neural crest cells using a transition medium comprising epidermal growth factor, fibroblast growth factor 2 and a cell apoptosis inhibitor and a mesenchymal stromal cell differentiation medium comprising epidermal growth factor, fibroblast growth factor 2 and platelet growth factor, thereby obtaining the desired stem cell-derived function-enhanced mesenchymal stromal cells. In combination with a specific embodiment, the obtained stem cell-derived function-enhanced mesenchymal stromal cells highly express CD47 and IL-10, wherein the expression of CD47 can help transplanted cells escape the attack of the immune system and prolong the survival time of cells, and the expression of IL-10 can be used for anti-inflammatory purposes.

[0104] In combination with specific embodiments, the mesenchymal stromal cells with high expression of IL-10 and CD47 prepared by the method for preparing functionally enhanced mesenchymal stromal cells derived from stem cells of the present invention can effectively alleviate the immune rejection reaction during cell therapy and effectively treat osteoarthritis.

[0105] Preferably, in the preparation method of stem cell-derived function-enhanced mesenchymal stromal cells of the present invention, the neural crest cell differentiation medium, the transition medium and the mesenchymal stromal cell differentiation medium do not contain animal-derived components (for example, FBS, BSA), and the entire cell culture process is in a serum-free culture medium and matrix. The differences between batches are small, and the obtained cell components are clear, thereby achieving the purpose of serum-free differentiation and reducing differentiation costs, providing an effective method for the treatment of skin injuries.

[0106] The preparation method of the stem cell-derived functionally enhanced mesenchymal stromal cells of the present invention is based on a non-animal-derived and chemically determined culture medium, and the obtained MSC has a strong proliferation ability and can effectively treat skin damage and osteoarthritis. The differentiation scheme does not use animal-derived components such as FBS and BSA, excludes allergens and pathogens that may be brought by animal components, and has higher safety. At the same time, the chemical composition of each culture medium is determined, and a more stable differentiation efficiency can be maintained between batches, providing a more high-quality MSCs resource for cell therapy. At the same time, the present invention can produce mesenchymal mechanism cells with high expression of IL-10 and CD47, which can effectively alleviate the immune rejection reaction during cell therapy, and effectively treat osteoarthritis.

[0107] The present invention also discloses a method for preparing the above-mentioned stem cell-derived function-enhanced mesenchymal stromal cells, which is a method for preparing the stem cell-derived function-enhanced mesenchymal stromal cells according to an embodiment of the present invention.

[0108] The stem cell-derived mesenchymal stromal cells with enhanced functions of the present invention have various uses.

[0109] Specifically, in combination with the specific embodiments, the stem cell-derived function-enhanced mesenchymal stromal cells of the present invention can effectively treat skin injuries and osteoarthritis.

[0110] Therefore, the stem cell-derived mesenchymal stromal cells with enhanced functions of the present invention can be used to prepare therapeutic devices and / or drugs for skin damage and / or osteoarthritis.

[0111] The following are specific embodiments.

[0112] In a specific embodiment, DMEM / F12 (Gibco, C11330500BT), Advanced DMEM / F12 (Gibco, 12634010), ncTarget Basal Medium (Shouning Biotechnology, RP01020), ncTarget Supplement A (Shouning Biotechnology, RP01020), ncTarget Supplement B (Shouning Biotechnology, RP01020), Matrigel (Corning Matrigel, 354277), hygromycin (Invivogen, ant-hg-1), ITS-X (growth additive insulin-transferrin-selenium-ethanolamine additive, Procell, PB180431-10mL), FBS (Gibco, 12664025), GlutaMAX (Gibco, 35050061), NEAA (Gibco, 11140050), N2 (Yeasen, 60706ES08), SB431542 (Taoshu Biotechnology, T1726), CHIR99021 (Taoshu Biotechnology, T2310), EGF (Sino Biological, 10605-HNAE), FGF2 (Sino Biological, 10605-HNAE), Y27632 (Taoshu Biotechnology, T1870), PDGFBB (Sino Biological, 10572-HNAE).

[0113] Example 1 Culture human pluripotent stem cells in an animal-free, chemically defined environment.

[0114] 1.1 Resuscitation of human pluripotent stem cells Table 4: Human pluripotent stem cell maintenance medium

[0115] 1) Prepare the human pluripotent stem cell maintenance medium according to Table 4. Then add Matrigel (Corning Matrix Gel, 354277) diluted with DMEM / F12 at a concentration of 1 μg / ml to the 6-well plate, gently shake the 6-well plate to evenly spread the solution in the wells, and coat the culture plate at room temperature for at least 30 minutes.

[0116] 2) Take out a tube of frozen hPSC cell line H9 (from Xiang Kuanhui's research group at Peking University School of Medicine) from liquid nitrogen. 3) Use tweezers to hold the cells and shake them quickly in a 37°C water bath to thaw them. Transfer the thawed cells to a 50 ml centrifuge tube.

[0117] 4) Use a 10 ml pipette to draw 10 ml of DMEM / F12 medium and slowly add it to the 50 ml centrifuge tube containing the thawed cells while continuously shaking the centrifuge tube to mix the solution. After all the solution has been added, centrifuge at 300 g for 3 minutes.

[0118] 5) Discard the supernatant, resuspend the cells in 4 ml human pluripotent stem cell maintenance medium, add Y27632 (Taoshu Biotechnology, T1870) at a final concentration of 5 μM to the cell suspension, and gently shake the centrifuge tube to mix the cells.

[0119] 6) Discard the DMEM / F12 in the coated culture plate, add 2 ml of the cell suspension in 5) to 2 wells of the 6-well plate, move the culture plate up and down and left and right 10 times to evenly spread the cells in the wells, and culture the cells in a 37℃ 5% CO2 cell culture incubator.

[0120] 1.2 Human pluripotent stem cell culture medium replacement Every 24 h, the medium in the 6-well plate was discarded and replaced with 2 ml of human pluripotent stem cell maintenance medium per well.

[0121] 1.3 Passaging of human pluripotent stem cell maintenance medium When the cell density reaches 80-90%, subculture with human pluripotent stem cell maintenance medium is performed.

[0122] 1) Coat a 6-well plate following the steps in 1.1.

[0123] 2) Discard the culture medium in the 6-well plate, add 2 ml of 1× PBS to each well for washing, gently shake the 6-well plate to wash away the dead cells in the wells, and then discard the PBS.

[0124] 3) Add 0.5 mM EDTA at a volume of 1 ml per well and place the cells back into a 37°C, 5% CO2 cell culture incubator.

[0125] 4) After 6 min, remove the cells, gently blow to remove the cells and form a uniform cell suspension, add 1 ml DMEM / F12 to each well to terminate digestion, transfer the cells to a centrifuge tube, and centrifuge at 300 g for 3 min.

[0126] 5) Discard the supernatant in the centrifuge tube and resuspend the cells in 12 ml of stem cell maintenance medium. Add Y27632 at a final concentration of 5 μM to the cell suspension and gently shake the centrifuge tube to mix the cells.

[0127] 6) Discard the DMEM / F12 in the coated culture plate, add 2 ml of the cell suspension in 5) to each well of the 6-well plate, move the culture plate up and down and left and right 10 times to evenly spread the cells in the wells, and culture the cells in a 37°C, 5% CO2 cell culture incubator.

[0128] Example 2 The coding sequences of IL-10 and CD47 were inserted into human pluripotent stem cells.

[0129] 1. Graphs such as Figure 2 Construction of the master plasmid pTLL98 is shown.

[0130] 1.1) A DNA product containing the initial vector sequence, the CDS sequence of human IL-10 and the CDS sequence of human CD47 is amplified and extracted by conventional molecular cloning methods.

[0131] Among them, the initial vector sequence is shown in SEQ ID NO.2, the CDS sequence of human IL-10 is shown in SEQ ID NO.3, and the CDS sequence of human CD47 is shown in SEQ ID NO.4.

[0132] 1.2) Referring to Table 5 below, the above DNA products were amplified using the following primers to obtain Fragment 1, Fragment 2 and Fragment 3 DNA purification products.

[0133] Table 5

[0134] 1.3) Use ClonExpress Ultra One Step Cloning Kit (purchased from Vazyme) to carry out recombination reaction according to the system in Table 6 below.

[0135] Table 6

[0136] When the total volume of the vector and insert is greater than 5 μl, the reaction system can be scaled up to 20 μl.

[0137] After the above system is configured, place it in a PCR instrument at 50°C for 30 min; then cool to 4°C or immediately place on ice to cool.

[0138] 1.4) Transformation of recombinant products Thaw the chemically competent cells for cloning on ice (DH5α Competent Cell, Vazyme #C502). Take 5 - 10 μl of the recombinant product and add it to 100 μl of competent cells, flick the tube wall to mix (do not oscillate to mix), and place on ice for 30 min.

[0139] After heat shock in a 42°C water bath for 45 sec, immediately cool on ice for 2 - 3 min.

[0140] Add 900 μl LB liquid medium (without antibiotics) and incubate at 37°C for 1 h (200 - 250 rpm).

[0141] Pre-warm the LB solid medium plates with the corresponding resistance in a 37°C incubator.

[0142] Centrifuge at 5,000 rpm (2,500 × g) for 5 min, discard 900 μl of supernatant, resuspend the cells in the remaining medium, and spread evenly on the plate containing ampicillin using a sterile spreader.

[0143] Incubate inverted at 37°C for 12 - 16 h.

[0144] Three monoclonal strains were selected for shaking and sequencing, and the strains with correct sequencing were stored at -80°C.

[0145] At this point, the construction of plasmid pTLL98 was completed.

[0146] 2. Graphs such as Figure 3 Construction of helper plasmid pTLL56 as shown.

[0147] 2.1) Using the vector plasmid pX459 V2.0 (Plasmid #62988, from addgene) as a template, use BbsⅠ (purchased from NEB) restriction endonuclease for enzyme digestion. The enzyme digestion system is shown in Table 7 below.

[0148] Table 7

[0149] After reacting at 37°C for 2 h, the product was purified according to 2.2.3 to obtain vector1.

[0150] 2.2) Oligo1 and Oligo2 were synthesized at a concentration of 10 μM.

[0151] Among them, the Oligo1 sequence is shown as SEQ ID NO.11, and the Oligo2 sequence is shown as SEQ ID NO.12.

[0152] According to the system configuration shown in Table 8, the reaction conditions were: 95°C, 4 min; 70°C 10 min; 25°C, 30 sec; cooling to 4°C, and the product anneal 1 was obtained after the reaction.

[0153] Table 8

[0154] According to the system configuration shown in Table 9, the reaction conditions were: 16°C overnight. The obtained product was transformed according to step 1.4) to obtain plasmid pTLL56.

[0155] Table 9

[0156] 3. Plasmid transfection and construction of IL10 and CD47 overexpressing cell lines.

[0157] After the plasmid is prepared, the plasmid is electroporated into human pluripotent stem cells: plasmid dosage: 2.5μg plasmid 1, 1.5μg plasmid 2; lonza 2B instrument electroporation parameter B-016; human pluripotent stem cell dosage: 1 million; electroporation volume: 100μl. After electroporation, the cells are cultured in a 6-well plate according to the stem cell passage method, and the liquid is changed every day. After three days, they are screened with stem cell maintenance medium containing 100μg / ml hygromycin (Invivogen, ant-hg-1) and the drug is added continuously for three days. After that, the screened stem cells are amplified to the number of 3-6 wells of the 6-well plate, the stem cells are digested and labeled with CD47-APC flow cytometry antibodies, and flow cytometry is performed to obtain CD47-positive human pluripotent stem cells (eiPS). Figure 4 . The CD47-positive human pluripotent stem cells (eiPS) were sorted and tested by IL-10 ELISA. Figure 5 .

[0158] Combination Figure 4 It can be seen that eiPSC highly expresses CD47, that is, the CD47 overexpressing cell line stem cell line was established.

[0159] Combination Figure 5 It can be seen that eiPSC highly expresses IL10, that is, the IL10 overexpressing cell line stem cell line was established.

[0160] Example 3 CD47-positive human pluripotent stem cells (eiPS) were induced to differentiate into mesenchymal stromal cells.

[0161] 1) Subculture stem cells according to the method in 1.3.

[0162] Neural crest cell differentiation medium, transition medium and mesenchymal stromal cell differentiation medium were prepared according to Table 1, Table 2 and Table 3. Mesenchymal stromal cell differentiation medium 1, mesenchymal stromal cell differentiation medium 2 and mesenchymal stromal cell differentiation medium 3 were prepared according to Table 10, Table 11 and Table 12 below, respectively.

[0163] Table 10: Mesenchymal stromal cell differentiation medium 1

[0164] Table 11: Mesenchymal stromal cell differentiation medium 2

[0165] Table 12: Mesenchymal stromal cell differentiation medium 3

[0166] 2) After 24 hours of subculturing, the culture medium was replaced with neural crest cell differentiation medium. The medium was changed every 24 hours for 7 days.

[0167] 3) After 7 days, the differentiated cells are neural crest cells (NCCs).

[0168] 4) Coat a 6-well plate by following the steps in 1.1.

[0169] 5) Digest the NCCs according to step 1) to step 5) in 1.3 and count them in a hemocytometer. Resuspend the neural crest cells in an appropriate volume of transition medium to a cell density of 1×10 5 Pieces / cm 2 -2.5×10 5 Pieces / cm 2 , gently shake the centrifuge tube to mix the cells.

[0170] 6) Discard the DMEM / F12 in the coated culture plate, add 2 ml of the cell suspension in 5) to each well of the 6-well plate, move the culture plate up and down and left and right 10 times to evenly spread the cells in the wells, and culture the cells in a 37°C, 5% CO2 cell culture incubator.

[0171] 7) On the first day, change the medium every 24 hours according to step 6) and step 7). From day 2 to day 15, divide the cells into three parts and use mesenchymal stromal cell differentiation medium, mesenchymal stromal cell differentiation medium 2 and mesenchymal stromal cell differentiation medium 3 respectively. Change the medium every 24 hours according to step 6) and step 7) for 14 days. During this period, the cell confluence reaches 100%, and the cells are passaged at a ratio of 1:6.

[0172] 8) After 14 days, flow cytometry was used to analyze the positive markers of mesenchymal stromal cells (CD73, CD90, CD105) and the negative markers of mesenchymal stromal cells (CD34, CD45). Figure 6 Among them, the red line is the negative control.

[0173] Combination Figure 6 It can be seen that the mesenchymal stromal cell differentiation medium can achieve the differentiation of NCCs into MSCs, the mesenchymal stromal cell differentiation medium 2 can achieve the differentiation of NCCs into MSCs, and the mesenchymal stromal cell differentiation medium 3 can achieve the differentiation of NCCs into MSCs.

[0174] It should be noted that the MSCs obtained in Example 3 used subsequently, unless otherwise specified, were all MSCs obtained using a mesenchymal stromal cell differentiation medium.

[0175] Test Example 1 Immunocytochemistry was used to further verify the marker genes of NCCs obtained in Example 3 1) Provide NCCs obtained in Example 3.

[0176] 2) Discard the culture supernatant and add 500 μl 1× PBS to wash three times.

[0177] 3) Add 500 μl 4% paraformaldehyde (Biyuntian, P0099-100ml) and fix at room temperature for 15 min.

[0178] 4) Discard the paraformaldehyde, add 500 μl 1× PBS to wash three times, and add 500 μl 2% BSA to block at room temperature for 30 minutes.

[0179] 5) Discard BSA, dilute HNK-1 primary antibody (biolegend, 359602), NGFR primary antibody (biolegend, 345102), AP2α primary antibody (santacruz, sc-12726) and SOX10 primary antibody (biolegend, 847201) in 2% BSA at a ratio of 1:200, add to the wells and stain for 30 min at room temperature.

[0180] 6) Discard the BSA containing the primary antibody, add 500 μl 1× PBS and wash three times, dilute Donkey anti-mouse Alexa Fluor 488 fluorescent secondary antibody (Invitrogen, R37114) in 2% BSA at a ratio of 1:500, add it to the wells and stain for 15 min at room temperature in the dark.

[0181] 7) Discard the BSA containing the secondary antibody, wash three times with 500 μl 1× PBS, add 500 μl 1× 4',6-diamidino-2-phenylindole (DAPI, Solarbio, C0060) and counterstain for 10 minutes at room temperature in the dark.

[0182] 8) Wash with 500 μl 1× PBS three times and observe under a fluorescence microscope. Figure 7 Among them, HNK-1, NGFR, AP2α and SOX10 are markers of neural crest cells.

[0183] Depend on Figure 7 It can be seen that Example 3 can effectively induce the production of neural crest cells.

[0184] Test Example 2 Flow cytometry was used to detect the expression of markers of NCCs obtained in Example 3. 1) Provide NCCs obtained in Example 3, and digest the cells according to steps 1) to 5) in 1.3.

[0185] 2) Centrifuge at 300 g for 3 min, discard the supernatant, resuspend the cells in FACS buffer (composition: 1× PBS, 5 g / LBSA, 2 mM EDTA), and repeat the centrifugation operation.

[0186] 3) Discard the supernatant, resuspend the cells in 100 μl FACS buffer, add 1 μl APC Anti-Human HNK-1 (BD, 560845) and 1 μl PE Anti-Human NGFR (BD, 557196), mix well, and stain at 4°C in the dark for 15 min.

[0187] 4) Add 1 ml FACS buffer and centrifuge at 300 g for 3 min.

[0188] 5) Discard the supernatant, resuspend the cells in 500 μl FACS buffer and filter using a flow cytometry tube with a filter.

[0189] 6) Turn on the Guava easyCyte flow cytometer and clean the instrument according to the operating instructions.

[0190] 7) After loading, adjust the voltage and gain intensity of the FSC-H, SSC-H, Yellow-B and Red-R channels so that the cells are located in the figure and circle the single cells.

[0191] 8) Analyze the signal intensity of the Yellow-B (i.e., fluorescent group PE signal) channel and the Red-R (i.e., fluorescent group APC signal) channel.

[0192] 9) Analyze using FlowJo software and get Figure 8 , among which HNK-1 and NGFR are neural crest cell markers.

[0193] Depend on Figure 8 It can be seen that Example 3 can effectively induce the generation of neural crest cells.

[0194] Test Example 3 Flow cytometry was used to detect the expression of MSC markers obtained in Example 3. 1) Referring to Example 3, MSCs were induced to differentiate using Mesenchymal Stromal Cell Differentiation Medium 1, and the cells were digested according to Steps 1) to 5) in 1.3.

[0195] 2) Centrifuge at 300 g for 3 min, discard the supernatant, resuspend the cells in FACS buffer (composition: 1× PBS, 5 g / LBSA, 2 mM EDTA), and repeat the centrifugation operation.

[0196] 3) Discard the supernatant, resuspend the cells in 300 μl FACS buffer, divide into three parts, add 1 μl PE Anti-Human CD105, 1 μl PE Anti-Human CD90, and 1 μl PE Anti-Human CD73 respectively, mix well, and stain at 4°C in the dark for 15 min.

[0197] 4) Add 1 ml FACS buffer and centrifuge at 300 g for 3 min.

[0198] 5) Discard the supernatant, resuspend the cells in 500 μl FACS buffer and filter using a flow cytometry tube with a filter.

[0199] 6) Turn on the Guava easyCyte flow cytometer and clean the instrument according to the operating instructions.

[0200] 7) After loading, adjust the voltage and gain intensity of the FSC-H, SSC-H, Yellow-B and Red-R channels so that the cells are located in the figure and circle the single cells.

[0201] 8) Analyze the signal intensity of the Yellow-B (i.e., fluorescent group PE signal) channel and the Red-R (i.e., fluorescent group APC signal) channel.

[0202] 9) Analyze using FlowJo software and get Fig. 9 Among them, CD73, CD90 and CD105 are all positive markers of mesenchymal stromal cells, and the red line is the negative control.

[0203] Depend on Fig. 9 It can be seen that the mesenchymal stromal cell differentiation medium 1 can effectively induce the generation of mesenchymal stromal cells.

[0204] Combination Figure 6 and Fig. 9 It can be seen that FBS is not necessary for MSCs differentiation, and there is no need to replace FBS with BSA and ITS-X. The use of mesenchymal stromal cell differentiation medium without animal-derived components can induce NCCs to differentiate into MSCs.

[0205] Test Example 4 Verification of the multipotency of MSCs obtained in Example 3 Mesenchymal Stromal Cell Chondrogenic Differentiation Medium (MCDM, ScienCell, 7551), Mesenchymal Stromal Cell Osteogenic Differentiation Medium (MODM, ScienCell, 7531) and Mesenchymal Stromal Cell Adipogenic Differentiation Medium (MCDM, ScienCell, 7541) were used to induce MSCs to differentiate into chondrocytes, adipocytes and osteoblasts according to the instructions of the reagents, and then stained with Alcian blue, Oil red O and Alizarin red to obtain Fig.10 .

[0206] Depend on Fig.10 It can be seen that the MSCs obtained in Example 3 have the ability to be differentiated and have certain multipotency.

[0207] The MSCs obtained in Example 3 were passaged and counted. The cells were planted in a six-well plate at a density of 1 million cells / well, and passaged every three days and counted to obtain Fig.11 .

[0208] Depend on Fig.11 It can be seen that the MSCs obtained in Example 3 have a strong proliferation ability. After the sixth generation, the cell proliferation ability decreases. As the number of generations increases, the cell proliferation rate changes from fast to slow. The sixth generation cell proliferation curve is basically parallel to the X-axis, indicating that the cells basically do not proliferate from the sixth generation onwards.

[0209] The MSCs obtained in Example 3 were mixed with non-fluorescent umbilical mesenchymal stromal cells (UCMSCs) (from the Stem Cell Research Center of Peking University Third Hospital) at a certain ratio (GFP ratio <50%). This was the first generation (P1). The cells were passaged every 4-6 days and analyzed by flow cytometry to obtain Fig.12 and Fig.13 .

[0210] Combination Fig.12 and Fig.13 , it can be seen that the proportion of MSCs (GFP positive) cells increases with each generation, and by the fourth generation, the proportion of MSCs cells reaches more than 99%.

[0211] It can be seen from this that the proliferation ability of MSCs is stronger than that of umbilical cord mesenchymal stromal cells (UCMSCs), and a large number of cells can be obtained.

[0212] Immunodeficient mice (NSG, Beijing Weitong, 10-week-old 25 g male mice) were subjected to 8 mm skin injury wound model and treated with MSCs and phosphate buffered saline (PBS) respectively. Fig.14 .

[0213] Combination Fig.14 , it can be seen that compared with the PBS control group, MSCs have a better therapeutic effect on skin damage.

[0214] Small animal imaging assays revealed that luciferase-expressing MSCs were transplanted into immunodeficient mice. Fig.15 Among them, MSCs carrying luciferase were obtained by the following operation: plasmids 80493 and 72833 were purchased from addgene, and the plasmids were electroporated into H9X cell lines (the same electroporation method as pTLL98) to obtain H9X cell lines carrying luciferase, which were then differentiated into MSCs.

[0215] Combination Fig.15 , it can be seen that MSCs can survive at the site of skin injury wound for 14 days until the wound is completely repaired.

[0216] Nude mice were treated with 8 mm skin injury wounds and treated with MSCs and NCC-eMSCs. Compared with the MSCs control group, NCC-eMSCs had a better therapeutic effect on skin injury.

[0217] That is, NCC-eMSCs (overexpressing CD47-IL-10) had a better therapeutic effect than MSCs (not overexpressing CD47-IL-10), and the MSC group had a better therapeutic effect than the PBS group.

[0218] MSCs carrying luciferase were injected into the C57 mouse joint cavity and detected by small animal in vivo imaging. Fig.16 .

[0219] Combination Fig.16 , it can be seen that MSCs can survive in the joint cavity of C57 mice for at least 8 days, indicating that MSCs have low immunogenicity.

[0220] Four groups of wild-type 8-week-old male SD rats were selected: sham operation group, PBS group, MSCs group and eMSCs group (MSCs differentiated from eiPS are called eMSCs (edited MSCs)). Each group had 5 rats, and they underwent sham operation (sham operation group) and ACLT operation (PBS group, MSCs group and eMSCs group) on the right hind limb.

[0221] ACLT surgery: After anesthesia, the skin is prepared, the skin, subcutaneous tissue and joint capsule are incised, the anterior cruciate ligament is exposed and cut, and after the knee joint examination confirms a positive anterior drawer test, the joint capsule and skin are sutured layer by layer.

[0222] Sham operation: After anesthesia, the skin was prepared, the skin and joint capsule were incised without cutting the anterior cruciate ligament, and the joint capsule and skin were sutured layer by layer.

[0223] One week after surgery, the PBS group, MSCs group, and eMSCs group were injected with 100ul PBS or 2×10 6 MSCs or 2 × 10 6 eMSCs were injected once a week for 4 times. Euthanasia was performed 6 weeks after modeling, and further imaging evaluation was performed to obtain Fig.17 and Fig.18 .

[0224] In this study, micro-CT was used to detect the subchondral bone and osteophyte formation of the knee joints of experimental mice. The knee joints of each group of experimental mice were removed as a whole for use (without fixation and can be stored at -80℃), and the specimens of each group were scanned using micro-CT. The micro-CT images were reconstructed three-dimensionally using Mimics Research software. Semi-quantitative analysis was performed using Inveon Research Workplace software, and the measured data included bone mineral density (BMD), trabecular bone volume per total volume (BV / TV), and trabecular bone pattern factor (Tb. Pf).

[0225] Combination Fig.17 and Fig.18 It can be seen that compared with the control group, the osteophyte formation was reduced, BMD was increased, BV / TV was increased, and Tb. Pf was reduced in the MSCs group and the eMSCs group, indicating that the MSCs group and the eMSCs group have a therapeutic effect on osteoarthritis.

[0226] The IL-10 expression of MSC and eMSC was detected using IL-10 ELISA kit (Solebo, SEKH0047). Fig.19 .

[0227] Combination Fig.19 , it can be seen that IL-10 is highly expressed in eMSCs derived from eiPSCs, proving that the stem cell line overexpressing IL-10 was successfully constructed.

[0228] In the description of the embodiments of the present invention, it should be noted that the terms "center", "longitudinal", "lateral", "up", "down", "front", "back", "left", "right", "vertical", "horizontal", "top", "bottom", "inside", "outside" and the like indicate positions or positional relationships based on the positions or positional relationships shown in the accompanying drawings, and are only for the convenience of describing the embodiments of the present invention and simplifying the description, and do not indicate or imply that the devices or elements referred to must have a specific orientation, be constructed and operate in a specific orientation, and therefore cannot be understood as limitations on the embodiments of the present invention. In addition, the terms "first", "second", and "third" are used for descriptive purposes only and cannot be understood as indicating or implying relative importance.

[0229] In the description of the embodiments of the present invention, it should be noted that, unless otherwise clearly specified and limited, the terms "connected" and "connection" should be understood in a broad sense, for example, it can be a fixed connection, a detachable connection, or an integral connection; it can be a mechanical connection or an electrical connection; it can be a direct connection or an indirect connection through an intermediate medium. For ordinary technicians in this field, the specific meanings of the above terms in the embodiments of the present invention can be understood according to specific circumstances.

[0230] In the embodiments of the present invention, unless otherwise clearly specified and limited, the first feature being "above" or "below" the second feature may mean that the first and second features are in direct contact, or the first and second features are in indirect contact through an intermediate medium. Moreover, the first feature being "above", "above" and "above" the second feature may mean that the first feature is directly above or obliquely above the second feature, or simply means that the first feature is higher in level than the second feature. The first feature being "below", "below" and "below" the second feature may mean that the first feature is directly below or obliquely below the second feature, or simply means that the first feature is lower in level than the second feature.

[0231] In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" etc. means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the embodiment of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art may combine and combine the different embodiments or examples described in this specification and the features of the different embodiments or examples, without contradiction.

[0232] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for preparing stem cell-derived function-enhanced mesenchymal stromal cells, characterized in that: The steps include: Providing human pluripotent stem cells; Inserting an IL-10 gene coding sequence and a CD47 gene coding sequence into the human pluripotent stem cells, and obtaining CD47-positive human pluripotent stem cells after screening, wherein the CD47-positive human pluripotent stem cells can also express IL-10; Using a neural crest cell differentiation medium to induce differentiation and culture the CD47-positive human pluripotent stem cells to obtain neural crest cells, wherein the neural crest cell differentiation medium includes a Wnt pathway agonist and a TGF-β receptor kinase inhibitor; The neural crest cells are induced to differentiate using a transition medium and a mesenchymal stromal cell differentiation medium in sequence to obtain the desired stem cell-derived function-enhanced mesenchymal stromal cells, wherein the transition medium includes epidermal growth factor, fibroblast growth factor 2 and a cell apoptosis inhibitor, and the mesenchymal stromal cell differentiation medium includes epidermal growth factor, fibroblast growth factor 2 and platelet growth factor.

2. The method for preparing stem cell-derived function-enhanced mesenchymal stromal cells according to claim 1, characterized in that: The neural crest cell differentiation medium, the transitional medium and the mesenchymal stromal cell differentiation medium do not contain animal-derived components.

3. The method for preparing stem cell-derived function-enhanced mesenchymal stromal cells according to claim 1, characterized in that: The operation of inserting the IL-10 gene coding sequence and the CD47 gene coding sequence into the human pluripotent stem cells is: electro-transforming the main plasmid including the IL-10 gene coding sequence and the CD47 gene coding sequence and the auxiliary plasmid for locating the insertion position into the human pluripotent stem cells.

4. The method for preparing stem cell-derived function-enhanced mesenchymal stromal cells according to claim 3, characterized in that: The master plasmid is prepared by the following operation: using CRISPR Cas9 to insert the IL-10 gene coding sequence and the CD47 gene coding sequence into the CLYBL site of the master plasmid.

5. The method for preparing stem cell-derived function-enhanced mesenchymal stromal cells according to claim 4, characterized in that: The main plasmid is a donor plasmid, and the auxiliary plasmid is a gRNA plasmid.

6. The method for preparing stem cell-derived function-enhanced mesenchymal stromal cells according to any one of claims 1 to 5, characterized in that: The operation of inducing differentiation culture of the CD47-positive human pluripotent stem cells using neural crest cell differentiation medium is as follows: after subculturing the CD47-positive human pluripotent stem cells for 24 hours, the medium is replaced with the neural crest cell differentiation medium, and the medium is replaced every 24 hours for 7 days.

7. The method for preparing stem cell-derived function-enhanced mesenchymal stromal cells according to claim 6, characterized in that: The operation of inducing differentiation culture of the neural crest cells by sequentially using a transition medium and a mesenchymal stromal cell differentiation medium is as follows: resuspending the neural crest cells in the transition medium to a cell density of 1×10 5 Pieces / cm 2 ~2.5×10 5 Pieces / cm 2 Then, the neural crest cells were cultured in a cell culture incubator at 37° C. and 5% CO 2 , with the medium changed every 24 hours for 1 day, and then the culture medium was replaced with the mesenchymal stromal cell differentiation medium, with the medium changed every 24 hours for 14 days.

8. The method for preparing stem cell-derived function-enhanced mesenchymal stromal cells according to claim 7, characterized in that: The Wnt pathway agonist is CHIR99021, the TGF-β receptor kinase inhibitor is SB431542, and the cell apoptosis inhibitor is Y27632; The formula of the neural crest cell differentiation medium is as follows: The formula of the transition medium is as follows: The formula of the mesenchymal stromal cell differentiation medium is as follows: 。 9. A stem cell-derived mesenchymal stromal cell with enhanced function, characterized in that: The cells are prepared by the method for preparing stem cell-derived function-enhanced mesenchymal stromal cells as claimed in any one of claims 1 to 8.

10. Use of stem cell-derived function-enhanced mesenchymal stromal cells for preparing therapeutic devices and / or drugs for skin damage and / or osteoarthritis.

Images