WO2023016246A1 - 间充质干细胞的快速诱导分化方法、试剂盒及其应用 - Google Patents

间充质干细胞的快速诱导分化方法、试剂盒及其应用 Download PDF

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WO2023016246A1
WO2023016246A1 PCT/CN2022/107902 CN2022107902W WO2023016246A1 WO 2023016246 A1 WO2023016246 A1 WO 2023016246A1 CN 2022107902 W CN2022107902 W CN 2022107902W WO 2023016246 A1 WO2023016246 A1 WO 2023016246A1
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cells
stem cells
mesenchymal stem
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吴理达
顾雨春
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呈诺再生医学科技(珠海横琴新区)有限公司
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Definitions

  • the invention belongs to the field of cell engineering, in particular, the invention relates to a method for rapidly inducing differentiation of mesenchymal stem cells, a kit and an application thereof.
  • Human induced pluripotent stem cells are pluripotent stem cells induced from human terminally differentiated cells through in vitro reprogramming technology, with in vitro self-renewal stability, and maintain normal karyotype and development of multiple stem cells. capability.
  • iPSC cells are very similar to embryonic stem cells (ES) in terms of morphology, proliferation and differentiation ability, cell surface antigens, gene expression patterns, etc., and because iPSCs can come from donor somatic cells, Therefore, it will not cause immune rejection in clinical application, and can avoid the medical ethics problems brought about by embryonic stem cells. Therefore, it plays an important role in disease modeling, drug discovery, and cell therapy. Developments in disciplines such as cell biology and regenerative medicine.
  • MSC Mesenchymal stem cell
  • iPSCs Mesenchymal stem cell
  • iPSC differentiation into MSC is mainly through the differentiation of embryoid body (Embryoid body, EB), that is, through hanging drop culture or suspension culture technology, iPSC is aggregated into a spherical structure, which is called embryoid body.
  • EB embryoid body
  • cytokines cytokines
  • the disadvantage of the embryoid body differentiation method is that the differentiation efficiency is low, the differentiation process is complicated and time-consuming, and more types of cytokines need to be added, which is costly. Therefore, the existing technology of iPSC differentiation into MSC has problems such as long time-consuming, low yield, high technical requirements, and limited cell purity, which restricts the large-scale cultivation of autologous mesenchymal stem cells and limits its industrialization and marketization. .
  • the present invention provides a method for rapidly inducing differentiation of mesenchymal stem cells.
  • the present invention provides a medium combination for inducing pluripotent stem cells to differentiate into mesenchymal stem cells.
  • the medium combination includes the first-stage early mesoderm cell-inducing differentiation medium, and the second-stage mesenchymal stem cell-inducing differentiation medium;
  • the medium for inducing differentiation of early mesoderm cells in the first stage comprises a combination of cytokines and small molecule compounds
  • the combination of cytokines and small molecule compounds includes: vitamin C or vitamin C derivatives, recombinant human bone morphogenetic protein, TGF- ⁇ type I Receptor inhibitors;
  • the vitamin C or vitamin C derivatives include: L-Ascorbic acid, calcium ascorbate, magnesium ascorbate, zinc ascorbate, potassium ascorbate, sodium ascorbate, dehydroascorbic acid, L-threonic acid, L-xylonic acid , L-lythreonic acid, L-ascorbic acid monostearate, L-ascorbic acid dipalmitate, L-ascorbic acid 6-hexadecanoic acid compound, L-ascorbic acid 2-0-phosphate, L-ascorbic acid 3- 0-phosphate, L-ascorbic acid 2-0-sulfate;
  • the vitamin C or vitamin C derivative is L-Ascorbic acid
  • the concentration of the L-Ascorbic acid is 50 ⁇ g/ml
  • the recombinant human bone morphogenetic proteins include: BMP4, BMP2, BMP6, BMP7, BMP9, BMP12, BMP1, BMP3, BMP5, BMP8, BMP13, BMP10, BMP11, BMP14, BMP15;
  • the recombinant human bone morphogenetic protein is BMP4;
  • the concentration of the BMP4 is 25ng/ml
  • the TGF- ⁇ type I Receptor inhibitors include: SB-431542, Dorsomorphin, A83-01, LDN193189, RepSox, SB525334, DMH-1, SB-505124, sb4, ITD-1, GW788388, LSKL, SD -208, LDN-212854, K02288, LDN-214117, R-268712, SM16, A77-01, BIO-013077-01;
  • the TGF- ⁇ type I Receptor inhibitor is SB-431542;
  • the concentration of said SB-431542 is 2uM
  • the first-stage early mesoderm cell induction differentiation medium further comprises 90% DMEM/F12, 10% Knockout serum replacement, 1% L-glutamine;
  • the first-stage early mesoderm cell induction differentiation medium comprises L-Ascorbic acid, BMP4, SB-431542, 90% DMEM/F12, 10% Knockout serum replacement, 1% L-glutamine;
  • the medium for inducing differentiation of mesenchymal stem cells in the second stage contains small molecule compounds
  • the small molecule compound is a TGF- ⁇ type I Receptor inhibitor
  • the TGF- ⁇ type I Receptor inhibitors include: SB-431542, Dorsomorphin, A83-01, LDN193189, RepSox, SB525334, DMH-1, SB-505124, sb4, ITD-1, GW788388, LSKL, SD -208, LDN-212854, K02288, LDN-214117, R-268712, SM16, A77-01, BIO-013077-01;
  • the TGF- ⁇ type I Receptor inhibitor is SB-431542;
  • the concentration of said SB-431542 is 2uM
  • the second-stage mesenchymal stem cell differentiation medium further comprises 90% DMEM/F12, 10% Knockout serum replacement, 1% L-glutamine;
  • the second stage mesenchymal stem cell induction differentiation medium comprises SB-431542, 90% DMEM/F12, 10% Knockout serum replacement, 1% L-glutamine.
  • the present invention provides a method for rapidly inducing differentiation of induced pluripotent stem cells into mesenchymal stem cells.
  • the method includes: using the medium combination described in the first aspect of the present invention to cultivate induced pluripotent stem cells;
  • the method comprises the steps of:
  • step (3) Induced differentiation of mesenchymal stem cells in the second stage: the early mesoderm cells obtained in step (2) are cultivated by using the second stage mesenchymal stem cell induction differentiation medium described in the first aspect of the present invention to obtain mesenchymal stem cells.
  • step (1) includes digestion, inoculation, and medium exchange of induced pluripotent stem cells
  • the digestion of the induced pluripotent stem cells comprises: adding a digestion solution to the induced pluripotent stem cells for incubation, discarding the digestion solution, and then adding a culture medium to terminate the digestion;
  • the digestion solution includes EDTA digestion solution, trypsin digestion solution, trypsin-EDTA digestion solution, collagenase digestion solution, accutase digestion solution;
  • the digestive juice is EDTA digestive juice
  • the dosage of the EDTA digestion solution is 0.5mM, 2mL;
  • the incubation conditions are 37°C, 5min;
  • the medium is E8 complete medium
  • the inoculation of the induced pluripotent stem cells includes: collecting the digested induced pluripotent stem cell suspension, resuspending the cells with a culture medium, inoculating them on a culture plate to continue culturing, and adjusting the cell density to 5 ⁇ 10 cells after inoculation. 4 /cm 2 ;
  • described culture medium is the E8 complete medium that adds rock inhibitor
  • the resuspension condition of the resuspended cells is 1000 rpm, 5min;
  • the conditions for continuing the culture are 37°C, 24h;
  • changing the medium of the induced pluripotent stem cells includes: changing the induced pluripotent stem cells after inoculation and culture for 24 hours with an equal volume of culture medium, and continuing to culture until the cell confluency is at least 30%;
  • the medium is the E8 complete medium without rock inhibitor
  • condition for continuing the culture is 37° C. for 24 hours.
  • step (2) includes adding the first-stage early mesoderm cell induction differentiation medium described in the first aspect of the present invention to the cultured induced pluripotent stem cells obtained in step (1), Continue to induce culture;
  • the consumption of the first stage early mesoderm cell induction differentiation medium described in the first aspect of the present invention is 3mL/well;
  • condition for continuing the induction culture is 37° C. for 4 days.
  • step (3) includes adding the second-stage mesenchymal stem cell induction differentiation medium described in the first aspect of the present invention to the early mesoderm cells obtained in step (2), and after continuing the induction culture , for subculture;
  • the amount of the second-stage mesenchymal stem cell-inducing differentiation medium described in the first aspect of the present invention is 2 mL/well;
  • the conditions for continuing the induction culture are 37°C, 4-10 days;
  • the subculturing includes adding digestive fluid to the mesenchymal stem cells induced to differentiate at a cell confluency reaching 80% for incubation, and adding the second-stage mesenchymal stem cells described in the first aspect of the present invention after incubation Stop the digestion of the induction differentiation medium, collect the cells, then add the mesenchymal stem cell maturation medium to resuspend the cells, inoculate in the culture plate and add the mesenchymal stem cell maturation medium for culturing to obtain mature mesenchymal stem cells;
  • the digestive fluid includes accutase digestive fluid, EDTA digestive fluid, trypsin digestive fluid, trypsin-EDTA digestive fluid and collagenase digestive fluid;
  • the digestive juice is accutase digestive juice
  • the amount of the accutase digestion solution is 4 times diluted, 0.5mL;
  • the incubation conditions are 37°C, 8min;
  • the amount of the second-stage mesenchymal stem cell differentiation medium described in the first aspect of the present invention is 1 mL;
  • the amount of the mesenchymal stem cell maturation medium for the resuspended cells is 1 mL;
  • the resuspension condition of the resuspended cells is 1000 rpm, 5min;
  • the cell density after inoculation is 3-8 ⁇ 10 3 /cm 2 ;
  • the amount of the mesenchymal stem cell maturation medium for the cultured cells is 2 mL;
  • the mesenchymal stem cell maturation medium contains 95% ⁇ -MEM, 5% HPL.
  • the present invention provides a mesenchymal stem cell or cell population derived from induced pluripotent stem cells.
  • mesenchymal stem cells or cell populations are induced and differentiated by the method described in the second aspect of the present invention.
  • the present invention provides a cell therapy product.
  • the cell therapy product includes the mesenchymal stem cells or cell populations described in the third aspect of the present invention.
  • the "cell therapy product” mentioned herein refers to the treatment, prevention and/or diagnosis and prevention of immune diseases, nervous system diseases, blood system diseases, cardiovascular and cerebrovascular system diseases, bone diseases, cartilage diseases, diabetes complicated diseases, liver and kidney injury, and tumors, including cells or tissues prepared from humans through isolation, culture, and specialized manipulation, and more specifically, the "cell therapy products” refer to drugs for the treatment, prevention and/or Or diagnosis and prevention of immune diseases, nervous system diseases, blood system diseases, cardiovascular and cerebrovascular system diseases, bone diseases, cartilage diseases, diabetic complications, liver and kidney damage, tumor drugs, which contain autologous, homologous or heterologous Proliferation or sorting of source in vitro living cells or preparation by any process of modifying the biological characteristics of cells by other methods in order to restore the function of cells or tissues; cell therapy products are mainly divided into somatic cell therapy products and stem cell therapy products according to the degree of cell differentiation, And the present invention is particularly aimed at mesenchymal stem cell therapy products.
  • the cell therapy product can be used to form other types of cells such as fat cells, bone cells, chondrocytes, muscle cells, nerve cells, and cardiomyocytes.
  • the present invention provides a feeder cell for culturing cells.
  • the feeder cells include the mesenchymal stem cells or cell populations described in the third aspect of the present invention.
  • the present invention provides a kit for rapidly inducing differentiation of induced pluripotent stem cells into mesenchymal stem cells.
  • kit includes the medium combination described in the first aspect of the present invention.
  • kit also includes induced pluripotent stem cells, digestive fluid, E8 complete medium with rock inhibitor added, E8 complete medium without rock inhibitor, mesenchymal stem cell maturation medium;
  • the digestion solution includes EDTA digestion solution, trypsin digestion solution, trypsin-EDTA digestion solution, collagenase digestion solution, accutase digestion solution;
  • the digestive juice includes EDTA digestive juice, accutase digestive juice;
  • the mesenchymal stem cell maturation medium contains 95% ⁇ -MEM, 5% HPL.
  • the present invention also provides a method for treating immune diseases, nervous system diseases, blood system diseases, cardiovascular and cerebrovascular system diseases, bone diseases, cartilage diseases, diabetes complications, liver and kidney damage, and tumors.
  • the method includes administering an effective amount of the cell therapy product described in the fourth aspect of the present invention or the mesenchymal stem cell or cell population described in the third aspect of the present invention to a subject in need.
  • the present invention provides any one of the following applications:
  • the mesenchymal stem cells or cell populations described in the third aspect of the present invention are prepared for the treatment of immune diseases, nervous system diseases, blood system diseases, cardiovascular and cerebrovascular system diseases, bone diseases, cartilage diseases, and complications of diabetes , liver and kidney injury, and application in cell therapy products for tumors;
  • the cell therapy product described in the fourth aspect of the present invention is used in the treatment of immune diseases, nervous system diseases, blood system diseases, cardiovascular and cerebrovascular system diseases, bone diseases, cartilage diseases, diabetic complications, liver and kidney damage, and tumors Applications;
  • kit according to the sixth aspect of the present invention in preparing induced pluripotent stem cell-derived mesenchymal stem cells or cell populations.
  • the immune diseases include rheumatoid arthritis, multiple sclerosis, systemic lupus erythematosus, inflammatory bowel disease, psoriasis, autoimmune uveitis, Sjogren's syndrome, polymyositis, dermatomyositis inflammation, type 1 diabetes, celiac disease, thyroiditis, hypophysitis, Crohn's disease;
  • the nervous system diseases include Parkinson's syndrome, Alzheimer's disease, amyotrophic lateral sclerosis, traumatic brain injury, spinal cord injury, spinal muscular atrophy, epilepsy, myasthenia gravis, Huntington's disease , Lewy body dementia;
  • the blood system diseases include graft-versus-host disease (GVHD) after cell transplantation, aplastic anemia, acute leukemia, thalassemia, immune cytopenia, hemophilia, myelodysplastic syndrome, thrombocytopenia ;
  • GVHD graft-versus-host disease
  • cardiovascular and cerebrovascular system diseases include myocardial infarction, heart failure, vascular disease, ischemic vascular disease, heart failure, coronary heart disease, ischemic heart disease, cerebral infarction, cerebral hemorrhage, ischemic stroke, stroke ;
  • the bone diseases include osteoarthritis, femoral head necrosis, lumbar disc degenerative disease, spondylosis, osteoporosis, degenerative arthritis, rheumatoid arthritis, bursitis, synovitis, cervical spondylosis, lumbar disease, frozen shoulder, ankylosing spondylitis;
  • the cartilage disease includes relapsing polychondritis, osteochondritis dissecans, costochondritis, osteochondrosis, pathological articular cartilage defect, Kohler's bone disease, juvenile osteochondritis deformity, tibial trochanteric bone Osteochondrosis, Scheuermann's disease;
  • diabetic complications include diabetic foot ulcer, diabetic retinopathy, diabetic cardiomyopathy, diabetic peripheral neuropathy, diabetic encephalopathy, diabetic kidney injury, and diabetic lower limb ischemic disease;
  • liver and kidney damage includes liver cirrhosis, liver damage, kidney damage, nephrosclerosis, hepatorenal syndrome, liver failure, and kidney failure;
  • the tumors include lymphoma, non-small cell lung cancer, melanoma, glioma, colon cancer, intrahepatic bile duct cancer, liver cancer, cervical cancer, esophageal cancer, multiple myeloma, gastric cancer, breast cancer, prostate cancer , lung cancer, glioma, bladder cancer, ovarian cancer.
  • the present invention has the following advantages and beneficial effects:
  • the present invention provides a technology for isolating mesenchymal stem cells by iPSC monolayer induction method, which has obvious advantages compared with the currently commonly used embryoid body differentiation method, mainly in the simple operation of the differentiation process, high differentiation efficiency and It is convenient for purification and separation, and does not involve animal serum and other non-human additives in the differentiation process, so it is very suitable for the production of a sufficient number of autologous mesenchymal stem cells to meet clinical needs.
  • the mesenchymal stem cells differentiated from iPSCs have sufficient cell sources, strong cell proliferation ability, vigorous cell vitality, and may have better damage repair and anti-aging effects after reinfusion;
  • iPSC-derived mesenchymal stem cells by firstly inducing iPSCs into early mesoderm cells, and then further inducing differentiation into mesenchymal stem cells, the differences between adult-derived mesenchymal stem cells and other unrestricted induction pathways iPSC-derived mesenchymal stem cells can be resolved.
  • the shortcomings of qualitative and mixed can solve the problem of limited sources of mesenchymal stem cells, and the obtained mesenchymal stem cells have stronger proliferation and immune regulation capabilities;
  • the technology for isolating mesenchymal stem cells by the iPSC monolayer induction method provided by the present invention can not only produce a sufficient number of mesenchymal stem cells that can meet clinical needs in a short period of time with a high yield, but also has no embryoid body formation Therefore, the preparation process is simple, and mesenchymal stem cells with homogeneity can be obtained, thereby having the effect of providing a cell therapeutic agent in a shorter time than the existing method.
  • Figure 1 shows the cell morphology of iPSCs used for induction, where A: 40 ⁇ , B: 100 ⁇ ;
  • Figure 2 shows the cell morphology of iPSCs 24h after inoculation
  • Figure 3 shows the colony morphology of iPSCs on the second day of induction (100 ⁇ );
  • Figure 4 shows the cell morphology of iPSCs on the sixth day of induction (100 ⁇ );
  • Figure 5 shows the cell morphological feature map of P2 generation cells
  • Figure 6 shows the cell morphology diagram after 21 days of differentiation
  • Figure 7 shows the cell morphological feature map of the P8 generation cells
  • Figure 8 shows the results of flow cytometry detection of MSC cell surface markers CD90 and CD105 obtained from iPSC differentiation
  • Figure 9 shows the results of flow cytometry detection of MSC cell surface markers CD34 and CD45 obtained from iPSC differentiation
  • Fig. 10 shows the results of detection of pluripotency of MSC cells differentiated from iPSCs, wherein, panel A: osteogenic differentiation, panel B: chondrogenic differentiation, and panel C: adipogenic differentiation.
  • Example 1 Induced differentiation of human iPSCs to obtain autologous MSC cells
  • the main reagent information used in the embodiment of the present invention is shown in Table 1.
  • 1Treatment of the culture plate Incubate the culture plate with matrigel at room temperature for more than 30 minutes;
  • 2Cell digestion and inoculation For iPSCs cultured in T25 flasks (Beijing Chengnuo Medical Biotechnology Co., Ltd.), discard the culture medium, wash twice with DPBS, 1 min each time; add 2 mL of 0.5 mM EDTA digestion solution; incubate at 37°C for 5 min, discard EDTA, add E8 complete medium to stop digestion;
  • Collect the cell suspension and count the cells take different amounts of the cell suspension according to the cell density and the amount of cells needed, and centrifuge at 1000 rpm for 5 minutes; resuspend the cells with E8 complete medium added with rock inhibitor, and adjust the cell density to 5 ⁇ after inoculation 10 4 cells/cm 2 , to ensure that the cell confluence reaches 30% to 50% the next day, inoculate in a 6-well plate, and place it back in the incubator for 24 hours;
  • 3Change the medium Take the cells cultured in step 2 for 24 hours, gently absorb the culture medium in the culture well, replace the medium with an equal volume of E8 medium without rock inhibitor, put it back in the incubator for 24 hours, and the cell confluence Need to be at least 30%.
  • iPSC cells Take the iPSC cells obtained above, gently absorb the culture medium in the culture well, gently wash the culture well once with 3mL DPBS in each well, add 3mL mesoderm differentiation medium to each well, and place them back in the incubator for induction culture for 4 days;
  • the composition of the mesoderm differentiation medium is: 90% DMEM/F12, 10% Knockout serum replacement, 1% L-glutamine, cytokines and a combination of small molecule compounds;
  • the combination of cytokines and small molecule compounds includes: vitamin C derivatives (L-Ascorbic acid), recombinant human bone morphogenetic protein (BMP4), TGF- ⁇ type I Receptor inhibitors;
  • TGF- ⁇ type I Receptor inhibitors include: SB-431542, Dorsomorphin, A83-01, LDN193189, RepSox, SB525334, DMH-1, SB-505124, sb4, ITD-1, GW788388, LSKL, SD-208, LDN -212854, K02288, LDN-214117, R-268712, SM16, A77-01, BIO-013077-01;
  • the concentration of the vitamin C derivative is 50 ⁇ g/ml.
  • the concentration of BMP4 is 25ng/ml.
  • the TGF- ⁇ type I Receptor inhibitor is SB-431542. More preferably, the concentration of SB-431542 is 2uM.
  • the MSC induction medium consists of: 90% DMEM/F12, 10% Knockout serum replacement, 1% L-glutamine and TGF- ⁇ type I Receptor inhibitor;
  • TGF- ⁇ type I Receptor inhibitors include: SB-431542, Dorsomorphin, A83-01, LDN193189, RepSox, SB525334, DMH-1, SB-505124, sb4, ITD-1, GW788388, LSKL, SD-208, LDN -212854, K02288, LDN-214117, R-268712, SM16, A77-01, BIO-013077-01;
  • the TGF- ⁇ type I Receptor inhibitor is SB-431542; more preferably, the concentration of said SB-431542 is 2uM.
  • Gently aspirate the culture medium in the culture well wash it twice with DPBS, 1 min each time; add 0.5 mL of 4-fold diluted accutase digestion solution; after incubating at 37°C for 8 min, add 1 mL of complete MSC medium to stop the digestion; collect the cell suspension to 15mL centrifuge tube; wash the original culture well gently with 1mL MSC maturation medium, and collect together into a 15mL centrifuge tube.
  • the above-mentioned MSC maturation medium includes: 95% ⁇ -MEM, 5% HPL;
  • the P2 generation cells continued to be induced and cultured, and the cells were observed every day; when the confluence of the cells reached 80%, the subculture was carried out, and the subculture method was consistent with the P1 to P2 subculture method, and the culture was about 21 days.
  • the collected cells were resuspended and mixed in 10 mL calcium and magnesium-free PBS, and 50 ⁇ L was sampled for cell counting, washed once at 300 g ⁇ 5 min, and the supernatant was discarded; then resuspended and mixed in 10 mL calcium and magnesium-free PBS, washed once at 300 g ⁇ 5 min, discarded supernatant; add 1mL 4% paraformaldehyde solution, fix at room temperature for 30min;
  • MSC cells differentiated from iPSCs expressed at least 70% positive marker CD90, and expressed at least 80% positive marker CD105 (see Figure 8); MSC cells differentiated from iPSCs did not express negative marker CD34, The negative marker CD45 was not expressed (see FIG. 9 ), indicating that the MSC cells differentiated from iPSC prepared by the present invention were consistent with the MSC cell surface markers.
  • the MSC cells differentiated from the expanded iPSCs in Example 1 were subjected to osteogenic differentiation experiments, chondrogenic differentiation experiments, and adipogenic differentiation experiments to verify the MSC cells differentiated from iPSCs in the present invention. of pluripotency.
  • the above-mentioned osteoblast differentiation medium includes: 90% DMEM, 10% FBS, small molecular compound cytokine combination;
  • the small molecule compound cytokine combination includes: glucocorticoid receptor agonists, vitamin C derivatives, phosphatase inhibitors, L-glutamine;
  • the glucocorticoid receptor agonist is dexamethasone
  • the vitamin C derivative is L-ascorbic acid
  • the phosphatase inhibitor is ⁇ -glycerophosphate
  • the above-mentioned chondrocyte differentiation medium includes: DMEM, small molecule compound cytokine combination;
  • the small molecule compound cytokine combination includes: glucocorticoid receptor agonists, vitamin C derivatives, sodium pyruvate, TGF- ⁇ , insulin, transferrin, sodium biselenite, L-glutamine;
  • the glucocorticoid receptor agonist is dexamethasone, and the vitamin C derivative is L-ascorbic acid.
  • the above-mentioned preadipocyte differentiation medium includes: 90% DMEM, 10% FBS, small molecular compound cytokine combination;
  • the small molecule compound cytokine combination includes: glucocorticoid receptor agonists, insulin, non-selective COX1 and COX2 inhibitors, phosphodiesterase (PDE) inhibitors, L-glutamine;
  • the glucocorticoid receptor agonist is dexamethasone
  • the non-selective COX1 and COX2 inhibitor is indomethacin
  • the phosphodiesterase (PDE) inhibitor is IBMX.
  • the experimental results of the adipogenic differentiation experiment showed that after 14 days of adipogenic differentiation of iPSC-differentiated MSC cells, oil red-O staining showed that the intracellular lipid vesicles of mature adipocytes were stained bright red, and the red lipid droplets were distributed in granular form in the result figure in the cytoplasm (see Figure 10C), indicating that MSC cells differentiate into adipocytes;

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Abstract

提供了间充质干细胞的快速诱导分化方法、试剂盒及其应用，提供的间充质干细胞的快速诱导分化方法不仅可以在短时间内以高产率制备可满足临床需求的足够数量的间充质干细胞，而且由于没有胚状体形成的步骤，因此制备工艺简单，具有分化效率高、便于纯化分离等优点，此外，在分化过程中不涉及动物血清和其它非人源添加物，更适合临床的需求，分化得到的间充质干细胞增殖能力强，细胞活力旺盛。

Description

间充质干细胞的快速诱导分化方法、试剂盒及其应用 技术领域
本发明属于细胞工程领域，特别地，本发明涉及一种间充质干细胞的快速诱导分化方法、试剂盒及其应用。
背景技术
人类诱导多能性干细胞(Human induced pluripotent stem cells，iPSC)是从人类终末分化细胞经过体外重编程技术诱导形成的多能性干细胞，具有体外自我更新稳定性，并维持正常核型和发育多能性。iPSC细胞不论是在形态、增殖分化能力、细胞表面抗原、基因表达模式等方面，均与胚胎干细胞(Embryonic stem cell，ES)有着极大的相似性，且由于iPSC可来自供者的体细胞，所以在临床应用时不会引起免疫排斥作用，又能够避开胚胎干细胞所带来的医学伦理学问题，因此在疾病建模、药物发现以及细胞疗法等各方面都发挥着重要的作用，促进了细胞生物学和再生医学等学科的发展。
间充质干细胞(Mesenchymal stem cell，MSC)是一种多潜能干细胞，可以分化为骨组织、结缔组织的多种结构，是在成人体内许多组织如骨髓、脐带血、脂肪和外周血等中都存在的一大类多能细胞，在正常时处于静止状态，当所在的组织或器官受到损伤时，MSC可以迅速进入增殖状态并保持极大的增殖潜力，可分化为不同的细胞类型以修复受损组织，在损伤修复和血管再生等方面有着重要的意义。由iPSC分化得到的MSC具有细胞来源于自体细胞，无免疫排斥，细胞来源充足，细胞增殖能力强，细胞活力旺盛，并可能在回输后能够有更好的损伤修复、免疫调节和抗衰老的功效。
由于MSC具有易于分离扩增及具有多向分化潜能等优点，其成为了目前本领域的研究热点，但是，MSC难以规模化扩增及异质性是目前限制MSC临床化应用的两大障碍。目前，iPSC分化为MSC的方法主要是通过拟胚体(Embryoid body，EB)途径分化，即通过悬滴培养或者悬浮培养技术，使iPSC聚集成球状结构，该结构称为拟胚体，在拟胚体分化的过程中，往往需要添加较多种类的细胞因子，并且在悬滴培养或者悬浮培养若干天后，再转入贴壁培养，此后会有间充质干细胞迁出。
拟胚体分化法的缺点是分化效率低，分化过程复杂，耗时长，而且需要添加较多种类的细胞因子，成本较高。因此，现有的iPSC分化为MSC的技术存在耗时长，产量低，技术要求高，细胞纯度有限等问题，制约了自体间充质干细胞的大规模培养，限制了其产业化和市场化的需求。
发明内容
基于此，为了克服上述现有技术存在的技术缺陷，本发明提供了一种间充质干细胞的快速诱导分化方法。
为了实现上述发明目的，本发明采取了如下技术方案：
第一方面，本发明提供了一种诱导多能干细胞分化为间充质干细胞的培养基组合。
进一步，所述培养基组合包括第一阶段早期中胚层细胞诱导分化培养基、第二阶段间充质干细胞诱导分化培养基；
优选地，所述第一阶段早期中胚层细胞诱导分化培养基包含细胞因子及小分子化合物组合；
更优选地，所述细胞因子及小分子化合物组合包括：维生素C或维生素C衍生物、重组人骨形态发生蛋白、TGF-βtype I Receptor抑制剂；
最优选地，所述维生素C或维生素C衍生物包括：L-Ascorbic acid、抗坏血酸钙、抗坏血酸镁、抗坏血酸锌、抗坏血酸钾、抗坏血酸钠、脱氢抗坏血酸、L-苏糖酸、L-木糖酸、L-来苏糖酸、L-抗坏血酸单硬脂酸化物、L-抗坏血酸二棕榈酸化物、L-抗坏血酸6-十六酸化合物、L-抗坏血酸2-0-磷酸化物、L-抗坏血酸3-0-磷酸化物、L-抗坏血酸2-0-硫酸化物；
最优选地，所述维生素C或维生素C衍生物为L-Ascorbic acid；
最优选地，所述L-Ascorbic acid的浓度为50μg/ml；
最优选地，所述重组人骨形态发生蛋白包括：BMP4、BMP2、BMP6、BMP7、BMP9、BMP12、BMP1、BMP3、BMP5、BMP8、BMP13、BMP10、BMP11、BMP14、BMP15；
最优选地，所述重组人骨形态发生蛋白为BMP4；
最优选地，所述BMP4的浓度为25ng/ml；
最优选地，所述TGF-βtype I Receptor抑制剂包括：SB-431542、Dorsomorphin、A83-01、LDN193189、RepSox、SB525334、DMH-1、SB-505124、sb4、ITD-1、GW788388、LSKL、SD-208、LDN-212854、K02288、LDN-214117、R-268712、SM16、A77-01、BIO-013077-01；
最优选地，所述TGF-βtype I Receptor抑制剂为SB-431542；
最优选地，所述SB-431542的浓度为2uM；
更优选地，所述第一阶段早期中胚层细胞诱导分化培养基还包含90％DMEM/F12、10％Knockout serum replacement、1％L-glutamine；
最优选地，所述第一阶段早期中胚层细胞诱导分化培养基包含L-Ascorbic acid、BMP4、SB-431542、90％DMEM/F12、10％Knockout serum replacement、1％L-glutamine；
优选地，第二阶段间充质干细胞诱导分化培养基包含小分子化合物；
更优选地，所述小分子化合物为TGF-βtype I Receptor抑制剂；
最优选地，所述TGF-βtype I Receptor抑制剂包括：SB-431542、Dorsomorphin、A83-01、LDN193189、RepSox、SB525334、DMH-1、SB-505124、sb4、ITD-1、GW788388、LSKL、SD-208、LDN-212854、K02288、LDN-214117、R-268712、SM16、A77-01、BIO-013077-01；
最优选地，所述TGF-βtype I Receptor抑制剂为SB-431542；
最优选地，所述SB-431542的浓度为2uM；
更优选地，所述第二阶段间充质干细胞诱导分化培养基还包含90％DMEM/F12、10％Knockout serum replacement、1％L-glutamine；
最优选地，所述第二阶段间充质干细胞诱导分化培养基包含SB-431542、90％DMEM/F12、10％Knockout serum replacement、1％L-glutamine。
第二方面，本发明提供了一种诱导多能干细胞快速诱导分化为间充质干细胞的方法。
进一步，所述方法包括：采用本发明第一方面所述的培养基组合对诱导多能干细胞进行培养；
优选地，所述方法包括如下步骤：
(1)提供诱导多能干细胞并进行培养，得到经培养后的诱导多能干细胞；
(2)第一阶段早期中胚层细胞的诱导分化：采用本发明第一方面中所述的第一阶段早期中胚层细胞诱导分化培养基对步骤(1)得到的经培养后的诱导多能干细胞进行培养，得到早期中胚层细胞；
(3)第二阶段间充质干细胞的诱导分化：采用本发明第一方面中所述的第二阶段间充质干细胞诱导分化培养基对步骤(2)得到的早期中胚层细胞进行培养，得到间充质干细胞。
进一步，步骤(1)中所述的培养包括诱导多能干细胞的消化、接种、换液；
优选地，所述诱导多能干细胞的消化包括：向诱导多能干细胞中加入消化液进行孵育，弃去消化液后，加入培养基终止消化；
更优选地，所述消化液包括EDTA消化液、胰蛋白酶消化液、胰蛋白酶-EDTA消化液、胶原酶消化液、accutase消化液；
最优选地，所述消化液为EDTA消化液；
最优选地，所述EDTA消化液的用量为0.5mM、2mL；
更优选地，所述孵育的条件为37℃、5min；
更优选地，所述培养基为E8完全培养基；
优选地，所述诱导多能干细胞的接种包括：收集经消化后的诱导多能干细胞细胞悬液，用培养基重悬细胞，接种于培养板中继续培养，调整细胞密度为接种后5×10 4/cm 2；
更优选地，所述培养基为添加rock inhibitor的E8完全培养基；
更优选地，所述重悬细胞的重悬条件为1000转/分、5min；
更优选地，所述继续培养的条件为37℃、24h；
优选地，所述诱导多能干细胞的换液包括：将经接种培养24h后的诱导多能干细胞用培养基等体积换液，继续培养至细胞汇合度至少为30％；
更优选地，所述培养基为不含rock inhibitor的E8完全培养基；
更优选地，所述继续培养的条件为37℃、24h。
进一步，步骤(2)中所述的培养包括在步骤(1)得到的经培养后的诱导多能干细胞中加入本发明第一方面中所述的第一阶段早期中胚层细胞诱导分化培养基，继续诱导培养；
优选地，所述本发明第一方面中所述的第一阶段早期中胚层细胞诱导分化培 养基的用量为3mL/孔；
优选地，所述继续诱导培养的条件为37℃、4天。
进一步，步骤(3)中所述的培养包括在步骤(2)得到的早期中胚层细胞中加入本发明第一方面中所述的第二阶段间充质干细胞诱导分化培养基，继续诱导培养后，进行传代培养；
优选地，所述本发明第一方面中所述的第二阶段间充质干细胞诱导分化培养基的用量为2mL/孔；
优选地，所述继续诱导培养的条件为37℃、4-10天；
优选地，所述传代培养包括在细胞汇合度达到80％的诱导分化得到的间充质干细胞中加入消化液进行孵育，孵育后加入本发明第一方面中所述的第二阶段间充质干细胞诱导分化培养基终止消化，收集细胞，再加入间充质干细胞成熟培养基重悬细胞，接种于培养板中并加入间充质干细胞成熟培养基进行培养，即得成熟的间充质干细胞；
更优选地，所述消化液包括accutase消化液、EDTA消化液、胰蛋白酶消化液、胰蛋白酶-EDTA消化液胶原酶消化液；
最优选地，所述消化液为accutase消化液；
最优选地，所述accutase消化液的用量为4倍稀释、0.5mL；
更优选地，所述孵育的条件为37℃、8min；
更优选地，所述本发明第一方面中所述的第二阶段间充质干细胞诱导分化培养基的用量为1mL；
更优选地，所述重悬细胞的间充质干细胞成熟培养基的用量为1mL；
更优选地，所述重悬细胞的重悬条件为1000转/分、5min；
更优选地，所述接种后的细胞密度为3-8×10 3/cm 2；
更优选地，所述培养细胞的间充质干细胞成熟培养基的用量为2mL；
最优选地，所述间充质干细胞成熟培养基包含95％α-MEM、5％HPL。
第三方面，本发明提供了一种诱导多能干细胞来源的间充质干细胞或细胞群体。
进一步，所述间充质干细胞或细胞群体为采用本发明第二方面所述的方法诱导分化得到的。
第四方面，本发明提供了一种细胞治疗产品。
进一步，所述细胞治疗产品包括本发明第三方面所述的间充质干细胞或细胞群体。
本文中所述的“细胞治疗产品”，是指用于治疗、预防和/或诊断以及预防免疫性疾病、神经系统疾病、血液系统疾病、心脑血管系统疾病、骨疾病、软骨疾病、糖尿病并发症、肝肾损伤、肿瘤的药物，包括通过分离、培养和专业化操作而由人类制备的细胞或组织，且更具体来说，所述“细胞治疗产品”是指用于治疗、预防和/或诊断以及预防免疫性疾病、神经系统疾病、血液系统疾病、心脑血管系统疾病、骨疾病、软骨疾病、糖尿病并发症、肝肾损伤、肿瘤的药物，其通过包含对自体、同源或异源体外活细胞进行增殖或分选或通过其它方法修饰细胞生物特性的任何过程制备，以便恢复细胞或组织的功能；细胞治疗产品根据细胞分化的程度主要分为体细胞治疗产品和干细胞治疗产品，且本发明特别针对的是间充质干细胞治疗产品。
进一步，所述细胞治疗产品可用于形成脂肪细胞、骨细胞、软骨细胞、肌细胞、神经细胞、心肌细胞等其他类型的细胞。
第五方面，本发明提供了一种用于培养细胞的饲养细胞。
进一步，所述饲养细胞包括本发明第三方面所述的间充质干细胞或细胞群体。
第六方面，本发明提供了一种用于将诱导多能干细胞快速诱导分化为间充质干细胞的试剂盒。
进一步，所述试剂盒包括本发明第一方面所述的培养基组合。
进一步，所述试剂盒还包括诱导多能干细胞、消化液、添加rock inhibitor的E8完全培养基、不含rock inhibitor的E8完全培养基、间充质干细胞成熟培养基；
优选地，所述消化液包括EDTA消化液、胰蛋白酶消化液、胰蛋白酶-EDTA消化液、胶原酶消化液、accutase消化液；
更优选地，所述消化液包括EDTA消化液、accutase消化液；
优选地，所述间充质干细胞成熟培养基包含95％α-MEM、5％HPL。
此外，本发明还提供了一种治疗免疫性疾病、神经系统疾病、血液系统疾病、心脑血管系统疾病、骨疾病、软骨疾病、糖尿病并发症、肝肾损伤、肿瘤的方法。
进一步，所述方法包括给有需要的受试者施用有效量的本发明第四方面所述的细胞治疗产品或本发明第三方面所述的间充质干细胞或细胞群体。
第七方面，本发明提供了如下任一方面应用：
(1)本发明第一方面所述的培养基组合在制备诱导多能干细胞来源的间充质干细胞或细胞群体中的应用；
(2)本发明第三方面所述的间充质干细胞或细胞群体在制备用于治疗免疫性疾病、神经系统疾病、血液系统疾病、心脑血管系统疾病、骨疾病、软骨疾病、糖尿病并发症、肝肾损伤、肿瘤的细胞治疗产品中的应用；
(3)本发明第四方面所述的细胞治疗产品在治疗免疫性疾病、神经系统疾病、血液系统疾病、心脑血管系统疾病、骨疾病、软骨疾病、糖尿病并发症、肝肾损伤、肿瘤中的应用；
(4)本发明第五方面所述的饲养细胞在制备成熟且商业化的细胞产品中的应用；
(5)本发明第六方面所述的试剂盒在制备诱导多能干细胞来源的间充质干细胞或细胞群体中的应用。
进一步，所述免疫性疾病包括类风湿性关节炎、多发性硬化、系统性红斑狼疮、炎症性肠病、银屑病、自身免疫性葡萄膜炎、干燥综合征、多发性肌炎、皮肌炎、I型糖尿病、乳糜泻、甲状腺炎、垂体炎、克罗恩病；
进一步，所述神经系统疾病包括帕金森氏综合征、阿尔兹海默症、肌萎缩性脊髓侧索硬化症、外伤性脑损伤、脊髓损伤、脊肌萎缩症、癫痫、重症肌无力、亨廷顿病、路易体痴呆；
进一步，所述血液系统疾病包括细胞移植后移植物抗宿主病(GVHD)、再生障碍性贫血、急性白血病、地中海贫血、免疫性血细胞减少症、血友病、骨髓增生异常综合症、血小板减少症；
进一步，所述心脑血管系统疾病包括心肌梗死、心力衰竭、血管病变、缺血性血管疾病、心脏衰竭、冠心病、缺血性心脏病、脑梗死、脑出血、缺血性脑卒 中、中风；
进一步，所述骨疾病包括骨关节炎、股骨头坏死、腰椎间盘退行性疾病、脊柱病、骨质疏松、退行性关节炎、风湿性关节炎、滑囊炎、滑膜炎、颈椎病、腰椎病、肩周炎、强直性脊柱炎；
进一步，所述软骨疾病包括复发性多发性软骨炎、剥脱性骨软骨炎、肋骨软骨炎、骨软骨症、病理性关节软骨缺损、科勒氏骨病、幼年畸形性骨软骨炎、胫骨粗隆骨软骨病、Scheuermann病；
进一步，所述糖尿病并发症包括糖尿病足溃疡、糖尿病视网膜病变、糖尿病心肌病、糖尿病周围神经病变、糖尿病性脑病、糖尿病性肾损伤、糖尿病下肢缺血性疾病；
进一步，所述肝肾损伤包括肝硬化、肝损伤、肾损伤、肾硬化、肝肾综合征、肝衰竭、肾衰竭；
进一步，所述肿瘤包括淋巴瘤、非小细胞肺癌、黑色素瘤、神经胶质瘤、结肠癌、肝内胆管癌、肝癌、宫颈癌、食管癌、多发性骨髓瘤、胃癌、乳腺癌、前列腺癌、肺癌、脑胶质瘤、膀胱癌、卵巢癌。
与现有技术相比，本发明具有如下的优点和有益效果：
(1)本发明提供了一种iPSC单层诱导法分离间充质干细胞的技术，该技术与目前常用的胚体分化法相比具有明显的优势，主要表现在分化过程操作简单，分化效率高且便于纯化分离，在分化过程中不涉及动物血清和其它非人源添加物，因此，非常适合生产满足临床需求的足够数量的自体间充质干细胞。由iPSC分化得到的间充质干细胞具有细胞来源充足，细胞增殖能力强，细胞活力旺盛，并可能在回输后能够有更好的损伤修复和抗衰老的功效；
(2)本发明通过先将iPSC诱导为早期中胚层细胞，再进一步诱导分化为间充质干细胞，可解决成人来源的间充质干细胞及其他非限定诱导途径iPSC来源的间充质干细胞的异质性与混杂的缺点，可解决间充质干细胞来源受限的问题，获得的间充质干细胞具有更强的增殖与免疫调节能力；
(3)本发明提供的iPSC单层诱导法分离间充质干细胞的技术，不仅可以在短时间内以高产率制备可满足临床需求的足够数量的间充质干细胞，而且由于没有胚状体形成的步骤，因此制备工艺简单，并且可以获得具有均质性的间充质干细胞，从而具有可以在比现有方法更短的时间内提供细胞治疗剂的效果。
附图说明
以下，结合附图来详细说明本发明的实施方案，其中：
图1显示诱导用iPSC的细胞形态图，其中，A图：40×，B图：100×；
图2显示接种24h后的iPSC的细胞形态图；
图3显示iPSC诱导第二天的集落形态图(100×)；
图4显示iPSC诱导第六天的细胞形态图(100×)；
图5显示P2代细胞的细胞形态特征图；
图6显示分化21天后的细胞形态图；
图7显示P8代细胞的细胞形态特征图；
图8显示流式细胞仪检测iPSC分化得到的MSC细胞表面标记物CD90、CD105的结果图；
图9显示流式细胞仪检测iPSC分化得到的MSC细胞表面标记物CD34、CD45的结果图；
图10显示iPSC分化得到的MSC细胞的多能性的检测结果图，其中，A图：成骨分化，B图：成软骨分化，C图：成脂分化。
具体实施方式
下面结合具体实施例，进一步阐述本发明，所述实施例仅用于解释本发明，而不能理解为对本发明的限制。本领域的普通技术人员可以理解为：在不脱离本发明的原理和宗旨的情况下可以对这些实施例进行多种变化、修改、替换和变型，本发明的范围由权利要求及其等同物限定。下列实施例中未注明具体条件的实验方法，通常按照常规条件或按照厂商所建议的条件实施检测，下列实施例中所用的材料、试剂等，如无特殊说明，均可从商业途径得到。
实施例1 人类iPSC诱导分化得到自体MSC细胞
1、实验材料
本发明实施例中所使用的主要试剂信息见表1。
表1 本发明实施例中使用的主要试剂信息
Figure PCTCN2022107902-appb-000001
Figure PCTCN2022107902-appb-000002
2、诱导前细胞的准备
(1)实验方法
①培养板的处理：培养板用matrigel室温孵育30分钟以上；
②细胞消化和接种：T25瓶中培养的iPSC(北京呈诺医学生物科技有限公司)弃培养基，DPBS洗两遍，每次1min；加入0.5mM EDTA消化液2mL；37℃孵育5min后，弃EDTA，加入E8完全培养基终止消化；
收集细胞悬液，细胞计数；根据细胞密度和需要细胞量取不同量细胞悬液，1000转/分离心5min；用添加rock inhibitor的E8完全培养基重悬细胞，调整细胞密度为接种后5×10 4cells/cm 2，以保证第二天细胞汇合度达到30％至50％，接种于6孔板中，放置回培养箱培养24小时；
③换液：将步骤②中培养24小时后的细胞，轻轻吸取培养孔中的培养液，用不含rock inhibitor的E8培养基等体积换液，放置回培养箱培养24小时，细胞汇合度需要在至少30％。
(2)实验结果
结果显示，iPSC呈集落状生长，集落边界清楚，集落内细胞核质比高，核仁明显(见图1)，换液培养24小时后的细胞汇合度在30％以上(见图2)。
3、早期中胚层细胞的诱导
(1)实验方法
取上述得到的iPSC细胞，轻轻吸取培养孔中的培养液，每孔用3mL DPBS轻轻洗培养孔一次，每孔添加3mL中胚层分化培养基，放置回培养箱中诱导培养4天；
所述中胚层分化培养基组成为：90％DMEM/F12、10％Knockout serum  replacement、1％L-glutamine、细胞因子以及小分子化合物组合；
其中，细胞因子以及小分子化合物组合包括：维生素C衍生物(L-Ascorbic acid)、重组人骨形态发生蛋白(BMP4)、TGF-βtype I Receptor抑制剂；
其中，TGF-βtype I Receptor抑制剂包括：SB-431542、Dorsomorphin、A83-01、LDN193189、RepSox、SB525334、DMH-1、SB-505124、sb4、ITD-1、GW788388、LSKL、SD-208、LDN-212854、K02288、LDN-214117、R-268712、SM16、A77-01、BIO-013077-01；
优选地，所述维生素C衍生物(L-Ascorbic acid)的浓度为50μg/ml。
优选地，所述BMP4的浓度为25ng/ml。
优选地，所述TGF-βtype I Receptor抑制剂为SB-431542。更优选地，所述SB-431542的浓度为2uM。
(2)实验结果
结果显示，在诱导培养第1天到第4天期间，iPSC集落内细胞出现典型的上皮间质转化的形态特征(见图3)，即细胞由多角形的上皮样细胞变为间质细胞样细胞，集落界限消失，细胞核质比明显减小。
4、MSC细胞的诱导
(1)实验方法
在上述步骤诱导培养第4天结束后，轻轻吸取培养孔中的培养液，每孔用1mL DPBS轻轻洗培养孔一次，每孔添加2mL完全MSC诱导培养基，放置回培养箱培养4-10天；
所述MSC诱导培养基组成为：90％DMEM/F12、10％Knockout serum replacement、1％L-glutamine以及TGF-βtype I Receptor抑制剂；
其中，TGF-βtype I Receptor抑制剂包括：SB-431542、Dorsomorphin、A83-01、LDN193189、RepSox、SB525334、DMH-1、SB-505124、sb4、ITD-1、GW788388、LSKL、SD-208、LDN-212854、K02288、LDN-214117、R-268712、SM16、A77-01、BIO-013077-01；
优选地，TGF-βtype I Receptor抑制剂为SB-431542；更优选地，所述SB-431542的浓度为2uM。
在诱导培养第4天到第10天期间，细胞增殖迅速，并且长满培养面，继续诱导培养，当细胞汇合度达到80％时，进行如下传代培养：
轻轻吸取培养孔中的培养基，DPBS洗两遍，每次1min；加入4倍稀释的accutase消化液0.5mL；37℃孵育8min后，加入1mL完全MSC培养基终止消化；收集细胞悬液至15mL离心管中；再用1mL MSC成熟培养基轻轻清洗原培养孔，并一起收集至15mL离心管中。1000转/分离心5min；用MSC成熟培养基重悬细胞，调整细胞密度为接种后3-8×10 3cells/cm 2，接种于matrigel处理的6孔板中，每孔2mL MSC成熟培养基，放置回培养箱中培养，此为第2代细胞记为P2；
上述MSC成熟培养基包括：95％α-MEM、5％HPL；
P2代细胞继续诱导培养，每天观察细胞；当细胞汇合度达到80％时即进行传代培养，传代方法与P1至P2传代方法一致，培养到21天左右。
(2)实验结果
结果显示，在诱导培养第4天到第10天期间，细胞增殖迅速，并且长满培养面(见图4)；从P1代细胞起，细胞形态为典型的MSC长梭形形态(见图5)；培养21天后，细胞呈典型的MSC细胞形态(见图6)，表明了本发明成功将iPSC诱导分化为MSC细胞。
5、iPSC诱导分化得到的MSC细胞的扩增
(1)实验方法
在上述诱导培养21天后细胞汇合度达到80％以上时，轻轻吸取培养孔中的培养基，DPBS洗两遍，每次1min；加入4倍稀释的accutase消化液0.5mL；37℃孵育8min后，加入含有5％HPL的α-MEM培养基终止消化；收集细胞悬液至15mL离心管中；800转/分离心5min；用含有5％HPL的α-MEM培养基重悬细胞，六孔板每孔接种至一个T25瓶中，此后细胞即按脐带来源MSC细胞的传代方法进行传代，冻存，实现iPSC诱导分化得到的MSC细胞的扩增。
(2)实验结果
结果显示，在扩增过程中，iPSC来源的MSC细胞形态与脐带来源MSC细胞形态类似(见图7)，进一步表明了本发明成功将iPSC诱导分化为MSC细胞。
实施例2 流式细胞仪检测iPSC分化得到的MSC细胞表面标记物
1、实验方法
(1)细胞消化
用移液器移去实施例1中得到的iPSC分化的MSC细胞扩增后的细胞培养液，加入无钙镁PBS，轻轻晃动培养瓶，弃无钙镁PBS，重复上述过程一次；后加入4倍稀释的Accutase，37℃放置，显微镜下观察细胞的消化情况；当细胞大部分脱壁时，轻拍培养皿，收集细胞悬液至50mL离心管中；
(2)单细胞悬液制备
30μm孔径筛网过滤上述50mL离心管中细胞悬液至新的50mL离心管中，300g×5min收集细胞，弃去上清液；
(3)细胞固定
收集的细胞用10mL无钙镁PBS重悬混匀，并取样50μL进行细胞计数，300g×5min洗涤一次，弃上清；再用10mL无钙镁PBS重悬混匀，300g×5min洗涤一次，弃上清；加入1mL 4％多聚甲醛溶液，室温固定30min；
(4)封闭
固定后的细胞300g×5min离心，弃上清，用1mL无钙镁PBS重悬混匀，300g×5min洗涤一次，弃上清；再用1mL无钙镁PBS重悬混匀，300g×5min洗涤一次，弃上清；第三次用1mL无钙镁PBS重悬混匀，300g×5min洗涤一次，弃上清；加入1mL 1％BSA封闭液室温封闭30min；
(5)荧光染色
封闭后的细胞300g×5min离心，弃上清，加入0.7mL无钙镁PBS重悬细胞，轻轻吹打混匀，分装至7个1.5mL EP管中，留1个做对照，其余6个分别 按比例加入抗体，4℃冰箱避光孵育30分钟，300g×5min离心，弃上清；用1mL无钙镁PBS重悬混匀，300g×5min洗涤一次，弃上清；再用1mL无钙镁PBS重悬混匀，300g×5min洗涤一次，弃上清；第三次用1mL无钙镁PBS重悬混匀，300g×5min洗涤一次，弃上清；加入200μL无钙镁PBS，轻轻吹打混匀后转移至流式细胞仪管，用于流式细胞仪检测。
2、实验结果
结果显示，由iPSC分化而来的MSC细胞表达至少70％以上阳性标记的CD90，表达至少80％以上阳性标记的CD105(见图8)；由iPSC分化而来的MSC细胞不表达阴性标记CD34，不表达阴性标记CD45(见图9)，表明了本发明制备得到的由iPSC分化而来的MSC细胞和MSC细胞表面标志物一致。
实施例3 iPSC分化得到的MSC细胞的多能性的检测
本实施例对实施例1中扩增后的iPSC分化得到的MSC细胞分别进行了成骨诱导分化实验、成软骨诱导分化实验和成脂诱导分化实验，以验证本发明中iPSC分化得到的MSC细胞的多能性。
1、成骨诱导分化实验
(1)在MSC成熟培养基中培养iPSC来源的MSC；
(2)在将MSC分化为成骨细胞之前，确保细胞达到100％的汇合度，使用成骨细胞分化培养基；
(3)培养细胞2-3周，每3-4天换一次培养基；
(4)PBS清洗后，使用4％多聚甲醛溶液固定半小时；
(5)吸去固定液，用蒸馏水清洗细胞；
(6)室温下，用2％的茜素红染色液去除水和污渍3分钟；用水重复清洗以去除多余的污渍；
上述成骨细胞分化培养基包括：90％DMEM、10％FBS、小分子化合物细胞因子组合；
其中，小分子化合物细胞因子组合包括：糖皮质激素受体激动剂、维生素C衍生物、磷酸酶抑制剂、L-glutamine；
优选地，糖皮质激素受体激动剂为地塞米松，维生素C衍生物为L-ascorbic acid，磷酸酶抑制剂为β-glycerophosphate。
2、成软骨诱导分化实验
(1)在T-25或T-75培养瓶中，MSC成熟培养基中，培养iPSC来源的MSC；
(2)在将MSC分化为软骨细胞之前，进行细胞计数，调整细胞浓度为5-10×10 6/mL，在软骨细胞分化培养基中重悬；
(3)将20-30μL上述细胞滴加到24孔板中，静置培养2-4小时；
(4)小心添加1mL软骨细胞分化培养基；
(5)每3天更换一次培养基，培养细胞2周；
(6)PBS清洗后，使用4％多聚甲醛溶液固定，并做成石蜡切片；
(7)对切片进行阿尔新蓝(alcian blue)染色，在光显微镜下可视化；
上述软骨细胞分化培养基包括：DMEM、小分子化合物细胞因子组合；
其中，小分子化合物细胞因子组合包括：糖皮质激素受体激动剂、维生素C衍生物、丙酮酸钠、TGF-β、胰岛素、转铁蛋白、亚硒酸氢钠、L-glutamine；
优选地，糖皮质激素受体激动剂为地塞米松，维生素C衍生物为L-ascorbic acid。
3、成脂诱导分化实验
(1)在MSC成熟培养基中培养iPSC来源的MSC；
(2)在将MSC分化为成脂细胞之前，确保细胞达到100％的汇合度，使用脂肪前体细胞分化培养基；
(3)培养细胞2-3周，每3-4天换一次培养基；
(4)PBS清洗后，使用4％多聚甲醛溶液固定半小时；
(5)吸去固定液，用蒸馏水清洗细胞，并添加足够量的60％异丙醇，以覆盖细胞，在室温下孵育5分钟；
(6)吸去60％异丙醇，并添加足够的油红O染色溶液，以覆盖细胞；在室温下孵育15分钟；
上述脂肪前体细胞分化培养基包括：90％DMEM、10％FBS、小分子化合物细胞因子组合；
其中，小分子化合物细胞因子组合包括：糖皮质激素受体激动剂、胰岛素、非选择性的COX1和COX2抑制剂、磷酸二酯酶(PDE)抑制剂、L-glutamine；
优选地，糖皮质激素受体激动剂为地塞米松，非选择性的COX1和COX2抑制剂为吲哚美辛，磷酸二酯酶(PDE)抑制剂为IBMX。
4、实验结果
成骨诱导分化实验的实验结果显示，iPSC分化的MSC细胞成骨分化28天后，茜素红染色，红橙色晶体表示为钙沉积物，由结果图中可见明显的红橙色着色的钙化结节(见图10A)，表明了MSC细胞分化为成骨细胞；
成软骨诱导分化实验的实验结果显示，iPSC分化的MSC细胞成软骨分化14天后，经酸化处理，阿尔新蓝染色，蓝色染色表示由软骨细胞合成蛋白多糖聚糖，由结果图中可见软骨骨质呈蓝色(见图10B)，表明了MSC细胞分化为成软骨细胞；
成脂诱导分化实验的实验结果显示，iPSC分化的MSC细胞成脂分化14天后，油红-O染色，成熟脂肪细胞的细胞内脂囊染色鲜红，由结果图中可见红色脂滴呈颗粒状分布于细胞质中(见图10C)，表明了MSC细胞分化为脂肪细胞；
以上结果表明了由iPS分化的MSC细胞经过扩增后仍具有分化为成骨、成软骨和成脂分化的能力，证明了本发明实施例1中制备的由iPSC诱导分化得到的MSC细胞具有多能性。
上述实施例的说明只是用于理解本发明的方法及其核心思想。应当指出，对于本领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以对本发明进行若干改进和修饰，这些改进和修饰也将落入本发明权利要求的保护范围内。

Claims (10)

  1. 一种诱导多能干细胞分化为间充质干细胞的培养基组合，其特征在于，所述培养基组合包括第一阶段早期中胚层细胞诱导分化培养基、第二阶段间充质干细胞诱导分化培养基；
    优选地，所述第一阶段早期中胚层细胞诱导分化培养基包含细胞因子及小分子化合物组合；
    更优选地，所述细胞因子及小分子化合物组合包括：维生素C或维生素C衍生物、重组人骨形态发生蛋白、TGF-βtype I Receptor抑制剂；
    最优选地，所述维生素C或维生素C衍生物包括：L-Ascorbic acid、抗坏血酸钙、抗坏血酸镁、抗坏血酸锌、抗坏血酸钾、抗坏血酸钠、脱氢抗坏血酸、L-苏糖酸、L-木糖酸、L-来苏糖酸、L-抗坏血酸单硬脂酸化物、L-抗坏血酸二棕榈酸化物、L-抗坏血酸6-十六酸化合物、L-抗坏血酸2-0-磷酸化物、L-抗坏血酸3-0-磷酸化物、L-抗坏血酸2-0-硫酸化物；
    最优选地，所述维生素C或维生素C衍生物为L-Ascorbic acid；
    最优选地，所述L-Ascorbic acid的浓度为50μg/ml；
    最优选地，所述重组人骨形态发生蛋白包括：BMP4、BMP2、BMP6、BMP7、BMP9、BMP12、BMP1、BMP3、BMP5、BMP8、BMP13、BMP10、BMP11、BMP14、BMP15；
    最优选地，所述重组人骨形态发生蛋白为BMP4；
    最优选地，所述BMP4的浓度为25ng/ml；
    最优选地，所述TGF-βtype I Receptor抑制剂包括：SB-431542、Dorsomorphin、A83-01、LDN193189、RepSox、SB525334、DMH-1、SB-505124、sb4、ITD-1、GW788388、LSKL、SD-208、LDN-212854、K02288、LDN-214117、R-268712、SM16、A77-01、BIO-013077-01；
    最优选地，所述TGF-βtype I Receptor抑制剂为SB-431542；
    最优选地，所述SB-431542的浓度为2uM；
    更优选地，所述第一阶段早期中胚层细胞诱导分化培养基还包含90％DMEM/F12、10％Knockout serum replacement、1％L-glutamine；
    最优选地，所述第一阶段早期中胚层细胞诱导分化培养基包含L-Ascorbic acid、BMP4、SB-431542、90％DMEM/F12、10％Knockout serum replacement、1％L-glutamine；
    优选地，第二阶段间充质干细胞诱导分化培养基包含小分子化合物；
    更优选地，所述小分子化合物为TGF-βtype I Receptor抑制剂；
    最优选地，所述TGF-βtype I Receptor抑制剂包括：SB-431542、Dorsomorphin、A83-01、LDN193189、RepSox、SB525334、DMH-1、SB-505124、sb4、ITD-1、GW788388、LSKL、SD-208、LDN-212854、K02288、LDN-214117、R-268712、SM16、A77-01、BIO-013077-01；
    最优选地，所述TGF-βtype I Receptor抑制剂为SB-431542；
    最优选地，所述SB-431542的浓度为2uM；
    更优选地，所述第二阶段间充质干细胞诱导分化培养基还包含90％DMEM/F12、10％Knockout serum replacement、1％L-glutamine；
    最优选地，所述第二阶段间充质干细胞诱导分化培养基包含SB-431542、90％DMEM/F12、10％Knockout serum replacement、1％L-glutamine。
  2. 一种诱导多能干细胞快速诱导分化为间充质干细胞的方法，其特征在于，所述方法包括：采用权利要求1所述的培养基组合对诱导多能干细胞进行培养；
    优选地，所述方法包括如下步骤：
    (1)提供诱导多能干细胞并进行培养，得到经培养后的诱导多能干细胞；
    (2)第一阶段早期中胚层细胞的诱导分化：采用权利要求1中所述的第一阶段早期中胚层细胞诱导分化培养基对步骤(1)得到的经培养后的诱导多能干细胞进行培养，得到早期中胚层细胞；
    (3)第二阶段间充质干细胞的诱导分化：采用权利要求1中所述的第二阶段间充质干细胞诱导分化培养基对步骤(2)得到的早期中胚层细胞进行培养，得到间充质干细胞。
  3. 根据权利要求2所述的方法，其特征在于，步骤(1)中所述的培养包括诱导多能干细胞的消化、接种、换液；
    优选地，所述诱导多能干细胞的消化包括：向诱导多能干细胞中加入消化液进行孵育，弃去消化液后，加入培养基终止消化；
    更优选地，所述消化液包括EDTA消化液、胰蛋白酶消化液、胰蛋白酶-EDTA消化液、胶原酶消化液、accutase消化液；
    最优选地，所述消化液为EDTA消化液；
    最优选地，所述EDTA消化液的用量为0.5mM、2mL；
    更优选地，所述孵育的条件为37℃、5min；
    更优选地，所述培养基为E8完全培养基；
    优选地，所述诱导多能干细胞的接种包括：收集经消化后的诱导多能干细胞细胞悬液，用培养基重悬细胞，接种于培养板中继续培养，调整细胞密度为接种后5×10 4/cm 2；
    更优选地，所述培养基为添加rock inhibitor的E8完全培养基；
    更优选地，所述重悬细胞的重悬条件为1000转/分、5min；
    更优选地，所述继续培养的条件为37℃、24h；
    优选地，所述诱导多能干细胞的换液包括：将经接种培养24h后的诱导多能干细胞用培养基等体积换液，继续培养至细胞汇合度至少为30％；
    更优选地，所述培养基为不含rock inhibitor的E8完全培养基；
    更优选地，所述继续培养的条件为37℃、24h。
  4. 根据权利要求2所述的方法，其特征在于，步骤(2)中所述的培养包括在步骤(1)得到的经培养后的诱导多能干细胞中加入权利要求1中所述的第一阶段早期中胚层细胞诱导分化培养基，继续诱导培养；
    优选地，所述权利要求1中所述的第一阶段早期中胚层细胞诱导分化培养基的用量为3mL/孔；
    优选地，所述继续诱导培养的条件为37℃、4天。
  5. 根据权利要求2所述的方法，其特征在于，步骤(3)中所述的培养包括在步骤(2)得到的早期中胚层细胞中加入权利要求1中所述的第二阶段间充质干细胞诱导分化培养基，继续诱导培养后，进行传代培养；
    优选地，所述权利要求1中所述的第二阶段间充质干细胞诱导分化培养基的 用量为2mL/孔；
    优选地，所述继续诱导培养的条件为37℃、4-10天；
    优选地，所述传代培养包括在细胞汇合度达到80％的诱导分化得到的间充质干细胞中加入消化液进行孵育，孵育后加入权利要求1中所述的第二阶段间充质干细胞诱导分化培养基终止消化，收集细胞，再加入间充质干细胞成熟培养基重悬细胞，接种于培养板中并加入间充质干细胞成熟培养基进行培养，即得成熟的间充质干细胞；
    更优选地，所述消化液包括accutase消化液、EDTA消化液、胰蛋白酶消化液、胰蛋白酶-EDTA消化液胶原酶消化液；
    最优选地，所述消化液为accutase消化液；
    最优选地，所述accutase消化液的用量为4倍稀释、0.5mL；
    更优选地，所述孵育的条件为37℃、8min；
    更优选地，所述权利要求1中所述的第二阶段间充质干细胞诱导分化培养基的用量为1mL；
    更优选地，所述重悬细胞的间充质干细胞成熟培养基的用量为1mL；
    更优选地，所述重悬细胞的重悬条件为1000转/分、5min；
    更优选地，所述接种后的细胞密度为3-8×10 3/cm 2；
    更优选地，所述培养细胞的间充质干细胞成熟培养基的用量为2mL；
    最优选地，所述间充质干细胞成熟培养基包含95％α-MEM、5％HPL。
  6. 一种诱导多能干细胞来源的间充质干细胞或细胞群体，其特征在于，所述间充质干细胞或细胞群体为采用权利要求2-5任一项所述的方法诱导分化得到的。
  7. 一种细胞治疗产品，其特征在于，所述细胞治疗产品包括权利要求6所述的间充质干细胞或细胞群体。
  8. 一种用于培养细胞的饲养细胞，其特征在于，所述饲养细胞包括权利要求6所述的间充质干细胞或细胞群体。
  9. 一种用于将诱导多能干细胞快速诱导分化为间充质干细胞的试剂盒，其特征在于，所述试剂盒包括权利要求1所述的培养基组合。
  10. 如下任一方面应用，其特征在于，所述应用包括：
    (1)权利要求1所述的培养基组合在制备诱导多能干细胞来源的间充质干细胞或细胞群体中的应用；
    (2)权利要求6所述的间充质干细胞或细胞群体在制备用于治疗免疫性疾病、神经系统疾病、血液系统疾病、心脑血管系统疾病、骨疾病、软骨疾病、糖尿病并发症、肝肾损伤、肿瘤的细胞治疗产品中的应用；
    (3)权利要求7所述的细胞治疗产品在治疗免疫性疾病、神经系统疾病、血液系统疾病、心脑血管系统疾病、骨疾病、软骨疾病、糖尿病并发症、肝肾损伤、肿瘤中的应用；
    (4)权利要求8所述的饲养细胞在制备成熟且商业化的细胞产品中的应用；
    (5)权利要求9所述的试剂盒在制备诱导多能干细胞来源的间充质干细胞或细胞群体中的应用。
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CN113512525A (zh) * 2020-04-10 2021-10-19 南京大学 一种间充质干细胞制剂及其应用
CN117683712A (zh) * 2024-02-02 2024-03-12 深圳市北科生物科技有限公司 多能干细胞分化获得间充质干细胞的三维诱导方法
CN117683712B (zh) * 2024-02-02 2024-05-10 深圳市北科生物科技有限公司 多能干细胞分化获得间充质干细胞的三维诱导方法

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