WO2023217130A1 - Formulation biologique contenant des cellules de type précurseur de muscle squelettique, son procédé de préparation et son application - Google Patents

Formulation biologique contenant des cellules de type précurseur de muscle squelettique, son procédé de préparation et son application Download PDF

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WO2023217130A1
WO2023217130A1 PCT/CN2023/092965 CN2023092965W WO2023217130A1 WO 2023217130 A1 WO2023217130 A1 WO 2023217130A1 CN 2023092965 W CN2023092965 W CN 2023092965W WO 2023217130 A1 WO2023217130 A1 WO 2023217130A1
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cells
skeletal muscle
muscle precursor
precursor
preparation containing
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周伸奥
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上海赛立维生物科技有限公司
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Definitions

  • the present invention relates to the field of biotechnology, and in particular to a biological agent containing skeletal muscle precursor-like cells and its preparation method and application.
  • Skeletal muscle is the largest organ of the human body, and its main functions are movement, shape maintenance, and respiration.
  • myofiber cell the English name of myofiber cell is: myofiber. It is composed of many parallel-arranged myofibrils (myofibril's English name: myofibril) and is surrounded by a myofibrillar membrane (myofibril's English name: sarcolemma, the cell membrane of myofiber cells).
  • myofibrillar membrane myofibril's English name: sarcolemma, the cell membrane of myofiber cells.
  • basement membrane the English name of basement membrane is: basement membrane).
  • each myofibril can be divided into multiple sarcomeres (the English name of sarcomeres is: sarcomeres), which generate tensile force through contraction.
  • each muscle fiber cell usually contains hundreds of cell nuclei (the English name of the cell nucleus is: myonucleus). They are distributed close to the muscle fiber membrane and jointly control the movement and metabolic processes of the muscle fiber cells.
  • myofiber cells skeletal muscle also contains a large amount of connective tissue (the English name of connective tissue is: connective tissue), Blood vessels, nerves, and connective tissue connect muscle fiber cells into an overall functional unit and are fixed to the bones through tendons (the English name of tendons is: tendon).
  • muscle stem cells can be labeled into two groups.
  • the first group of muscle stem cells accounts for about 80% of the total number. They proliferate, differentiate, fuse and eventually form myofiber cells; the scientists will The second group of muscle stem cells is called "reserve cells”.
  • the second group of muscle stem cells divides at a significantly slower rate and eventually becomes a muscle stem cell bank in muscle tissue.
  • follow-up studies have shown that the first group of muscle stem cells and the second group of muscle stem cells come from the asymmetric division of muscle stem cells (the English name of asymmetric division is: asymmetric division).
  • Duchenne muscular dystrophy is a common fatal neuromuscular disease in children. It is inherited in an X-linked recessive manner and is caused by mutations in the DMD gene. Children often develop the disease in childhood. The disease is hidden and can easily be missed or misdiagnosed in the early stages. Because defects at the genetic level are irreversible, the body cannot repair the damage through muscle cell regeneration and compensation, which gradually leads to late changes in massive fibrosis in skeletal muscles, myocardium and other parts of the body, and ultimately leads to death due to severe impact on the cardiopulmonary function of the child.
  • the DMD gene is located at Xp21, with a genome span of 2.4Mb.
  • Dystrophin deficiency leads to at least two results. The first result is that muscle cells are destroyed by mechanical force during eccentric contraction, and the ion channels that are sensitive to mechanical force are dysregulated. The second result is that muscle cells are damaged by mechanical force during eccentric contraction. Because the muscle cell membrane cannot remain intact, abnormal protein and calcium influx, creatine kinase efflux, etc. occur, eventually causing muscle cell degeneration and necrosis.
  • Peripheral nerve injury (English name: peripheral nerve injury, PNI) is a common clinical trauma, which can be caused by various reasons such as tearing, traction, cutting, compression, ischemia, high temperature, freezing, infection and nutritional and metabolic disorders. Peripheral nerves can regenerate to a certain extent after damage, but the recovery rate is very slow. After nerves are severely damaged, the skeletal muscles they control atrophy within a few days. As the atrophy time prolongs, cell apoptosis gradually increases, and the number of myocyte nuclei gradually decreases, eventually causing permanent loss of skeletal muscle function.
  • PNI peripheral nerve injury
  • Muscle atrophy caused by genetic mutations such as Duchenne muscular dystrophy and muscle atrophic changes caused by peripheral nerve injury (PNI) will cause muscle atrophic lesions, causing the number of muscle cells to decrease until the permanent loss of skeletal muscle function thereby threatening the patient's life. If muscle atrophy can be controlled, muscle degeneration can be reduced and movement and respiratory functions can be saved. Therefore, there are several ideas for the treatment of muscle diseases: cell therapy, gene editing therapy and traditional hormone therapy.
  • MTT myoblast transfer therapy
  • the myofiber cells fuse and then express normal DMD, correcting the degeneration, necrosis and other changes of muscle fibers; mesenchymal stem cells (English name: mesenchymal stem cells, English abbreviation: MSC) are a type of adult stem cells with multiple differentiation potentials , MSCs cultured in vitro for more than 3 generations have good uniformity, low immune response to vein transplantation, and 5 When MSCs were transferred to gene knockout mice, the intensity of DMD immunofluorescence increased with time, confirming the feasibility of introducing and expressing the DMD gene through this approach.
  • mesenchymal stem cells English name: mesenchymal stem cells, English abbreviation: MSC
  • the biggest difficulty in muscle stem cell therapy is the lack of donor cells.
  • the patient's own muscle stem cells need to be removed, and the expression of Dystrophin can be obtained through infection in vitro.
  • Injection back into the patient's body but there is currently no method that can expand large amounts of human muscle stem cells in vitro;
  • research on combined mesenchymal stem cell therapy shows that this method is only suitable for patients with mild to moderate muscle atrophy, but not for patients with extreme muscle mass. The curative effect is minimal, and the long-term efficacy is unknown.
  • Gene editing therapy In vivo gene editing mediated by adeno-associated virus (English abbreviation: AAV) is a promising treatment idea. By injecting adeno-associated virus, the expression of dystrophin can be restored in myofibroblasts to a certain extent, thus To increase the strength of muscle fiber cells and avoid necrosis, the advantages of AAV treatment are as follows: first, the virus can be amplified in vitro and is easy to obtain; second, AAV has been proven to have low tumorigenicity in vivo and is relatively safe; third, because the virus Due to its diffusibility, AAV can infect a larger range of tissues through blood transport; other gene editing therapies also include exon skipping and stop codon readthrough.
  • Exon skipping uses exon skipping splicing technology to frameshift DMD. Changing the mutation type to an integer mutation type may transform DMD with severe symptoms into mild symptoms; terminate DMD Code reading: The aminoglycoside antibiotic gentamicin can reduce the ability of ribosomes to recognize abnormal terminators during the translation process of dystrophin, and skip these abnormal terminators to continue translation. This result was found in mouse animal experiments It also achieved significant curative effect.
  • Gene therapy has the following shortcomings: First, the biggest obstacle to adeno-associated virus therapy is that it cannot break through the microenvironmental barrier to infect muscle stem cells. Myofibroblasts that have been infected by the virus and acquire Dystrophin expression will die due to lifespan and metabolic replacement, so AAV therapy cannot fundamentally solve the problem of muscle diseases and can only treat patients multiple times and continuously; secondly, exon skipping can only target specific gene defect types, and the energy efficiency of oligonucleotides entering the cell membrane is low, which may It is immunogenic; thirdly, stop codon read-through therapy only targets nonsense mutations because gentamicin has low read-through energy efficiency and long-term large-scale application has large toxic side effects, and the drug may have short-term or long-term side effects.
  • Glucocorticoids have achieved certain success in the treatment of DMD due to their anti-inflammatory effects and are most commonly used in clinical applications. However, the side effects of glucocorticoids often outweigh their benefits and can produce long-term inflammatory reactions that require combined anti-inflammatory treatment.
  • the purpose of the present invention is to provide a biological preparation containing skeletal muscle precursor-like cells and its preparation method and application, so as to solve the problem of immune rejection in the treatment of muscle atrophy caused by gene mutations in the prior art.
  • the present invention provides a biological preparation containing skeletal muscle precursor-like cells that negatively express at least one of CD34, CD45, and HLA-DRPQ.
  • the beneficial effect of the biological preparation containing skeletal muscle precursor-like cells of the present invention is that the negative expression of at least one of CD34, CD45, and HLA-DRPQ by the skeletal muscle precursor-like cells allows the human body to respond to the skeletal muscle.
  • Precursor-like cells are well accepted, so this application solves the problem of immune rejection in the existing technology for treating muscle atrophy caused by gene mutations.
  • the skeletal muscle precursor-like cells positively express at least one of CD29, CD56, CD24 and PAX7.
  • the present invention also provides a method for preparing a biological preparation containing skeletal muscle precursor-like cells, which includes the following steps:
  • S0 Provides skeletal muscle precursor-like cells
  • S1 Mix the skeletal muscle precursor-like cells with a pharmaceutically acceptable carrier to obtain a biological preparation containing skeletal muscle precursor-like cells, wherein the skeletal muscle precursor-like cells positively express CD29, CD56, At least one of CD24 and PAX7, and the skeletal muscle precursor-like cells negatively express at least one of CD34, CD45, and HLA-DRPQ.
  • the beneficial effect of the preparation method of the biological preparation containing skeletal muscle precursor-like cells of the present invention is that the preparation method of the biological preparation containing skeletal muscle precursor-like cells is simple, and the obtained biological preparation containing skeletal muscle precursor-like cells It can reduce the impact of muscle damage on the human body, adapt well to the human body, and will not cause immune rejection with the recipient.
  • the preparation method of skeletal muscle precursor-like cells includes the following steps:
  • S01 Place the primary skeletal muscle cells into a reprogramming medium for dedifferentiation culture until the fusion degree of the skeletal muscle precursor-like cells is not less than 80%, and use trypsin digestion solution to treat the skeletal muscle
  • the precursor-like cells are digested to obtain skeletal muscle precursor-like cells, wherein the reprogramming medium includes basal medium, tumor necrosis factor, interleukin and oncostatin.
  • the preparation method of the skeletal muscle precursor-like cells also includes: S02: placing the skeletal muscle precursor-like cells into the reprogramming medium for expansion and culture until the skeletal muscle precursor-like cells After the fusion degree of the cells is not less than 80%, the skeletal muscle precursor-like cells are digested using the trypsin digestion solution to obtain passaged skeletal muscle precursor-like cells.
  • S02 placing the skeletal muscle precursor-like cells into the reprogramming medium for expansion and culture until the skeletal muscle precursor-like cells After the fusion degree of the cells is not less than 80%, the skeletal muscle precursor-like cells are digested using the trypsin digestion solution to obtain passaged skeletal muscle precursor-like cells.
  • the beneficial effect is that skeletal muscle precursor-like cells can be continuously expanded in the reprogramming medium and the obtained skeletal muscle precursor-like cells can express skeletal muscle precursor-like cell markers.
  • the reprogramming medium also includes growth factors, ROCK kinase inhibitors, Wnt signaling pathway agonists, TGF- ⁇ signaling inhibitors and nutritional supplements.
  • the content of tumor necrosis factor is 2-20ng/mL
  • the content of interleukin is 20-50ng/mL
  • the content of oncostatin is based on the volume of the reprogramming medium. is 1-10ng/mL.
  • the content of the growth factor is 40-80ng/mL
  • the content of the ROCK kinase inhibitor is 5-20 ⁇ M
  • the content of the Wnt signaling pathway agonist is is 1-10 ⁇ M
  • the content of the TGF- ⁇ signal inhibitor is 0.1-5 ⁇ M
  • the content of the nutritional supplement is 0.5-10% of the final volume of the reprogramming medium.
  • the present invention also provides an application of a biological preparation containing skeletal muscle precursor-like cells, using the biological preparation containing skeletal muscle precursor-like cells to intervene in an in vivo animal model.
  • the beneficial effects of the application of the skeletal muscle precursor-like cells of the present invention are: after infusion of biological agents containing skeletal muscle precursor-like cells, local inflammation can be well controlled, muscle fibrosis can be controlled, and muscle degeneration can be alleviated. sexual lesions so that skeletal muscles can recover better.
  • the animal model includes a cytotoxin-induced muscle injury model.
  • Figure 1 is a photographic schematic diagram of the morphology of the first generation skeletal muscle precursor-like cells according to an embodiment of the present invention
  • Figure 2 is a photographic schematic diagram of the morphology of fourth-generation skeletal muscle precursor-like cells according to an embodiment of the present invention
  • Figure 3 is a schematic diagram of the proliferation curve of skeletal muscle precursor-like cells according to an embodiment of the present invention.
  • Figure 4 is a schematic diagram of the gene expression marker CD34 of skeletal muscle precursor-like cells according to an embodiment of the present invention.
  • Figure 5 is a schematic diagram of the gene expression marker CD45 of skeletal muscle precursor-like cells according to an embodiment of the present invention.
  • Figure 6 shows gene expression markers of skeletal muscle precursor-like cells according to the embodiment of the present invention. Schematic diagram of HLA-DRPQ situation
  • Figure 7 is a schematic diagram of the gene expression marker CD29 of skeletal muscle precursor-like cells according to an embodiment of the present invention.
  • Figure 8 is a schematic diagram of the gene expression marker CD56 of skeletal muscle precursor-like cells according to an embodiment of the present invention.
  • Figure 9 is a schematic diagram of the gene expression marker CD24 of skeletal muscle precursor-like cells according to an embodiment of the present invention.
  • Figure 10 is a schematic diagram of the gene expression marker PAX7 in skeletal muscle precursor-like cells according to an embodiment of the present invention.
  • Figure 11 is a schematic diagram comparing photos of pathological tissue sections of the drug-induced muscle injury model mice in the experimental group and the control group at different times according to the embodiment of the present invention
  • Figure 12 is a comparative diagram of the area distribution of new myofibroblasts in the experimental group and the control group of drug-induced muscle injury model mice according to the embodiment of the present invention.
  • Fibrosis is a significant pathological feature seen in muscle biopsy in patients with muscle atrophy, which can lead to muscle function disorders and ultimately fatality; anti-fibrosis treatment can not only improve muscle function, but also promote muscle regeneration, gene introduction and stem cells The colonization efficiency will become a necessary supplement for future gene and cell therapy.
  • anti-fibrosis treatment can not only improve muscle function, but also promote muscle regeneration, gene introduction and stem cells The colonization efficiency will become a necessary supplement for future gene and cell therapy.
  • drugs targeting fibrosis were tested and shown to improve muscle function and muscle damage phenotypes.
  • the biological agent containing skeletal muscle precursor-like cells of the present invention after being reinfused into the body, can effectively control local inflammation, control muscle fibrosis, and reduce muscle degeneration, so that skeletal muscles can be better treated. recover.
  • the present invention provides a biological preparation comprising skeletal muscle precursor-like cells that negatively express at least one of CD34, CD45, and HLA-DRPQ.
  • the skeletal muscle precursor-like cells negatively express at least one of CD34, CD45, and HLA-DRPQ, so that the human body can accept the skeletal muscle precursor-like cells well, so that this application solves the problem of the existing technology.
  • the HLA-DRPQ is the abbreviation of HLA-DR/DP/DQ.
  • the skeletal muscle precursor-like cells positively express at least one of CD29, CD56, CD24 and PAX7.
  • the present invention also provides a method for preparing a biological preparation containing skeletal muscle precursor-like cells, which includes the following steps:
  • S0 Provides skeletal muscle precursor-like cells
  • S1 Mix the skeletal muscle precursor-like cells with a pharmaceutically acceptable carrier to obtain a biological preparation containing skeletal muscle precursor-like cells, wherein the skeletal muscle precursor-like cells positively express CD29, CD56, At least one of CD24 and PAX7, and the skeletal muscle precursor-like cells negatively express at least one of CD34, CD45, and HLA-DRPQ.
  • the preparation method of the biological preparation containing skeletal muscle precursor-like cells is simple, and the obtained biological preparation containing skeletal muscle precursor-like cells can reduce the impact of muscle damage on the human body, and can adapt well to the human body without affecting the human body. Can cause immune rejection in the recipient.
  • the pharmaceutically acceptable carrier includes but is not limited to physiological saline and compound electrolyte injection.
  • the method for preparing skeletal muscle precursor-like cells includes the following steps:
  • S01 Place the primary skeletal muscle cells into a reprogramming medium for dedifferentiation culture until the fusion degree of the skeletal muscle precursor-like cells is not less than 80%, and use trypsin digestion solution to treat the skeletal muscle
  • the precursor-like cells are digested to obtain skeletal muscle precursor-like cells, wherein the reprogramming medium includes basal medium, tumor necrosis factor, interleukin and oncostatin.
  • the skeletal muscle primary cells are put into a reprogramming medium for dedifferentiation culture until the fusion degree of the skeletal muscle precursor-like cells is not less than 80%, and the trypsin digestion solution is used to digest the cells.
  • Skeletal muscle precursor-like cells are digested to obtain skeletal muscle precursors -like cells, wherein the reprogramming medium includes basal medium, tumor necrosis factor, interleukin and oncostatin, so that the skeletal muscle precursor-like cells continuously achieve self-renewal and proliferation, thereby solving the existing problem
  • the problem is that culture technology cannot culture skeletal muscle precursor-like cells in large quantities in vitro.
  • the preparation method of the skeletal muscle precursor-like cells further includes: S02: Put the skeletal muscle precursor-like cells into the reprogramming medium for expansion and culture until the skeletal muscle precursor-like cells are After the fusion degree of the muscle precursor-like cells is not less than 80%, the skeletal muscle precursor-like cells are digested using the trypsin digestion solution to obtain passaged skeletal muscle precursor-like cells. Skeletal muscle precursor-like cells can be continuously expanded in the reprogramming medium and the resulting skeletal muscle precursor-like cells can express skeletal muscle precursor-like cell markers.
  • the reprogramming medium further includes growth factors, ROCK kinase inhibitors, Wnt signaling pathway agonists, TGF- ⁇ signaling inhibitors and nutritional supplements.
  • the content of the tumor necrosis factor is 2-20ng/mL
  • the content of the interleukin is 20-50ng/mL
  • the inhibitory factor is 20-50ng/mL.
  • the content of oncostin is 1-10ng/mL.
  • the content of the tumor necrosis factor is 5-15ng/mL
  • the content of the interleukin is 25-40ng/mL
  • the inhibitory factor is 25-40ng/mL.
  • the content of oncostin is 2-8ng/mL.
  • the English name of the tumor necrosis factor is tumor necrosis factor
  • the English abbreviation of the tumor necrosis factor is TNF
  • the tumor necrosis factor TNF originates from the coastal area of Shanghai.
  • the interleukin includes interleukin-1 ⁇ and interleukin-6.
  • the English abbreviation of interleukin-1 ⁇ is IL-1 ⁇ .
  • the source of interleukin-1 ⁇ is From Huamei Biotechnology; the English abbreviation of interleukin-6 is IL-6, and the interleukin-6 is derived from Huamei Biotechnology.
  • the English name of the oncostatin is oncostatin M
  • the English abbreviation of the oncostatin is OSM
  • the oncostatin is derived from Yisheng Biotechnology.
  • the content of the growth factor is 40-80ng/mL
  • the content of the ROCK kinase inhibitor is 5-20 ⁇ M
  • the Wnt signaling pathway The content of the agonist is 1-10 ⁇ M
  • the content of the TGF- ⁇ signaling inhibitor is 0.1-5 ⁇ M
  • the content of the nutritional supplement is 0.5-10% of the final volume of the reprogramming medium.
  • the content of the growth factor is 60-75ng/mL
  • the content of the ROCK kinase inhibitor is 5-15 ⁇ M
  • the Wnt signal The content of the pathway agonist is 2-6 ⁇ M
  • the content of the TGF- ⁇ signaling inhibitor is 1-3 ⁇ M
  • the content of the nutritional supplement is 1-7% of the final volume of the reprogramming medium.
  • the reprogramming medium further includes growth factors, ROCK kinase inhibitors, Wnt signaling pathway agonists, TGF- ⁇ signaling inhibitors, nutritional supplements and buffers.
  • the growth factors include epidermal growth factor and alkaline Fibroblast growth factor, the epidermal growth factor is derived from nearshore organisms, and the basic fibroblast growth factor is derived from nearshore organisms; the ROCK kinase inhibitor Y-27632 is derived from Taoshu organisms, and the Wnt signaling pathway
  • the agonist CHIR99021 is derived from Taoshu Biotech
  • the TGF- ⁇ signaling inhibitor A8301 is derived from Taoshu Biotech
  • the nutritional supplements include N2 nutritional supplements and B27 nutritional supplements, the N2 nutritional supplements are derived from Yisheng biology.
  • the present invention also provides an application of a biological preparation containing skeletal muscle precursor-like cells, using the biological preparation containing skeletal muscle precursor-like cells to intervene in an in vivo animal model.
  • skeletal muscle precursor-like cells Through the infusion of biological agents containing skeletal muscle precursor-like cells, local inflammation can be well controlled, muscle fibrosis can be controlled, and muscle degeneration can be alleviated, so that skeletal muscles can be better recovered.
  • the animal model includes a cytotoxin-induced muscle injury model.
  • Human skeletal muscle precursor-like cells that positively express CD29, CD56 and PAX7 are obtained from skeletal muscle tissue removed after clinical orthopedic surgery or donated by donors.
  • the skeletal muscle tissue was found to be normal skeletal muscle tissue upon pathological examination.
  • the skeletal muscle tissue removed after clinical orthopedic surgery or the skeletal muscle tissue donated by a donor is a surgical sample derived from a patient aged no more than 40 years old.
  • the patient has no infectious virus infection after medical examination, and the patient has no infectious virus infection before surgery. No steroid use within 6 months drug.
  • the patient was fully informed about the purpose of obtaining surgical samples before surgery and signed an informed consent form.
  • sterile PBS buffer (0.01M) to clean and sterilize the skeletal muscle tissue
  • cell digestion solution 3 ml of cell digestion solution to digest the skeletal muscle tissue at 37 degrees Celsius for 90 minutes to obtain primary skeletal muscle cells.
  • the cell digestion solution is composed of type III collagenase, sterile PBS buffer and trypsin digestion solution, and based on the volume of the cell digestion solution, it includes 49.5% sterile PBS buffer (0.01M) and Trypsin digestion solution (1X) with a content of 49.5%, type III collagenase with a content of 1%, type III collagenase is from Shanghai Maokang Biotechnology Co., Ltd., and sterile PBS buffer is from Wuhan Pronosai Life Technology Co., Ltd., trypsin digestion liquid (containing 0.25% trypsin) comes from Biyuntian;
  • the skeletal muscle primary cell suspension was screen sorted with the assistance of sterile PBS buffer using a 70-micron sterile mesh to remove mucus and undigested tissue and collect the filtrate to complete the screen sorting ;
  • the filtrate is centrifuged and the supernatant is removed to obtain a precipitate.
  • Red blood cell lysate (from Soleba) is added to the precipitate for resuspension. The process is centrifuged again and the above process is repeated until the precipitate is centrifuged again. Until no red blood cells are observed in the sediment, the red blood cells are lysed and removed to finally obtain primary skeletal muscle cells.
  • the centrifugation speed for each centrifugation process is 1000g, and the centrifugation time for each centrifugation process is 3 minutes.
  • the composition of the reprogramming medium includes: based on the volume of the reprogramming medium, basic culture Base Ham F10 (from Wuhan Punosai Life Technology Co., Ltd.) accounts for 91% of the total volume of the reprogramming medium, with a content of 20 ng/ml of epithelial cell growth factor EGF and a content of 50 ng/ml of alkaline Fibroblast Growth Factor bFGF, 1% Nutritional Supplement N2 (1X), 1% Nutritional Supplement B27 (1X), 10 ⁇ M ROCK Kinase Inhibitor Y-27632, 3 ⁇ M Wnt Signaling pathway agonist CHIR99021, TGF- ⁇ signaling inhibitor A8301 at 1 ⁇ M, Tumor necrosis factor TNF at 10 ng/ml, Interleukin-1b at 20 ng/ml, 5 ng /ml of oncostatin OSM, 10 ng/ml of interleukin-6, 5% fetal bovine serum (the English abbrevi
  • Control medium Based on the volume of the control medium, the basal medium Ham F10 (from Wuhan Prosai Life Technology Co., Ltd.) accounts for 91% of the total volume of the control medium, with a content of 20 ng/ml of epithelial cells.
  • EGF EGF
  • basic fibroblast growth factor bFGF at 50 ng/ml
  • 1% nutritional supplement N2 (1X) 1% nutritional supplement B27 (1X) at 10 ⁇ M ROCK kinase inhibitor Y-27632, Wnt signaling pathway agonist CHIR99021 at 3 ⁇ M, TGF- ⁇ signaling inhibitor A8301 at 1 ⁇ M
  • 5% fetal bovine serum the English abbreviation of fetal bovine serum is: FBS , from Beijing Baiolaibo Technology Co., Ltd.).
  • the difference between reprogramming medium and control medium contains tumor necrosis factor TNF at 10 ng/ml, interleukin-1b at 20 ng/ml, and 5 ng/ml.
  • ml of oncostatin OSM containing 10 ng/ml of interleukin-6, while the control medium did not contain tumor necrosis factor TNF, interleukin-1b, Oncostatin OSM and interleukin-6.
  • Figure 1 is a schematic photographic diagram of the morphology of the first-generation skeletal muscle precursor-like cells according to an embodiment of the present invention
  • Figure 2 is a schematic photographic diagram of the morphology of the fourth-generation skeletal muscle precursor-like cells according to an embodiment of the present invention
  • Figure 3 is a schematic diagram of the implementation of the present invention. Schematic diagram of the proliferation curve of skeletal muscle precursor-like cells of an example.
  • skeletal muscle primary cells Place skeletal muscle primary cells in a 6-well plate at a seeding area of 10,000 cells/cm2, add 2 ml of reprogramming medium to each well for dedifferentiation culture to obtain skeletal muscle precursor-like cells until skeletal muscle precursor-like cells are obtained. After the confluence of the cells is not less than 80%, the skeletal muscle precursor-like cells are digested using trypsin digestion solution. The digestion time is 5 minutes.
  • the trypsin digestion solution (containing 0.25% trypsin) is derived from Biyuntian;
  • the skeletal muscle precursor-like cells will continue to be subcultured using reprogramming medium to obtain the first generation (P1) skeletal muscle precursor-like cells, the second generation (P2) skeletal muscle precursor-like cells, and the second generation (P2) skeletal muscle precursor-like cells.
  • Third generation (P3) skeletal muscle precursor-like cells skeletal muscle precursor-like cells.
  • Tenth generation (P10) skeletal muscle precursor-like cells, skeletal muscle precursor-like cells can be continuously passaged in reprogramming culture medium.
  • the digestion time is 5 minutes, in which the trypsin digestion solution (containing 0.25% trypsin) Sourced from Biyuntian; the control skeletal muscle precursor-like cells will continue to be subcultured in the control medium to obtain the first
  • the second generation (P1) controls skeletal muscle precursor-like cells
  • the second generation (P2) controls skeletal muscle precursor-like cells
  • the third generation (P3) controls skeletal muscle precursor-like cells
  • the fourth generation (P4) controls skeletal muscle precursor-like cells.
  • Somatic-like cells, fifth passage (P5) control skeletal muscle precursor-like cells.
  • the control skeletal muscle precursor-like cells can be passaged in the control medium, when they are passaged to the fifth passage, the control skeletal muscle precursor-like cells have Unable to proliferate in control medium, see Figure 3.
  • Figure 1 shows the P1 generation skeletal muscle precursor-like cells
  • Figure 2 shows the P4 generation skeletal muscle precursor-like cells.
  • the skeletal muscle precursor-like cells remain consistent in shape and are polygonal.
  • the reprogramming medium can allow the skeletal muscle precursor-like cells to continue to expand, and the skeletal muscle precursor-like cells
  • the expansion ability of cells has always been enhanced; using control medium for subculture, the expansion ability of control skeletal muscle precursor-like cells increased first and then decreased with the increase in the number of passages, and in the second generation , has the strongest amplification ability.
  • the abscissa in Figure 3 is the number of cell generations, the ordinate is the cell amplification multiple, and the number of generations is the number of passages.
  • Figure 4 is a schematic diagram of the gene expression marker CD34 of skeletal muscle precursor-like cells according to an embodiment of the present invention
  • Figure 5 is a schematic diagram of the gene expression marker CD45 of skeletal muscle precursor-like cells according to an embodiment of the present invention
  • Figure 6 It is a schematic diagram of the gene expression marker HLA-DRPQ of skeletal muscle precursor-like cells according to an embodiment of the present invention
  • Figure 7 is a schematic diagram of the gene expression marker CD29 of skeletal muscle precursor-like cells according to an embodiment of the present invention
  • Figure 8 It is the gene expression marker CD56 of skeletal muscle precursor-like cells according to the embodiment of the present invention.
  • Figure 9 is a schematic diagram of the gene expression marker CD24 of skeletal muscle precursor-like cells according to an embodiment of the present invention
  • Figure 10 is a schematic diagram of the gene expression marker PAX7 of skeletal muscle precursor-like cells according to an embodiment of the present invention.
  • the skeletal muscle precursor-like cells were washed with sterile PBS buffer (from Wuhan Prosai Life Technology Co., Ltd.), and then digested with trypsin digestion solution (Pancreatic Pancreas). Enzyme digestion solution (containing 0.25% trypsin) derived from Biyuntian) digested skeletal muscle precursor-like cells and then centrifuged to collect the cell pellet; add 600 microliters of staining buffer to the cell pellet to resuspend the cells.
  • sterile PBS buffer from Wuhan Prosai Life Technology Co., Ltd.
  • trypsin digestion solution Pancreatic Pancreas
  • FIG 4 Figure 5
  • the flow cytometry analysis results shown in Figure 6, Figure 7, Figure 8, Figure 9, and Figure 10 in which the staining buffer is from Thermo Fisher Scientific, and the flow cytometry antibody to be tested is from Thermo Fisher Scientific. company.
  • the specific operations and analysis steps of flow cytometric detection are routine technical means for those skilled in the art and will not be described in detail here.
  • the positivity rate of the positive peak of marker CD34 is 0.54%
  • the positivity rate of the positive peak of marker CD45 is 0.97%
  • the positivity rate of the positive peak of marker HLA-DRPQ is 0.68% , when the positive rate of the positive peak is greater than 70%, it means that the skeletal muscle precursor-like cells positively express the marker.
  • the skeletal muscle precursor-like cells of the present invention negatively express CD34, CD45 and HLA-DRPQ; refer to Figure 7, Figure 8, Figure 9 and Figure 10, the positive rate of the negative peak of the marker CD29 is 99.9%, and the negative rate of the positive peak of the marker CD56 100%, the positive rate of the negative peak of the marker CD24 is 99.2%, and the positive rate of the negative peak of the marker PAX7 is 93.3%.
  • the positive rate of the positive peak is greater than 70%, it means that skeletal muscle precursor-like cells positively express the marker. Therefore, the skeletal muscle precursor-like cells of the present invention positively express CD29, CD56, CD24 and PAX7.
  • the abscissas and ordinates in Figure 4, Figure 5, Figure 6, Figure 7, Figure 8, Figure 9 and Figure 10 have the same meaning.
  • the abscissa in Figure 4 represents the relative fluorescence intensity, and the ordinate represents the number of cells.
  • the skeletal muscle precursor-like cells are mixed with a pharmaceutically acceptable carrier to prepare a biological preparation containing the skeletal muscle precursor-like cells.
  • the biological preparation containing the skeletal muscle precursor-like cells is used to repair muscle damage, including skeletal muscle.
  • the specific steps of the biological preparation of precursor-like cells include: mixing the skeletal muscle precursor-like cells with physiological saline to prepare a biological preparation containing skeletal muscle precursor-like cells, that is, a biological preparation containing skeletal muscle precursor-like cells. It is a skeletal muscle precursor-like cell injection, wherein each microliter of the skeletal muscle precursor-like cell injection contains 2 ⁇ 10 4 skeletal muscle precursor-like cells.
  • Figure 11 is a schematic diagram comparing photos of pathological tissue sections of the drug-induced muscle injury model mice in the experimental group and the control group at different times according to the embodiment of the present invention
  • Figure 12 is a schematic diagram of the drug-induced muscle injury model mice in the experiment according to the embodiment of the present invention. Comparison of the area distribution of new myofibroblasts in the group and control group.
  • Animal models refer to drug-induced muscle injury models
  • mice 8-week-old male mice were selected.
  • cyclophosphamide dry powder was diluted to 10 ⁇ M with sterile PBS buffer to obtain cyclophosphamide injection. After aliquoting, store the solution at minus 80 degrees Celsius to avoid repeated freezing and thawing of cyclophosphamide injection; mice were intraperitoneally injected with 10% chloral hydrate. After injecting chloral hydrate, after one to two minutes, the mice were leveled.
  • cyclophosphamide Lying on a clean table, use a shaver to remove the hair outside the tibialis anterior muscle of the mouse, and use an insulin injection needle to vertically inject 100 microliters of cyclophosphamide injection into the tibialis anterior muscle of the mouse in 3 injections.
  • One needle was positioned at the center of the tibialis anterior muscle, and the remaining two needles were positioned at both ends of the tibialis anterior muscle to obtain a drug-induced muscle injury model.
  • the English abbreviation of cyclophosphamide is CTX
  • cyclophosphamide dry powder comes from Shanghai. Yuanxi Biotechnology Co., Ltd.
  • sterile PBS buffer was from Wuhan Pronosai Life Technology Co., Ltd.
  • chloral hydrate was from Shanghai Bevanta Biotechnology Co., Ltd.
  • mice On day 0, 50 ⁇ l of skeletal muscle precursor-like cell injection was injected into the tibialis anterior muscle of mice. On day 7, 50 ⁇ l of skeletal muscle precursor-like cell injection was injected into the tibialis anterior muscle of mice again. Cell injection, and a part of the tibialis anterior muscles of the experimental mice were collected, and the tibialis anterior muscles were sampled for pathological HE staining to examine the damage to the tibialis anterior muscles; on the 15th day, the mice were sacrificed to collect the tibialis anterior muscles of the mice, and the tibialis anterior muscles were collected. Muscle samples were taken for pathological HE staining to examine the damage to the tibialis anterior muscle. Among them, HE staining is hematoxylin-eosin staining.
  • Control group On day 0, 50 ⁇ l of normal saline was injected into the tibialis anterior muscle of mice. On day 7, 50 ⁇ l of normal saline was injected into the tibialis anterior muscle of mice again, and a part of the tibia of the experimental mice was collected.
  • samples of the tibialis anterior muscle were sampled for pathological HE staining to examine the damage to the tibialis anterior muscle; on the 15th day, the mice were sacrificed to collect the tibialis anterior muscles of the mice, and samples of the tibialis anterior muscle were sampled for pathological HE staining to examine the damage to the tibialis anterior muscle.
  • HE dye The color is hematoxylin-eosin staining.
  • the tibialis anterior muscle of the mice in the experimental group recovered better.
  • the visual field was filled with new myofibroblasts with nuclei in the center.
  • the number of immune cells was smaller, indicating the immune response of the tibialis anterior muscle after muscle injury. It has basically ended; while in the tibialis anterior muscle of the control mice, there were a large number of immune cells, and at the same time, the number of new myofibroblasts was small and the cross-sectional area was small.
  • the skeletal muscle precursor-like cell treatment method provided by the present invention can better solve muscle diseases. Because skeletal muscle precursor-like cells can proliferate and differentiate to produce new myofibroblasts in the body, and can also home in to form a muscle stem cell bank, they can theoretically provide patients with a steady supply of cells. Use reprogramming medium to culture skeletal muscle precursor-like cells so that the skeletal muscle precursor-like cells have the ability to continue to proliferate in vitro.
  • mice in the experimental group recovered better.
  • the visual field was filled with new myofibroblasts with nuclei in the center.
  • the number of immune cells was smaller, indicating that the tibialis anterior muscle was damaged.
  • the immune response after muscle injury has basically ended; while in the tibialis anterior muscle of mice in the control group, there were a large number of immune cells, and at the same time, the number of new myofibroblasts was small and the cross-sectional area was small.
  • the surface of the skeletal muscle precursor-like cells obtained by the present invention does not express immune rejection reactions, In response to the related protein HLA-DRPQ, allogeneic reinfusion of skeletal muscle precursor-like cells can be achieved.
  • the skeletal muscle precursor-like cells obtained by the present invention positively express CD29, CD56, CD24 and PAX7, and the skeletal muscle precursor-like cells can After being expanded in vitro to establish a cell seed bank, skeletal muscle precursor-like cells can be directly injected in situ into skeletal muscle precursor-like cell preparations when used to treat clinical skeletal muscle atrophy.

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Abstract

L'invention concerne une formulation biologique contenant des cellules de type précurseur de muscle squelettique, et son procédé de préparation et son application. Les cellules de type précurseur de muscle squelettique expriment au moins négativement l'un parmi CD34, CD45 et HLA-DRPQ. Par conséquent, le corps humain peut bien accepter les cellules de type précurseur de muscle squelettique. La présente demande résout le problème, dans l'état de la technique, du rejet immunitaire dans le traitement de l'atrophie musculaire provoquée par une mutation génétique.
PCT/CN2023/092965 2022-05-10 2023-05-09 Formulation biologique contenant des cellules de type précurseur de muscle squelettique, son procédé de préparation et son application WO2023217130A1 (fr)

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