CN117018032A

CN117018032A - Biological agent containing skeletal muscle precursor-like cells, and preparation method and application thereof

Info

Publication number: CN117018032A
Application number: CN202310522452.5A
Authority: CN
Inventors: 周伸奥
Original assignee: Shanghai Celliver Biotechnology Co Ltd
Current assignee: Shanghai Celliver Biotechnology Co Ltd
Priority date: 2022-05-10
Filing date: 2023-05-09
Publication date: 2023-11-10
Also published as: WO2023217132A1; WO2023217123A1; CN117025503A; WO2023217126A1; CN117025505A; CN117025502A; WO2023217128A1; WO2023217129A1; CN117025506A; WO2023217130A1; CN117025508A; WO2023217134A1; CN117018033A; CN117025507A; WO2023217125A1; CN117025504A; WO2023217121A1

Abstract

The application provides a biological agent containing skeletal muscle precursor cells, a preparation method and application thereof, wherein the skeletal muscle precursor cells negatively express at least one of CD34, CD45 and HLA-DRPQ. So that the human body can well accept the skeletal muscle precursor-like cells, and the application solves the problem of immune rejection in the treatment mode of muscular atrophy caused by gene mutation in the prior art.

Description

Biological agent containing skeletal muscle precursor-like cells, and preparation method and application thereof

Technical Field

The application relates to the field of biotechnology, in particular to a biological agent containing skeletal muscle precursor-like cells, and a preparation method and application thereof.

Background

Skeletal muscle is the largest organ of the human body, and the main functions of skeletal muscle are locomotion, morphological maintenance and respiration. In adult muscle tissue, the smallest functional unit constituting skeletal muscle is myofibroblasts (the english name of myofibroblasts is myofibber). It is formed by stacking a plurality of myofibrils (the English name of myofibrils is myoflibl) which are arranged in parallel, and the periphery of the myofiber is wrapped by a myofiber membrane (the English name of the myofiber membrane is sarcolema, namely the cell membrane of myofiber cells). There is a basal membrane (the English name of basal membrane is basement membrane) in addition to the membrane of myofibroblast. At the same time, each myofibril can be divided into a plurality of muscle segments (the English name of the muscle segments is sarcomeres), and the tension is generated by contraction. For mammals, each myofibroblast typically contains hundreds of nuclei (the english name of nuclei: myonucleus) distributed against the myofiber membrane, which together control the movement and metabolic processes of the myofiber cell. In addition to myofibroblasts, skeletal muscle contains a large amount of connective tissue (connective tissue is called connective tissue in english), blood vessels and nerves, and connective tissue connects myofibroblasts into an integral functional unit and is fixed to skeletal bone by tendons (tendon is called tendon).

As early as a century before muscle stem cells were discovered, pathologists have discovered the muscle's self-repair function. Tracking experiments show that during the muscle development of rats, muscle stem cells can be labeled as two populations, the first population of muscle stem cells accounting for about 80% of the total number, which proliferate, differentiate, fuse and ultimately form myofibroblasts; scientists refer to the second group of muscle stem cells as "reserve cells," which divide at a significantly slower rate, ultimately becoming a pool of muscle stem cells in muscle tissue. Subsequent studies have shown that the first and second populations of muscle stem cells result from asymmetric division of muscle stem cells (asymmetric division, english name: asymmetric division) and that when skeletal muscle is affected by such things as exercise strain, cold, heavy metals, biotoxins, etc., the repair process of muscle tissue is violent and rapid.

Duchenne muscular dystrophy (English name: duchenne muscular dystrophy, DMD) is a common dying pediatric neuromuscular disease, is X-linked recessive inheritance, is caused by DMD gene mutation, and is frequently caused in childhood, and is easy to miss and misdiagnose in early stage due to hidden onset. Since the defect of gene level is irreversible, the organism can not repair the injury through the regeneration and compensation of muscle cells, so that the advanced change of a great deal of fibrosis of skeletal muscle, cardiac muscle and other parts is gradually caused, and finally the injury is fatal due to serious influence on the heart and lung functions of the sick children. The DMD gene is located at Xp21, whose genome spans 2.4Mb, is the largest gene found in humans, and encodes dystrophin (the English name of dystrophin is dystophin). The lack of dystrophin results in at least two consequences, the first of which is that muscle cells are destroyed by mechanical forces during centrifugal contraction and ion channels that are sensitive to mechanical forces are deregulated, and the second of which is that muscle cell membranes are not intact, abnormal proteins and calcium influx, creatine kinase efflux, etc., occur, eventually leading to degeneration, necrosis of muscle cells.

Peripheral nerve injury (PNI, english name: peripheral nerve injury) is a clinically common wound and can be caused by various reasons such as tearing, traction, cutting, compression, ischemia, high temperature, freezing, infection, and nutritional and metabolic disorder, and the peripheral nerve can be regenerated to a certain extent after being damaged, but the recovery speed is very slow. After severe damage to the nerve, the innervated skeletal muscle is atrophic within days, with progressive increase in apoptosis and progressive decrease in the number of nuclei with prolonged atrophy time, ultimately resulting in permanent loss of skeletal muscle function.

Muscle atrophy due to gene mutation, such as Duchenne muscular dystrophy and muscular dystrophy changes due to peripheral nerve injury (peripheral nerve injury, PNI), can all occur with muscular dystrophy lesions, resulting in a decrease in muscle cell count until skeletal muscle function is permanently lost, thus threatening patient life. If the muscular atrophy can be controlled, the degenerative changes of muscles can be relieved, and the exercise and respiratory functions can be saved. Thus, the treatment of muscle diseases has the following ideas: cell therapy, gene editing therapy, and traditional hormonal therapy modalities.

Cell therapy is mainly based on cell transplantation, including myoblast transplantation and mesenchymal stem cell transplantation. Myoblast transplantation (English name: myoblast transfer therapy, english name: MTT): partrige et al firstly put forward MTT as one of strategies of DMD gene therapy, and proved by model mouse experiments that the transplanted myoblasts can be fused with host myofibroblasts, so as to express normal DMD, and correct the changes of degeneration, necrosis and the like of myofibers; the mesenchymal stem cells (English name: mesenchymal stem cells, english name: MSC for short) are adult stem cells with various differentiation potential, the uniformity of the MSC which is cultured in vitro and transmitted for more than 3 generations is good, the vein transplantation immune response is low, 5 generations of MSC are transplanted to a gene knockout mouse through a tail vein, the intensity of DMD immunofluorescence increases with time, and the feasibility of introducing and expressing the DMD gene by the route is verified.

Cell therapy suffers from the following drawbacks: firstly, the greatest difficulty in muscle stem cell therapy is that donor cells are insufficient, in order to avoid immune rejection, muscle stem cells of a patient need to be taken out, expression of Dystrophin is obtained in vitro through infection, and the obtained Dystrophin is reinfused into the patient after being greatly amplified, and no method capable of greatly amplifying human muscle stem cells in vitro exists at present; secondly, the research of the combined treatment of the mesenchymal stem cells shows that the method is only suitable for patients with light and medium muscular atrophy, has very little curative effect on patients with extremely reduced muscular capacity and has unknown long-term curative effect, so that a plurality of difficulties needing to be solved simultaneously in the stem cell transplantation treatment, such as the administration route, the suitability for case selection, the survival rate of cells after transplantation and the like, exist; third, myoblasts are used in clinical trials to treat muscle diseases, and although expression of Dystrophin is detected in patients, the effect is not ideal because myoblasts cannot home and replenish the bank of muscle stem cells in the body as they do.

Gene editing therapy: adeno-associated virus (AAV for short) mediated in vivo gene editing is a promising treatment idea, and by injecting adeno-associated virus, myofibroblasts can recover expression of Dystrophin to a certain extent, so that the intensity of myofibroblasts is increased, necrosis of myofibroblasts is avoided, and AAV treatment has the following advantages: the first virus can be amplified in vitro, so that convenience is brought to acquisition; the second AAV has low tumorigenicity and is relatively safe in vivo; thirdly, AAV can infect a wide range of tissues through blood transport due to the spread of the virus; other gene editing therapies also include exon skipping and stop codon readthrough, wherein exon skipping is by adopting an exon skipping splicing technology, and the frame shift mutation type of the DMD is modified to be a whole code mutation type, so that the DMD with heavier symptoms can be converted into the DMD with lighter symptoms; stop codon read-through: the aminoglycoside antibiotics gentamicin can reduce the capability of ribosome to recognize abnormal terminators in the process of dystrophin translation, and skip the abnormal terminators to continue translation, and the result also has obvious curative effect in the mouse animal experiment.

Gene therapy suffers from the following drawbacks: firstly, the biggest obstacle of the adeno-associated virus therapy is that muscle stem cells cannot break through the barrier of the microenvironment, and muscle fibroblasts expressed by Dystrophin obtained by virus infection die due to life and metabolism replacement, so that the AAV therapy cannot fundamentally solve the problem of muscle diseases, and can only treat patients for multiple times and continuously; second, exon skipping can only be targeted to specific gene defect types, and oligonucleotides are less energy efficient at entering the cell membrane, possibly immunogenic; third, stop codon read-through therapy has a large number of toxic side effects due to low gentamicin read-through energy efficiency and long-term large-scale application, and only aims at nonsense mutations, and medicines may have short-term or long-term side effects.

Traditional hormone therapy mode: glucocorticoids have found greatest clinical use in the treatment of DMD due to their anti-inflammatory effect with some success. However, the side effects of glucocorticoids often outweigh their benefits, producing long-term inflammatory responses, requiring combination anti-inflammatory therapy.

In the prior art, there is a defect of immune rejection in the treatment mode of muscular dystrophy caused by gene mutation, so that it is necessary to develop a biological preparation containing skeletal muscle precursor-like cells, and a preparation method and application thereof to solve the problems in the prior art.

Disclosure of Invention

The application aims to provide a biological agent containing skeletal muscle precursor-like cells, and a preparation method and application thereof, so as to solve the problem of immune rejection in the treatment mode of muscular atrophy caused by gene mutation in the prior art.

To achieve the above object, the present application provides a biological agent comprising skeletal muscle precursor-like cells that negatively express at least one of CD34, CD45, HLA-DRPQ.

The biological agent comprising skeletal muscle precursor-like cells of the present application has the beneficial effects that: the skeletal muscle precursor-like cells negatively express at least one of CD34, CD45 and HLA-DRPQ, so that the human body can well accept the skeletal muscle precursor-like cells, and the application solves the problem of immune rejection in the treatment mode of muscular atrophy caused by gene mutation in the prior art.

Preferably, the skeletal muscle precursor-like cells positively express at least one of CD29, CD56, CD24 and PAX7.

The application also provides a preparation method of the biological agent containing skeletal muscle precursor-like cells, which comprises the following steps:

s0: providing skeletal muscle precursor-like cells;

s1: mixing the skeletal muscle precursor-like cells with a pharmaceutically acceptable carrier to obtain a biological agent comprising skeletal muscle precursor-like cells, wherein the skeletal muscle precursor-like cells positively express at least one of CD29, CD56, CD24 and PAX7, and the skeletal muscle precursor-like cells negatively express at least one of CD34, CD45, HLA-DRPQ.

The preparation method of the biological agent containing skeletal muscle precursor cells has the beneficial effects that: the preparation method of the biological agent containing the skeletal muscle precursor-like cells is simple, and the obtained biological agent containing the skeletal muscle precursor-like cells can reduce the influence of muscle injury on a human body, can well adapt to the human body and can not generate immune rejection with a recipient.

Preferably, the method for preparing skeletal muscle precursor-like cells comprises the steps of:

s00: providing skeletal muscle primary cells;

s01: and placing the skeletal muscle primary cells into a reprogramming culture medium for performing dedifferentiation culture until the fusion degree of the skeletal muscle precursor-like cells is not lower than 80%, and performing digestion treatment on the skeletal muscle precursor-like cells by using pancreatin digestive juice to obtain the skeletal muscle precursor-like cells, wherein the reprogramming culture medium comprises a basal culture medium, tumor necrosis factor, interleukin and tumor suppressor.

Preferably, the method for preparing skeletal muscle precursor-like cells further comprises: s02: and placing the skeletal muscle precursor-like cells into the reprogramming culture medium for amplification culture until the fusion degree of the skeletal muscle precursor-like cells is not lower than 80%, and then performing digestion treatment on the skeletal muscle precursor-like cells by using the pancreatin digestive juice to obtain the passaged skeletal muscle precursor-like cells. The beneficial effects are that: skeletal muscle precursor-like cells can be continuously expanded in reprogramming media and the resulting skeletal muscle precursor-like cells can express skeletal muscle precursor-like cell markers.

Preferably, the reprogramming media further comprises growth factors, ROCK kinase inhibitors, wnt signaling pathway agonists, TGF- β signaling inhibitors, and nutritional supplements.

Preferably, the tumor necrosis factor is contained in an amount of 2-20ng/mL, the interleukin is contained in an amount of 20-50ng/mL, and the tumor suppressor is contained in an amount of 1-10ng/mL, based on the volume of the reprogramming medium.

Preferably, the growth factor is present in an amount of 40-80ng/mL, the ROCK kinase inhibitor is present in an amount of 5-20. Mu.M, the Wnt signaling pathway agonist is present in an amount of 1-10. Mu.M, the TGF-beta signaling inhibitor is present in an amount of 0.1-5. Mu.M, and the nutritional supplement is present in an amount of 0.5-10% of the final volume of the reprogramming media.

The application also provides application of the biological agent containing the skeletal muscle precursor-like cells, and the biological agent containing the skeletal muscle precursor-like cells is used for intervening in an in vivo animal model.

The skeletal muscle precursor-like cells of the present application have the beneficial effects of: after the biological agent containing skeletal muscle precursor cells is returned, the local inflammation can be well controlled, the muscle fibrosis can be controlled, and the muscle degenerative disease can be relieved, so that skeletal muscle can be better recovered.

Preferably, the animal model comprises a model of cytotoxin-induced muscle injury.

Drawings

FIG. 1 is a schematic photograph of a first generation skeletal muscle precursor-like cell morphology in accordance with an embodiment of the present application;

FIG. 2 is a schematic photograph of a fourth generation skeletal muscle precursor-like cell morphology in accordance with an embodiment of the present application;

FIG. 3 is a schematic representation of the proliferation of skeletal muscle precursor-like cells in accordance with an embodiment of the present application;

FIG. 4 is a schematic diagram showing the case of CD34, a gene expression marker of skeletal muscle precursor-like cells of an embodiment of the present application;

FIG. 5 is a schematic diagram showing the case of CD45, a gene expression marker of skeletal muscle precursor-like cells of an embodiment of the present application;

FIG. 6 is a schematic diagram showing the case of HLA-DRPQ, which is a gene expression marker of skeletal muscle precursor-like cells according to an embodiment of the present application;

FIG. 7 is a schematic diagram showing the case of CD29, a gene expression marker of skeletal muscle precursor-like cells of an embodiment of the present application;

FIG. 8 is a schematic diagram showing the case of CD56, a gene expression marker of skeletal muscle precursor-like cells of an embodiment of the present application;

FIG. 9 is a schematic diagram showing the case of CD24, a gene expression marker of skeletal muscle precursor-like cells of an embodiment of the present application;

FIG. 10 is a schematic diagram showing the case of the gene expression marker PAX7 of skeletal muscle precursor-like cells of the present application;

FIG. 11 is a schematic diagram showing pathological tissue section contrast photographs of a drug-induced muscle injury model mouse according to an embodiment of the present application at different times in an experimental group and a control group;

FIG. 12 is a graph showing the comparison of the area distribution of neomyofibroblasts in experimental and control groups in drug-induced muscle injury model mice according to the example of the present application.

Detailed Description

For the purpose of making the objects, technical solutions and advantages of the embodiments of the present application more apparent, the technical solutions in the embodiments of the present application will be clearly and completely described below, and it is apparent that the described embodiments are some embodiments of the present application, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the application without making any inventive effort, are intended to be within the scope of the application. Unless otherwise defined, technical or scientific terms used herein should be given the ordinary meaning as understood by one of ordinary skill in the art to which this application belongs. As used herein, the word "comprising" and the like means that elements or items preceding the word are included in the element or item listed after the word and equivalents thereof without precluding other elements or items.

Fibrosis is a significant pathological feature seen in muscle biopsies of patients with muscle atrophy, which can lead to a disturbance of muscle function and ultimately to death; the anti-fibrosis treatment not only can improve muscle function, but also can promote muscle regeneration, gene introduction and stem cell implantation efficacy, and becomes a necessary supplement for future gene and cell treatment. In mouse experiments, several drugs against fibrosis were tested, and the results indicate that they can improve muscle function and the phenotype of muscle damage. The biological agent containing skeletal muscle precursor-like cells can well control local inflammation after being infused back into the body, can control muscle fibrosis, and lighten muscle degenerative diseases, so that skeletal muscles can be recovered well.

The application provides a biological agent comprising skeletal muscle precursor-like cells that negatively express at least one of CD34, CD45, HLA-DRPQ.

Specifically, at least one of CD34, CD45 and HLA-DRPQ is expressed negatively by the skeletal muscle precursor-like cells, so that the human body can well accept the skeletal muscle precursor-like cells, and the application solves the problem of immune rejection in the treatment mode of muscular atrophy caused by gene mutation in the prior art.

In some embodiments of the application, the HLA-DRPQ is an abbreviation for HLA-DR/DP/DQ.

In some embodiments of the application, the skeletal muscle precursor-like cells positively express at least one of CD29, CD56, CD24, and PAX7.

s0: providing skeletal muscle precursor-like cells;

Specifically, the preparation method of the biological agent containing skeletal muscle precursor-like cells is simple, and the obtained biological agent containing skeletal muscle precursor-like cells can reduce the influence of muscle injury on human body, can well adapt to the human body, and can not generate immune rejection with a recipient.

In some embodiments of the application, the pharmaceutically acceptable carrier includes, but is not limited to, physiological saline and compound electrolyte injection.

Some embodiments of the application, the method for preparing skeletal muscle precursor-like cells comprises the steps of:

s00: providing skeletal muscle primary cells;

Specifically, the skeletal muscle precursor-like cells are put into a reprogramming culture medium for performing dedifferentiation culture until the fusion degree of the skeletal muscle precursor-like cells is not lower than 80%, and digestive treatment is performed on the skeletal muscle precursor-like cells by using pancreatin digestive juice to obtain the skeletal muscle precursor-like cells, wherein the reprogramming culture medium comprises a basal culture medium, tumor necrosis factor, interleukin and tumor suppressor, so that the skeletal muscle precursor-like cells continuously realize self-renewal and proliferation, and the problem that the skeletal muscle precursor-like cells cannot be cultured in a large amount in vitro in the prior art is solved.

Some embodiments of the application, the method for preparing skeletal muscle precursor-like cells further comprises: s02: and placing the skeletal muscle precursor-like cells into the reprogramming culture medium for amplification culture until the fusion degree of the skeletal muscle precursor-like cells is not lower than 80%, and then performing digestion treatment on the skeletal muscle precursor-like cells by using the pancreatin digestive juice to obtain the passaged skeletal muscle precursor-like cells. Skeletal muscle precursor-like cells can be continuously expanded in reprogramming media and the resulting skeletal muscle precursor-like cells can express skeletal muscle precursor-like cell markers.

Some embodiments of the application, the reprogramming media further comprise a growth factor, a ROCK kinase inhibitor, a Wnt signaling pathway agonist, a TGF- β signaling inhibitor, and a nutritional supplement.

In some embodiments of the application, the tumor necrosis factor is present in an amount of 2-20ng/mL, the interleukin is present in an amount of 20-50ng/mL, and the Oncoinhibin is present in an amount of 1-10ng/mL, based on the volume of the reprogramming media.

In some embodiments of the application, the tumor necrosis factor is present in an amount of 5-15ng/mL, the interleukin is present in an amount of 25-40ng/mL, and the Oncoinhibin is present in an amount of 2-8ng/mL, based on the volume of the reprogramming media.

In some embodiments of the present application, the english name of the tumor necrosis factor is tumor necrosis factor, the english name of the tumor necrosis factor is TNF, and the TNF is derived from offshore.

In still other embodiments of the present application, the interleukins include interleukin-1 beta and interleukin-6, the english language of interleukin-1 beta is abbreviated as IL-1 beta, and the interleukin-1 beta is derived from a magnificent organism; the English of the interleukin-6 is called IL-6, and the interleukin-6 is derived from a Huamei organism.

In other embodiments of the present application, the english name of the Oncostatin is oncostatin M, the english name of the Oncostatin is abbreviated as OSM, and the Oncostatin is derived from the living organism of the next holy.

In some embodiments of the application, the growth factor is present in an amount of 40-80ng/mL, the ROCK kinase inhibitor is present in an amount of 5-20. Mu.M, the Wnt signaling pathway agonist is present in an amount of 1-10. Mu.M, the TGF-beta signaling inhibitor is present in an amount of 0.1-5. Mu.M, and the nutritional supplement is present in an amount of 0.5-10% of the final volume of the reprogramming media.

In some embodiments of the application, the growth factor is present in an amount of 60-75ng/mL, the ROCK kinase inhibitor is present in an amount of 5-15. Mu.M, the Wnt signaling pathway agonist is present in an amount of 2-6. Mu.M, the TGF-beta signaling inhibitor is present in an amount of 1-3. Mu.M, and the nutritional supplement is present in an amount of 1-7% of the final volume of the reprogramming media, based on the volume of the reprogramming media.

In some embodiments of the application, the reprogramming media further comprises a growth factor, a ROCK kinase inhibitor, a Wnt signaling pathway agonist, a TGF- β signaling inhibitor, a nutritional supplement, and a buffer.

In some embodiments of the application, the growth factors include an epidermal growth factor derived from a near-shore organism and a basic fibroblast growth factor derived from a near-shore organism; the ROCK kinase inhibitor Y-27632 is derived from Tao Shu organisms, the Wnt signal pathway agonist CHIR99021 is derived from Tao Shu organisms, the TGF-beta signal inhibitor A8301 is derived from Tao Shu organisms, the nutritional supplements comprise an N2 nutritional supplement and a B27 nutritional supplement, and the N2 nutritional supplement is derived from the following holy organism.

Specifically, by reinfusion of a biological agent comprising skeletal muscle precursor-like cells, local inflammation can be well controlled, and muscle fibrosis can be controlled, thereby reducing muscle degeneration and allowing better recovery of skeletal muscle.

In some embodiments of the application, the animal model comprises a model of cytotoxin-induced muscle injury.

Examples

1. Initial organizational nature and source legitimacy declaration:

human skeletal muscle precursor-like cells positive for CD29, CD56 and PAX7 expression were obtained from skeletal muscle tissue resected after clinical orthopedic surgery or donor donated skeletal muscle tissue.

Specifically, the skeletal muscle tissue was shown to be normal skeletal muscle tissue by pathological examination.

Specifically, the skeletal muscle tissue resected after the clinical orthopedic surgery or the skeletal muscle tissue donated by the donor is a surgical sample derived from a patient with the age of not more than 40 years, the patient is not infected by infectious viruses after medical examination, and the patient does not use steroid hormone medicine within 6 months before the surgery. The patient was fully informed of the purpose of the acquisition of the surgical sample prior to surgery and signed an informed consent form.

2. Acquisition of skeletal muscle primary cells

Firstly, after washing and sterilizing skeletal muscle tissues by using a sterile PBS buffer (0.01M), subjecting the skeletal muscle tissues to digestion treatment for 90 minutes at 37 ℃ by using 3 ml of a cell digestion solution, so as to obtain a skeletal muscle primary cell suspension, wherein the cell digestion solution consists of type III collagenase, a sterile PBS buffer and pancreatin digestion solution, and the cell digestion solution comprises the sterile PBS buffer (0.01M) with the content of 49.5% and pancreatin digestion solution (1X) with the content of 49.5% by volume of the cell digestion solution, the type III collagenase is derived from Shanghai health biotechnology Co Ltd, the sterile PBS buffer is derived from marpranopsis life technologies Co., and the pancreatin digestion solution (containing 0.25% pancreatin) is derived from Biyun;

then, the skeletal muscle primary cell suspension was screened using a 70 micron sterile screen with the aid of sterile PBS buffer, mucus and undigested tissue were removed and filtrate was collected to complete the screening;

then, after the filtrate was subjected to centrifugation and the supernatant was removed to obtain a precipitate, after the suspension was carried out by adding a erythrocyte lysate (derived from solebao) to the precipitate, the above procedure was repeated again by centrifugation until no erythrocyte was observed in the precipitate after the re-centrifugation to complete erythrocyte lysis removal, and finally skeletal muscle primary cells were obtained, wherein the centrifugation speed per centrifugation was 1000g and the centrifugation time per centrifugation was 3 minutes.

3. Acquisition of skeletal muscle precursor-like cells

The reprogramming media composition includes: based on the volume of the reprogramming media, the basal medium Ham F10 (from the Wohaze life technologies Co., ltd.) accounts for 91% of the total volume of the reprogramming media, the epithelial cell growth factor EGF is 20 nanograms/ml, the basic fibroblast growth factor bFGF is 50 nanograms/ml, the nutritional supplement N2 (1X) is 1%, the B27 nutritional supplement (1X) is 1%, the ROCK kinase inhibitor Y-27632 is 10 mu M, the Wnt signal pathway agonist CHIR99021 is 3 mu M, the TGF-beta signal inhibitor A8301 is 1 mu M, the tumor necrosis factor TNF is 10 nanograms/ml, the interleukin-1B is 20 nanograms/ml, the Oncoinhibin OSM is 5 nanograms/ml, the interleukin-6 is 10 nanograms/ml, and the fetal bovine serum (the English technology of fetal bovine serum is called FBS, beijing technologies Co., ltd.).

Control medium: based on the volume of the control medium, the basal medium Ham F10 (from the Living Tech Co., ltd.) accounts for 91% of the total volume of the control medium, and has an epithelial cell growth factor EGF content of 20ng/ml, a basic fibroblast growth factor bFGF content of 50ng/ml, a nutritional supplement N2 (1X) content of 1%, a B27 nutritional supplement (1X) content of 1%, a ROCK kinase inhibitor Y-27632 content of 10 mu M, a Wnt signal pathway agonist CHIR99021 content of 3 mu M, a TGF-beta signal inhibitor A8301 content of 1 mu M, and fetal bovine serum (English of fetal bovine serum is abbreviated as: FBS from the Litsche Co., ltd.) content of 5%.

Differences between reprogramming media and control media: the reprogramming culture medium contains tumor necrosis factor TNF with the content of 10 nanograms/milliliter, interleukin-1 b with the content of 20 nanograms/milliliter, oncostatin OSM with the content of 5 nanograms/milliliter and interleukin-6 with the content of 10 nanograms/milliliter, and the control culture medium does not contain tumor necrosis factor TNF, interleukin-1 b, oncostatin OSM and interleukin-6.

FIG. 1 is a schematic photograph of a first generation skeletal muscle precursor-like cell morphology in accordance with an embodiment of the present application; FIG. 2 is a schematic photograph of a fourth generation skeletal muscle precursor-like cell morphology in accordance with an embodiment of the present application; FIG. 3 is a graphical representation of skeletal muscle precursor-like cell proliferation in accordance with an embodiment of the present application.

Reprogramming media culture:

placing skeletal muscle primary cells in a 6-hole plate at an inoculation area of 10000 per square centimeter, adding 2 milliliters of reprogramming culture medium into each hole to carry out dedifferentiation culture to obtain skeletal muscle precursor-like cells until the fusion degree of the skeletal muscle precursor-like cells is not lower than 80%, and then carrying out digestion treatment on the skeletal muscle precursor-like cells by using pancreatin digestion solution for 5 minutes, wherein the pancreatin digestion solution (containing 0.25% pancreatin) is derived from Biyun; the skeletal muscle precursor-like cells are further subcultured with a reprogramming medium to obtain first-generation (P1) skeletal muscle precursor-like cells, second-generation (P2) skeletal muscle precursor-like cells, third-generation (P3) skeletal muscle precursor-like cells … … tenth-generation (P10) skeletal muscle precursor-like cells, and skeletal muscle precursor-like cells are continuously passaged in the reprogramming medium.

Control medium culture:

placing skeletal muscle primary cells in a 6-hole plate at an inoculation area of 10000 per square centimeter, adding 2 milliliter of control culture medium into each hole to carry out dedifferentiation culture to obtain control skeletal muscle precursor-like cells, and carrying out digestion treatment on the control skeletal muscle precursor-like cells by using pancreatin digestive juice until the fusion degree of the control skeletal muscle precursor-like cells is not lower than 80%, wherein the digestion treatment time is 5 minutes, and the pancreatin digestive juice (containing 0.25% pancreatin) is derived from Biyun; the control skeletal muscle precursor-like cells were subcultured again with the control medium to obtain first-generation (P1) control skeletal muscle precursor-like cells, second-generation (P2) control skeletal muscle precursor-like cells, third-generation (P3) control skeletal muscle precursor-like cells, fourth-generation (P4) control skeletal muscle precursor-like cells, fifth-generation (P5) control skeletal muscle precursor-like cells, and control skeletal muscle precursor-like cells were passable in the control medium but were unable to proliferate in the control medium when passaged to the fifth-generation, see fig. 3.

Referring to fig. 1 and 2, fig. 1 is a P1 generation skeletal muscle precursor-like cell, fig. 2 is a P4 generation skeletal muscle precursor-like cell, and the morphology of the skeletal muscle precursor-like cell is kept uniform and polygonal by increasing the culture times.

Referring to fig. 3, as the number of passages increases, i.e., from P0 to P1, from P1 to P2 … …, and from P9 to P10, the reprogramming media is able to continue to expand skeletal muscle precursor-like cells, which have been increasingly amplified; subculturing is performed by using a control culture medium, the amplification capacity of the control skeletal muscle precursor-like cells increases and decreases with the increase of the number of passages, and the amplification capacity is strongest at the second passage, wherein the abscissa in fig. 3 represents the cell number, the ordinate represents the cell expansion multiple, and the number of passages is the number of passages.

4. Flow assay for skeletal muscle precursor-like cells

FIG. 4 is a schematic diagram showing the case of CD34, a gene expression marker of skeletal muscle precursor-like cells of an embodiment of the present application; FIG. 5 is a schematic diagram showing the case of CD45, a gene expression marker of skeletal muscle precursor-like cells of an embodiment of the present application; FIG. 6 is a schematic diagram showing the case of HLA-DRPQ, which is a gene expression marker of skeletal muscle precursor-like cells according to an embodiment of the present application; FIG. 7 is a schematic diagram showing the case of CD29, a gene expression marker of skeletal muscle precursor-like cells of an embodiment of the present application; FIG. 8 is a schematic diagram showing the case of CD56, a gene expression marker of skeletal muscle precursor-like cells of an embodiment of the present application; FIG. 9 is a schematic diagram showing the case of CD24, a gene expression marker of skeletal muscle precursor-like cells of an embodiment of the present application; FIG. 10 is a schematic diagram showing the case of the gene expression marker PAX7 of skeletal muscle precursor-like cells of the present application.

Separating skeletal muscle precursor-like cells from the reprogramming media, rinsing the skeletal muscle precursor-like cells with sterile PBS buffer (from the GmbH of the life technologies of Gymnosiren), then subjecting the skeletal muscle precursor-like cells to digestion with pancreatin digestion solution (containing 0.25% pancreatin) derived from Biyun, and centrifuging to collect cell pellets; adding 600 microliters of staining buffer to the cell sediment, sub-packaging the cell sediment into 6 1.5mL centrifuge tubes according to 100 microliters, adding 5 microliters of CD34, CD45, HLA-DRPQ, CD29, CD56, CD24 and PAX7 streaming antibodies into the 6 centrifuge tubes respectively, incubating for 20 minutes, adding 400 microliters of staining buffer to the streaming tube after re-suspending to obtain a sample to be tested, and performing on-machine detection on the sample to be tested in Accuri C6 streaming cytometer of BD bioscience in U.S. to obtain streaming analysis results shown in FIG. 4, FIG. 5, FIG. 6, FIG. 7, FIG. 8, FIG. 9 and FIG. 10, wherein the staining buffer is derived from Simer's femto, and the streaming antibodies to be tested are derived from Simer's femto. The specific operation and analysis steps of the flow detection are conventional technical means for those skilled in the art, and are not described herein.

Referring to fig. 4, 5 and 6, the positive rate of the positive peak of the marker CD34 is 0.54%, the positive rate of the positive peak of the marker CD45 is 0.97%, the positive rate of the positive peak of the marker HLA-DRPQ is 0.68%, and when the positive rate of the positive peak is more than 70%, it is indicated that skeletal muscle precursor-like cells positively express the marker, and thus, skeletal muscle precursor-like cells of the present application negatively express CD34, CD45 and HLA-DRPQ; referring to fig. 7, 8, 9 and 10, the positive rate of the negative peak of the marker CD29 was 99.9%, the negative rate of the positive peak of the marker CD56 was 100%, the positive rate of the negative peak of the marker CD24 was 99.2%, the positive rate of the negative peak of the marker PAX7 was 93.3%, and when the positive rate of the positive peak was more than 70%, it was demonstrated that the skeletal muscle precursor-like cells positively expressed the marker, and therefore, the skeletal muscle precursor-like cells positively expressed CD29, CD56, CD24 and PAX7 of the present application. The abscissas in fig. 4, 5, 6, 7, 8, 9, and 10 represent the same meaning, wherein the abscissas in fig. 4 represent relative fluorescence intensities, and the abscissas represent cell numbers.

5. Preparation of biological agents comprising skeletal muscle precursor-like cells

Mixing skeletal muscle precursor-like cells with a pharmaceutically acceptable carrier to produce a biological agent comprising skeletal muscle precursor-like cells, the biological agent comprising skeletal muscle precursor-like cells for repairing muscle damage, the specific steps of the biological agent comprising skeletal muscle precursor-like cells comprising: mixing skeletal muscle precursor-like cells with physiological saline to obtain a biological preparation containing skeletal muscle precursor-like cells, namely a skeletal muscle precursor-like cell injection, wherein each microliter of skeletal muscle precursor-like cell injection contains 2×10 ⁴ And skeletal muscle precursor-like cells.

6. Application of skeletal muscle precursor-like cell preparation in animal model and effect demonstration is carried out

FIG. 11 is a schematic diagram showing pathological tissue section contrast photographs of a drug-induced muscle injury model mouse according to an embodiment of the present application at different times in an experimental group and a control group; FIG. 12 is a graph showing the comparison of the area distribution of neomyofibroblasts in experimental and control groups in drug-induced muscle injury model mice according to the example of the present application.

The animal model refers to a drug-induced muscle injury model;

establishment of drug-induced muscle injury model: selecting 8-week-old male mice, diluting cyclophosphamide dry powder to 10 mu M with sterile PBS buffer solution in a biosafety cabinet to obtain cyclophosphamide injection, subpackaging the cyclophosphamide injection, and preserving at-80 ℃ to avoid repeated freeze thawing of the cyclophosphamide injection; the method comprises the steps of injecting 10% of chloral hydrate into an abdominal cavity of a mouse, lying the mouse on an ultra clean bench after injecting the chloral hydrate for one to two minutes, removing the fur outside the tibialis anterior muscle of the mouse by a shaver, vertically injecting 100 microliters of cyclophosphamide injection into the tibialis anterior muscle of the mouse by an insulin injection needle in 3 needles, wherein 1 needle is positioned at the central position of the tibialis anterior muscle, and the other two needles are positioned at two ends of the tibialis anterior muscle to obtain a drug-induced muscle injury model, wherein the English abbreviation of cyclophosphamide is CTX, cyclophosphamide dry powder is obtained from Shanghai source stream biotechnology Co, sterile PBS buffer is obtained from Wuhanplauosai life technologies Co, and the chloral hydrate is obtained from Shanghai Bei Moda biotechnology Co.

Experimental group: injecting 50 microliter of skeletal muscle precursor-like cell injection into the tibialis anterior of the mouse on day 0, injecting 50 microliter of skeletal muscle precursor-like cell injection into the tibialis anterior of the mouse again on day 7, collecting a part of tibialis anterior of the experimental mouse, sampling the tibialis anterior, and performing pathological HE staining to examine the injury condition of the tibialis anterior; on day 15, mice were sacrificed to collect tibialis anterior, samples of tibialis anterior were taken and pathological HE staining was performed to investigate tibialis anterior injury. Wherein the HE staining is hematoxylin-eosin staining.

Control group: injecting 50 microliter of physiological saline into the tibialis anterior muscle of the mouse on day 0, injecting 50 microliter of physiological saline into the tibialis anterior muscle of the mouse again on day 7, collecting a part of tibialis anterior muscle of the experimental mouse, and performing pathological HE staining on tibialis anterior muscle sampling to investigate the injury condition of the tibialis anterior muscle; on day 15, mice were sacrificed to collect tibialis anterior, samples of tibialis anterior were taken and pathological HE staining was performed to investigate tibialis anterior injury. Wherein the HE staining is hematoxylin-eosin staining.

Referring to fig. 11, the tibial anterior muscle of the mice in the experimental group was better recovered, showing that the visual field was filled with the neomyofibroblasts with the nucleus in the center, and the number of immune cells was small, indicating that the immune response after the muscle injury of the tibial anterior muscle had been substantially completed; whereas the control mice had a large number of immune cells in the tibialis anterior muscle, while the number of neomyofibroblasts was small and the cross-sectional area was small.

Referring to fig. 12, statistical analysis of the neomyofibroblasts from the different groups of mice showed that the area distribution of the neomyofibroblasts from the experimental group of mice was significantly different from that of the control group of mice. The abscissa in fig. 12 represents the area of the muscle fiber in square micrometers (i.e., μm ² ) The ordinate represents the myofiber frequency in 100%.

The skeletal muscle precursor cell treatment method provided by the application can better solve muscle diseases. Since skeletal muscle precursor cells can proliferate and differentiate to generate new myofibroblasts in vivo and can home to form a muscle stem cell bank, a continuous cell supply can be provided for patients in theory. Skeletal muscle precursor-like cells cultured using a reprogramming medium to provide sustained proliferation of skeletal muscle precursor-like cells in vitro.

In a mouse muscle injury model experiment, the muscle of the tibialis anterior of a mouse in an experimental group is found to be better recovered, the visual field is filled by neomyofibroblasts with nuclei in the center, the number of immune cells is small, and the immune response of the tibialis anterior after muscle injury is basically finished; whereas the control mice had a large number of immune cells in the tibialis anterior muscle, while the number of neomyofibroblasts was small and the cross-sectional area was small.

Because the surface of the skeletal muscle precursor-like cells obtained by the application is not expressed in protein HLA-DRPQ related to immune rejection reaction, the allogenic feedback of the skeletal muscle precursor-like cells can be realized, the skeletal muscle precursor-like cells obtained by the application positively express CD29, CD56, CD24 and PAX7, and can be amplified in vitro to be used for establishing a cell seed bank, and the skeletal muscle precursor-like cells can be directly injected in situ with the skeletal muscle precursor-like cell preparation when being used for treating clinical skeletal muscle atrophy.

The foregoing examples are illustrative only and serve to explain some features of the method of the application. The appended claims are intended to claim the broadest possible scope and the embodiments presented herein are merely illustrative of selected implementations based on combinations of all possible embodiments. It is, therefore, not the intention of the applicant that the appended claims be limited by the choice of examples illustrating the features of the application. Some numerical ranges used in the claims also include sub-ranges within which variations in these ranges should also be construed as being covered by the appended claims where possible.

Claims

1. A biologic comprising skeletal muscle precursor-like cells that negatively express at least one of CD34, CD45, HLA-DRPQ.

2. The biologic comprising skeletal muscle precursor-like cells of claim 1, wherein the skeletal muscle precursor-like cells positively express at least one of CD29, CD56, CD24, and PAX7.

3. A method of preparing a biologic comprising skeletal muscle precursor-like cells, comprising the steps of:

s0: providing skeletal muscle precursor-like cells;

4. A method of preparing a biologic comprising skeletal muscle precursor-like cells according to claim 3, comprising the steps of:

s00: providing skeletal muscle primary cells;

5. The method of claim 4, wherein the method of preparing skeletal muscle precursor-like cells further comprises:

s02: and placing the skeletal muscle precursor-like cells into the reprogramming culture medium for amplification culture until the fusion degree of the skeletal muscle precursor-like cells is not lower than 80%, and then performing digestion treatment on the skeletal muscle precursor-like cells by using the pancreatin digestive juice to obtain the passaged skeletal muscle precursor-like cells.

6. The method of claim 5, wherein the reprogramming media further comprises growth factors, ROCK kinase inhibitors, wnt signaling pathway agonists, TGF- β signaling inhibitors, and nutritional supplements.

7. The method according to claim 6, wherein the tumor necrosis factor is contained in an amount of 2-20ng/mL, the interleukin is contained in an amount of 20-50ng/mL, and the tumor suppressor is contained in an amount of 1-10ng/mL, based on the volume of the reprogramming media.

8. The method of claim 7, wherein the growth factor is present in an amount of 40-80ng/mL, the ROCK kinase inhibitor is present in an amount of 5-20 μm, the Wnt signaling pathway agonist is present in an amount of 1-10 μm, the TGF- β signaling inhibitor is present in an amount of 0.1-5 μm, and the nutritional supplement is present in an amount of 0.5-10% of the final volume of the reprogramming media.

9. Use of a biological agent comprising skeletal muscle precursor-like cells according to any one of claims 1-2 for intervention in an in vivo animal model using the biological agent comprising skeletal muscle precursor-like cells.

10. The use of a biologic comprising skeletal muscle precursor-like cells according to claim 9, wherein the animal model comprises a model of cytotoxin-induced muscle injury.