Method for differentiating human pluripotent stem cells into macrophages
阅读说明：本技术 一种人类多能干细胞分化巨噬细胞的方法 (Method for differentiating human pluripotent stem cells into macrophages ) 是由 那洁 段福宇 于 2019-09-10 设计创作，主要内容包括：本发明公开了一种人类多能干细胞分化巨噬细胞的方法。本发明提供了一种用于将多能干细胞分化为巨噬细胞的试剂盒,包括能够将多能干细胞分化为巨噬细胞的成套培养液。本发明采用单层细胞培养法首先分化获得造血干细胞,并通过进一步加入IL3和M-CSF刺激获得CD14和CD163阳性巨噬细胞。该方法化学成分确定、无动物源成分,大大提高了制备细胞的安全性,且具有耗时短、分化效率高、成本较低等特点。采用本发明提供的制备方法可大规模生产人巨噬细胞,质量稳定,安全性高,为组织工程、药物研发和细胞治疗提供大量细胞来源。(The invention discloses a method for differentiating human pluripotent stem cells into macrophages. The invention provides a kit for differentiating pluripotent stem cells into macrophages, which comprises a complete set of culture solution capable of differentiating the pluripotent stem cells into the macrophages. The invention adopts monolayer cell culture method to firstly differentiate and obtain hematopoietic stem cells, and further adds IL3 and M-CSF to stimulate and obtain CD14 and CD163 positive macrophages. The method has the advantages of definite chemical components, no animal-derived components, greatly improved safety of cell preparation, short time consumption, high differentiation efficiency, and low cost. The preparation method provided by the invention can be used for producing human macrophages in a large scale, has stable quality and high safety, and provides a large amount of cell sources for tissue engineering, drug research and development and cell therapy.)
1. A kit for differentiating pluripotent stem cells into macrophages comprises a culture solution II, a culture solution III, a culture solution IV, a culture solution V, a culture solution VI and a culture solution VII;
the culture solution II is any one of the following (a1) - (a 6):
(a1) a culture broth containing insulin-free B27supplement and human bone morphogenic protein 4;
(a2) a culture broth containing insulin-free B27supplement and human bone morphogenic protein 4; the volume percentage of the insulin-free B27 additive in the culture solution II is 1-2%; the concentration of the human bone morphogenetic protein 4 in the culture solution II is 5-10 ng/ml;
(a3) a culture broth containing insulin-free B27supplement and human bone morphogenic protein 4; the volume percentage content of the insulin-free B27 additive in the culture solution II is 2%; the concentration of the human bone morphogenetic protein 4 in the culture solution II is 5 ng/ml;
(a4) a culture broth comprising insulin-free B27supplement, L-glutamine or a substitute therefor, glycine, L-alanine, L-aspartic acid, L-glutamic acid, L-proline, L-serine, penicillin, streptomycin, vitamin C, and human bone morphogenic protein 4;
(a5) a culture broth comprising insulin-free B27supplement, L-glutamine or a substitute therefor, glycine, L-alanine, L-aspartic acid, L-glutamic acid, L-proline, L-serine, penicillin, streptomycin, vitamin C, and human bone morphogenic protein 4; the volume percentage of the insulin-free B27 additive in the culture solution II is 1-2%; the concentration of the L-glutamine or the substitute thereof in the culture solution II is 1-2 mM; the concentration of the glycine in the culture solution II is 7.5 mu g/mL; the concentration of the L-alanine in the culture solution II is 8.9 mu g/mL; the concentration of the L-aspartic acid in the culture solution II is 13.2 mug/mL; the concentration of the L-aspartic acid in the culture solution II is 13.30 mu g/mL; the concentration of the L-glutamic acid in the culture solution II is 14.70 mu g/mL; the concentration of the L-proline in the culture solution II is 11.50 mu g/mL; the concentration of the L-serine in the culture solution II is 10.5 mu g/mL; the concentration of the penicillin in the culture solution II is 50-100U/ml; the concentration of the streptomycin in the culture solution II is 50-100 mug/ml; the concentration of the vitamin C in the culture solution II is 25-50 ng/ml; the concentration of the human bone morphogenetic protein 4 in the culture solution II is 5-10 ng/ml;
(a6) a culture broth comprising insulin-free B27supplement, L-glutamine or a substitute therefor, glycine, L-alanine, L-aspartic acid, L-glutamic acid, L-proline, L-serine, penicillin, streptomycin, vitamin C, and human bone morphogenic protein 4; the volume percentage content of the insulin-free B27 additive in the culture solution II is 2%; the concentration of the L-glutamine or the substitute thereof in the culture solution II is 2 mM; the concentration of the glycine in the culture solution II is 7.5 mu g/mL; the concentration of the L-alanine in the culture solution II is 8.9 mu g/mL; the concentration of the L-aspartic acid in the culture solution II is 13.2 mug/mL; the concentration of the L-aspartic acid in the culture solution II is 13.30 mu g/mL; the concentration of the L-glutamic acid in the culture solution II is 14.70 mu g/mL; the concentration of the L-proline in the culture solution II is 11.50 mu g/mL; the concentration of the L-serine in the culture solution II is 10.5 mu g/mL; the concentration of the penicillin in the culture solution II is 100U/ml; the concentration of the streptomycin in the culture solution II is 100 mug/ml; the concentration of the vitamin C in the culture solution II is 50 ng/ml; the concentration of the human bone morphogenetic protein 4 in the culture solution II is 5 ng/ml;
the culture solution III is any one of the following (b1) - (b 3):
(b1) culture solution II containing GSK3 inhibitor;
(b2) culture solution II containing 1-2 μ M GSK3 inhibitor;
(b3) culture solution II containing 2. mu.M GSK3 inhibitor;
the culture solution IV is any one of the following (c1) - (c 6):
(c1) culture solution containing insulin-added B27 additive, human vascular endothelial growth factor VEGF-165 and human fibroblast growth factor;
(c2) culture solution containing insulin-added B27 additive, human vascular endothelial growth factor VEGF-165 and human fibroblast growth factor; the volume percentage of the B27 additive added with insulin in the culture solution IV is 1-2%; the concentration of the human vascular endothelial growth factor VEGF-165 in the culture solution IV is 25-50 ng/ml; the concentration of the human fibroblast growth factor in the culture solution IV is 5-10 ng/ml;
(c3) culture solution containing insulin-added B27 additive, human vascular endothelial growth factor VEGF-165 and human fibroblast growth factor; the volume percentage of the B27 additive added with insulin in the culture solution IV is 2%; the concentration of the human vascular endothelial growth factor VEGF-165 in the culture solution IV is 50 ng/ml; the concentration of the human fibroblast growth factor in the culture solution IV is 10 ng/ml;
(c4) a culture solution containing insulin-added B27 additive, L-glutamine or its substitute, glycine, L-alanine, L-aspartic acid, L-glutamic acid, L-proline, L-serine, penicillin, streptomycin, vitamin C, human vascular endothelial growth factor VEGF-165 and human fibroblast growth factor;
(c5) a culture solution containing insulin-added B27 additive, L-glutamine or its substitute, glycine, L-alanine, L-aspartic acid, L-glutamic acid, L-proline, L-serine, penicillin, streptomycin, vitamin C, human vascular endothelial growth factor VEGF-165 and human fibroblast growth factor; the volume percentage of the B27 additive added with insulin in the culture solution IV is 1-2%; the concentration of the L-glutamine or the substitute thereof in the culture solution IV is 1-2 mM; the concentration of the glycine in the culture solution IV is 7.5 mu g/mL; the concentration of the L-alanine in the culture solution IV is 8.9 mu g/mL; the concentration of the L-aspartic acid in the culture solution IV is 13.2 mug/mL; the concentration of the L-aspartic acid in the culture solution IV is 13.30 mu g/mL; the concentration of the L-glutamic acid in the culture solution IV is 14.70 mu g/mL; the concentration of the L-proline in the culture solution IV is 11.50 mu g/mL; the concentration of the L-serine in the culture solution IV is 10.5 mu g/mL; the concentration of the penicillin in the culture solution II is 50-100U/ml; the concentration of the streptomycin in the culture solution II is 50-100 mug/ml; the concentration of the vitamin C in the culture solution IV is 25-50 ng/ml; the concentration of the human vascular endothelial growth factor VEGF-165 in the culture solution IV is 25-50 ng/ml; the concentration of the human fibroblast growth factor in the culture solution IV is 5-10 ng/ml;
(c6) a culture solution containing insulin-added B27 additive, L-glutamine or its substitute, glycine, L-alanine, L-aspartic acid, L-glutamic acid, L-proline, L-serine, penicillin, streptomycin, vitamin C, human vascular endothelial growth factor VEGF-165 and human fibroblast growth factor; the volume percentage of the B27 additive added with insulin in the culture solution IV is 2%; the concentration of the L-glutamine or the substitute thereof in the culture solution IV is 2 mM; the concentration of the glycine in the culture solution IV is 7.5 mu g/mL; the concentration of the L-alanine in the culture solution IV is 8.9 mu g/mL; the concentration of the L-aspartic acid in the culture solution IV is 13.2 mug/mL; the concentration of the L-aspartic acid in the culture solution IV is 13.30 mu g/mL; the concentration of the L-glutamic acid in the culture solution IV is 14.70 mu g/mL; the concentration of the L-proline in the culture solution IV is 11.50 mu g/mL; the concentration of the L-serine in the culture solution IV is 10.5 mu g/mL; the concentration of the penicillin in the culture solution IV is 100U/ml; the concentration of the streptomycin in the culture solution IV is 100 mug/ml; the concentration of the vitamin C in the culture solution IV is 50 ng/ml; the concentration of the human vascular endothelial growth factor VEGF-165 in the culture solution IV is 50 ng/ml; the concentration of the human fibroblast growth factor in the culture solution IV is 10 ng/ml;
the culture solution V is any one of the following (d1) - (d 3):
(d1) a culture solution IV containing a TGF beta inhibitor;
(d2) a culture solution IV containing 5-10 mu M of TGF beta inhibitor;
(d3) a culture solution IV containing 10 mu M of a TGF beta inhibitor;
the culture solution VI is any one of the following (e1) - (e 6):
(e1) culture medium containing insulin-added B27 additive, human interleukin-3 and human macrophage colony stimulating factor;
(e2) culture medium containing insulin-added B27 additive, human interleukin-3 and human macrophage colony stimulating factor; the volume percentage of the B27 additive added with insulin in the culture solution VI is 1-2%; the concentration of the human interleukin-3 in the culture solution VI is 10-20 ng/ml; the concentration of the human macrophage colony stimulating factor in the culture solution VI is 50-100 ng/ml;
(e3) culture medium containing insulin-added B27 additive, human interleukin-3 and human macrophage colony stimulating factor; the volume percentage of the B27 additive added with insulin in the culture solution VI is 2 percent; the concentration of the human interleukin-3 in the culture solution VI is 10 ng/ml; the concentration of the human macrophage colony stimulating factor in the culture solution VI is 50 ng/ml;
(e4) a culture solution containing insulin-added B27 additive, L-glutamine or its substitute, glycine, L-alanine, L-aspartic acid, L-glutamic acid, L-proline, L-serine, penicillin, streptomycin, vitamin C, human interleukin-3 and human macrophage colony stimulating factor;
(e5) a culture solution containing insulin-added B27 additive, L-glutamine or its substitute, glycine, L-alanine, L-aspartic acid, L-glutamic acid, L-proline, L-serine, penicillin, streptomycin, vitamin C, human interleukin-3 and human macrophage colony stimulating factor; the volume percentage of the B27 additive added with insulin in the culture solution VI is 1-2%; the concentration of the L-glutamine or the substitute thereof in the culture solution VI is 1-2 mM; the concentration of the glycine in the culture solution VI is 7.5 mu g/mL; the concentration of the L-alanine in the culture solution VI is 8.9 mu g/mL; the concentration of the L-aspartic acid in the culture solution VI is 13.2 mug/mL; the concentration of the L-aspartic acid in the culture solution VI is 13.30 mu g/mL; the concentration of the L-glutamic acid in the culture solution VI is 14.70 mu g/mL; the concentration of the L-proline in the culture solution VI is 11.50 mu g/mL; the concentration of the L-serine in the culture solution VI is 10.5 mu g/mL; the concentration of the penicillin in the culture solution VI is 50-100U/ml; the concentration of the streptomycin in the culture solution VI is 50-100 mug/ml; the concentration of the vitamin C in the culture solution VI is 25-50 ng/ml; the concentration of the human interleukin-3 in the culture solution VI is 10-20 ng/ml; the concentration of the human macrophage colony stimulating factor in the culture solution VI is 50-100 ng/ml;
(e6) a culture solution containing insulin-added B27 additive, L-glutamine or its substitute, glycine, L-alanine, L-aspartic acid, L-glutamic acid, L-proline, L-serine, penicillin, streptomycin, vitamin C, human interleukin-3 and human macrophage colony stimulating factor; the volume percentage of the B27 additive added with insulin in the culture solution VI is 2 percent; the concentration of the L-glutamine or the substitute thereof in the culture solution VI is 2 mM; the concentration of the glycine in the culture solution VI is 7.5 mu g/mL; the concentration of the L-alanine in the culture solution VI is 8.9 mu g/mL; the concentration of the L-aspartic acid in the culture solution VI is 13.2 mug/mL; the concentration of the L-aspartic acid in the culture solution VI is 13.30 mu g/mL; the concentration of the L-glutamic acid in the culture solution VI is 14.70 mu g/mL; the concentration of the L-proline in the culture solution VI is 11.50 mu g/mL; the concentration of the L-serine in the culture solution VI is 10.5 mu g/mL; the concentration of the penicillin in the culture solution VI is 100U/ml; the concentration of the streptomycin in the culture solution VI is 100 mug/ml; the concentration of the vitamin C in the culture solution VI is 50 ng/ml; the concentration of the human interleukin-3 in the culture solution VI is 10 ng/ml; the concentration of the human macrophage colony stimulating factor in the culture solution VI is 50 ng/ml;
the culture solution VII is any one of the following (f1) - (f 6):
(f1) a culture solution containing insulin-supplemented B27supplement and human macrophage colony stimulating factor;
(f2) a culture solution containing insulin-supplemented B27supplement and human macrophage colony stimulating factor; the volume percentage of the B27 additive added with insulin in the culture solution VII is 1-2%; the concentration of the human macrophage colony stimulating factor in a culture solution VII is 50-100 ng/ml;
(f3) a culture solution containing insulin-supplemented B27supplement and human macrophage colony stimulating factor; the volume percentage of the B27 additive added with insulin in the culture solution VII is 2%; the concentration of the human macrophage colony stimulating factor in a culture solution VII is 50 ng/ml;
(f4) a culture solution containing insulin-supplemented B27 additive, L-glutamine or a substitute therefor, glycine, L-alanine, L-aspartic acid, L-glutamic acid, L-proline, L-serine, penicillin, streptomycin, vitamin C, and human macrophage colony stimulating factor;
(f5) a culture solution containing insulin-supplemented B27 additive, L-glutamine or a substitute therefor, glycine, L-alanine, L-aspartic acid, L-glutamic acid, L-proline, L-serine, penicillin, streptomycin, vitamin C, and human macrophage colony stimulating factor; the volume percentage of the B27 additive added with insulin in the culture solution VII is 1-2%; the concentration of the L-glutamine or the substitute thereof in the culture solution VII is 1-2 mM; the concentration of the glycine in the culture solution VII is 7.5 mu g/mL; the concentration of the L-alanine in the culture solution VII is 8.9 mu g/mL; the concentration of the L-aspartic acid in the culture solution VII is 13.2 mug/mL; the concentration of the L-aspartic acid in the culture solution VII is 13.30 mu g/mL; the concentration of the L-glutamic acid in the culture solution VII is 14.70 mu g/mL; the concentration of the L-proline in the culture solution VII is 11.50 mu g/mL; the concentration of the L-serine in the culture solution VII is 10.5 mu g/mL; the concentration of the penicillin in a culture solution VII is 50-100U/ml; the concentration of the streptomycin in the culture solution VII is 50-100 mug/ml; the concentration of the vitamin C in the culture solution VII is 25-50 ng/ml; the concentration of the human macrophage colony stimulating factor in a culture solution VII is 50-100 ng/ml;
(f6) a culture solution containing insulin-supplemented B27 additive, L-glutamine or a substitute therefor, glycine, L-alanine, L-aspartic acid, L-glutamic acid, L-proline, L-serine, penicillin, streptomycin, vitamin C, and human macrophage colony stimulating factor; the volume percentage of the B27 additive added with insulin in the culture solution VII is 2%; the concentration of the L-glutamine or the substitute thereof in the culture solution VII is 2 mM; the concentration of the glycine in the culture solution VII is 7.5 mu g/mL; the concentration of the L-alanine in the culture solution VII is 8.9 mu g/mL; the concentration of the L-aspartic acid in the culture solution VII is 13.2 mug/mL; the concentration of the L-aspartic acid in the culture solution VII is 13.30 mu g/mL; the concentration of the L-glutamic acid in the culture solution VII is 14.70 mu g/mL; the concentration of the L-proline in the culture solution VII is 11.50 mu g/mL; the concentration of the L-serine in the culture solution VII is 10.5 mu g/mL; the concentration of the penicillin in a culture solution VII is 100U/ml; the concentration of the streptomycin in the culture solution VII is 100 mug/ml; the concentration of the vitamin C in the culture solution VII is 50 ng/ml; the concentration of the human macrophage colony stimulating factor in the culture solution VII is 50 ng/ml.
2. A kit for differentiating hematopoietic stem cells into macrophages, comprising said culture solution VI and said culture solution VII of claim 1.
3. The kit of claim 1 or 2, wherein: the GSK3 inhibitor is any one of the following (g1) - (g 4):
(g1)CHIR-99021；
(g2)B216763；
(g3)BIO；
(g4)TWS119；
and/or
The TGF beta inhibitor is SB 431542.
4. The kit of any one of claims 1 to 3, wherein: the kit also comprises a culture solution I; the culture solution I is any one of the following (h1) - (h 6):
(h1) a stem cell culture fluid comprising a ROCK inhibitor;
(h2) a stem cell culture fluid containing 5-10 μ M ROCK inhibitor;
(h3) a stem cell culture broth containing 10 μ M ROCK inhibitor;
(h4) stem cell culture broth containing Y27632;
(h5) a stem cell culture fluid containing 5-10 μ M Y27632;
(h6) stem cell culture broth containing 10. mu. M Y27632.
5. The kit of claim 4, wherein: the stem cell culture solution is TeSR-E8 culture solution.
6. A method of differentiating pluripotent stem cells into macrophages, comprising the steps of:
(2) inoculating pluripotent stem cells into the culture solution II of claim 1, and culturing for 0.5-1.5 days;
(3) replacing the culture solution of the step (2) with the culture solution III described in claim 1 or 3, and culturing for 1.5-2.5 days;
(4) transferring the cells of the step (3) to the culture solution IV of claim 1, and culturing for 1.5-2.5 days;
(5) replacing the culture solution of the step (4) with the culture solution V described in claim 1 or 3, and culturing for 2-4 days;
(6) transferring the cells obtained in the step (5) into a culture solution VI as described in any one of claims 1 to 3, and culturing for 4 to 6 days;
(7) transferring the cells obtained in step (6) to the culture solution VII according to any of claims 1 to 3, and culturing for 2 to 4 days.
7. The method of claim 6, wherein: the method also comprises the step (1) before the step (2): culturing pluripotent stem cells in a culture medium I according to claim 4 or 5;
further, the step (1) is as follows: (a) inoculating pluripotent stem cells into the culture solution I according to claim 4 or 5, and culturing for 0.5 to 1.5 days; (b) replacing the culture solution of step (a) with the stem cell culture solution of claim 4 or 5 for 0.5 to 1.5 days.
8. A method of differentiating hematopoietic stem cells into macrophages comprising the steps of:
(6) inoculating hematopoietic stem cell cells into the culture solution VI according to any one of claims 1 to 3, and culturing for 4 to 6 days;
(7) transferring the cells obtained in step (6) to the culture solution VII according to any of claims 1 to 3, and culturing for 2 to 4 days.
9. Use of a kit according to any one of claims 1 to 5 for the preparation of macrophages.
10. Use according to claim 9, characterized in that: the application is to prepare the macrophage by taking the pluripotent stem cell or the hematopoietic stem cell as a starting cell.
Technical Field
The invention relates to the technical field of biology, in particular to a method for differentiating human pluripotent stem cells into macrophages.
Background
The human pluripotent stem cells comprise human embryonic stem cells and human induced pluripotent stem cells, can be differentiated into various types of cells in a human body, can be used for manufacturing disease models and carrying out drug toxicity tests, and can replace damaged cells through cell transplantation, promote body wound repair and treat diseases.
The macrophage is an important natural immune cell of a human body, and has important application value in aspects of resisting infection of pathogenic microorganism, removing necrotic cells and tissue debris, researching occurrence and development of atherosclerosis and the like. At present, a large amount of human macrophages are difficult to obtain, and amplification and gene manipulation are difficult to perform, so that related research and clinical application are greatly limited.
The main method currently used for differentiating human pluripotent stem cells into macrophages is the embryoid body (embryoid body) differentiation method. The method adopts human pluripotent cells cultured on mouse trophoblasts to generate embryoid bodies, and divides the embryoid bodies with proper size in a culture solution containing macrophage stimulating factor and interleukin 3 for about 3-4 weeks. The main problems of this method are long differentiation period, low efficiency and the animal-derived components, which have hindered their potential clinical application.
Disclosure of Invention
The method aims to originally create a differentiation method, and promotes the human pluripotent stem cells to be quickly and efficiently differentiated into high-purity macrophages through a cell culture additive with determined chemical components and low cost.
In a first aspect, the invention claims a kit for differentiating pluripotent stem cells into macrophages.
The kit for differentiating the pluripotent stem cells into the macrophages comprises a culture solution II, a culture solution III, a culture solution IV, a culture solution V, a culture solution VI and a culture solution VII;
the culture solution II is any one of the following (a1) - (a 6):
(a1) a culture broth containing insulin-free B27supplement and human bone morphogenic protein 4;
(a2) a culture broth containing insulin-free B27supplement and human bone morphogenic protein 4; the volume percentage of the insulin-free B27 additive in the culture solution II is 1-2%; the concentration of the human bone morphogenetic protein 4 in the culture solution II is 5-10 ng/ml;
(a3) a culture broth containing insulin-free B27supplement and human bone morphogenic protein 4; the volume percentage content of the insulin-free B27 additive in the culture solution II is 2%; the concentration of the human bone morphogenetic protein 4 in the culture solution II is 5 ng/ml;
(a4) a culture broth comprising insulin-free B27supplement, L-glutamine or a substitute therefor, glycine, L-alanine, L-aspartic acid, L-glutamic acid, L-proline, L-serine, penicillin, streptomycin, vitamin C, and human bone morphogenic protein 4;
(a5) a culture broth comprising insulin-free B27supplement, L-glutamine or a substitute therefor, glycine, L-alanine, L-aspartic acid, L-glutamic acid, L-proline, L-serine, penicillin, streptomycin, vitamin C, and human bone morphogenic protein 4; the volume percentage of the insulin-free B27 additive in the culture solution II is 1-2%; the concentration of the L-glutamine or the substitute thereof in the culture solution II is 1-2 mM; the concentration of the glycine in the culture solution II is 7.5 mu g/mL; the concentration of the L-alanine in the culture solution II is 8.9 mu g/mL; the concentration of the L-aspartic acid in the culture solution II is 13.2 mug/mL; the concentration of the L-aspartic acid in the culture solution II is 13.30 mu g/mL; the concentration of the L-glutamic acid in the culture solution II is 14.70 mu g/mL; the concentration of the L-proline in the culture solution II is 11.50 mu g/mL; the concentration of the L-serine in the culture solution II is 10.5 mu g/mL; the concentration of the penicillin in the culture solution II is 50-100U/ml; the concentration of the streptomycin in the culture solution II is 50-100 mug/ml; the concentration of the vitamin C in the culture solution II is 25-50 ng/ml; the concentration of the human bone morphogenetic protein 4 in the culture solution II is 5-10 ng/ml;
(a6) a culture broth comprising insulin-free B27supplement, L-glutamine or a substitute therefor, glycine, L-alanine, L-aspartic acid, L-glutamic acid, L-proline, L-serine, penicillin, streptomycin, vitamin C, and human bone morphogenic protein 4; the volume percentage content of the insulin-free B27 additive in the culture solution II is 2%; the concentration of the L-glutamine or the substitute thereof in the culture solution II is 2 mM; the concentration of the glycine in the culture solution II is 7.5 mu g/mL; the concentration of the L-alanine in the culture solution II is 8.9 mu g/mL; the concentration of the L-aspartic acid in the culture solution II is 13.2 mug/mL; the concentration of the L-aspartic acid in the culture solution II is 13.30 mu g/mL; the concentration of the L-glutamic acid in the culture solution II is 14.70 mu g/mL; the concentration of the L-proline in the culture solution II is 11.50 mu g/mL; the concentration of the L-serine in the culture solution II is 10.5 mu g/mL; the concentration of the penicillin in the culture solution II is 100U/ml; the concentration of the streptomycin in the culture solution II is 100 mug/ml; the concentration of the vitamin C in the culture solution II is 50 ng/ml; the concentration of the human bone morphogenetic protein 4 in the culture solution II is 5 ng/ml.
The culture solution III is any one of the following (b1) - (b 3):
(b1) culture solution II containing GSK3 inhibitor;
(b2) culture solution II containing 1-2 μ M GSK3 inhibitor;
(b3) culture II containing 2. mu.M of GSK3 inhibitor.
The culture solution IV is any one of the following (c1) - (c 6):
(c1) culture solution containing insulin-added B27 additive, human vascular endothelial growth factor VEGF-165 and human fibroblast growth factor bFGF;
(c2) culture solution containing insulin-added B27 additive, human vascular endothelial growth factor VEGF-165 and human fibroblast growth factor bFGF; the volume percentage of the B27 additive added with insulin in the culture solution IV is 1-2%; the concentration of the human vascular endothelial growth factor VEGF-165 in the culture solution IV is 25-50 ng/ml; the concentration of the human fibroblast growth factor (bFGF) in the culture solution IV is 5-10 ng/ml;
(c3) culture solution containing insulin-added B27 additive, human vascular endothelial growth factor VEGF-165 and human fibroblast growth factor bFGF; the volume percentage of the B27 additive added with insulin in the culture solution IV is 2%; the concentration of the human vascular endothelial growth factor VEGF-165 in the culture solution IV is 50 ng/ml; the concentration of the human fibroblast growth factor (bFGF) in the culture solution IV is 10 ng/ml;
(c4) a culture solution containing insulin-added B27 additive, L-glutamine or its substitute, glycine, L-alanine, L-aspartic acid, L-glutamic acid, L-proline, L-serine, penicillin, streptomycin, vitamin C, human vascular endothelial growth factor VEGF-165 and human fibroblast growth factor bFGF;
(c5) a culture solution containing insulin-added B27 additive, L-glutamine or its substitute, glycine, L-alanine, L-aspartic acid, L-glutamic acid, L-proline, L-serine, penicillin, streptomycin, vitamin C, human vascular endothelial growth factor VEGF-165 and human fibroblast growth factor bFGF; the volume percentage content of the B27 additive added with insulin in the culture solution IV is 1-2%; the concentration of the L-glutamine or the substitute thereof in the culture solution IV is 1-2 mM; the concentration of the glycine in the culture solution IV is 7.5 mu g/mL; the concentration of the L-alanine in the culture solution IV is 8.9 mu g/mL; the concentration of the L-aspartic acid in the culture solution IV is 13.2 mug/mL; the concentration of the L-aspartic acid in the culture solution IV is 13.30 mu g/mL; the concentration of the L-glutamic acid in the culture solution IV is 14.70 mu g/mL; the concentration of the L-proline in the culture solution IV is 11.50 mu g/mL; the concentration of the L-serine in the culture solution IV is 10.5 mu g/mL; the concentration of the penicillin in the culture solution IV is 50-100U/ml; the concentration of the streptomycin in the culture solution IV is 50-100 mug/ml; the concentration of the vitamin C in the culture solution IV is 25-50 ng/ml; the concentration of the human vascular endothelial growth factor VEGF-165 in the culture solution IV is 25-50 ng/ml; the concentration of the human fibroblast growth factor (bFGF) in the culture solution IV is 5-10 ng/ml;
(c6) a culture solution containing insulin-added B27 additive, L-glutamine or its substitute, glycine, L-alanine, L-aspartic acid, L-glutamic acid, L-proline, L-serine, penicillin, streptomycin, vitamin C, human vascular endothelial growth factor VEGF-165 and human fibroblast growth factor bFGF; the volume percentage content of the B27 additive added with insulin in the culture solution IV is 2%; the concentration of the L-glutamine or the substitute thereof in the culture solution IV is 2 mM; the concentration of the glycine in the culture solution IV is 7.5 mu g/mL; the concentration of the L-alanine in the culture solution IV is 8.9 mu g/mL; the concentration of the L-aspartic acid in the culture solution IV is 13.2 mug/mL; the concentration of the L-aspartic acid in the culture solution IV is 13.30 mu g/mL; the concentration of the L-glutamic acid in the culture solution IV is 14.70 mu g/mL; the concentration of the L-proline in the culture solution IV is 11.50 mu g/mL; the concentration of the L-serine in the culture solution IV is 10.5 mu g/mL; the concentration of the penicillin in the culture solution IV is 100U/ml; the concentration of the streptomycin in the culture solution IV is 100 mug/ml; the concentration of the vitamin C in the culture solution IV is 50 ng/ml; the concentration of the human vascular endothelial growth factor VEGF-165 in the culture solution IV is 50 ng/ml; the concentration of the human fibroblast growth factor bFGF in the culture solution IV is 10 ng/ml.
The culture solution V is any one of the following (d1) - (d 3):
(d1) a culture solution IV containing a TGF beta inhibitor;
(d2) a culture solution IV containing 5-10 mu M of TGF beta inhibitor;
(d3) culture broth IV containing 10. mu.M of a TGF-beta inhibitor.
The culture solution VI is any one of the following (e1) - (e 6):
(e1) culture medium containing insulin-added B27 additive, human interleukin-3 and human macrophage colony stimulating factor;
(e2) culture medium containing insulin-added B27 additive, human interleukin-3 and human macrophage colony stimulating factor; the volume percentage of the B27 additive added with insulin in the culture solution VI is 1-2%; the concentration of the human interleukin-3 in the culture solution VI is 10-20 ng/ml; the concentration of the human macrophage colony stimulating factor in the culture solution VI is 50-100 ng/ml;
(e3) culture medium containing insulin-added B27 additive, human interleukin-3 and human macrophage colony stimulating factor; the volume percentage of the B27 additive added with insulin in the culture solution VI is 2 percent; the concentration of the human interleukin-3 in the culture solution VI is 10 ng/ml; the concentration of the human macrophage colony stimulating factor in the culture solution VI is 50 ng/ml;
(e4) a culture solution containing insulin-added B27 additive, L-glutamine or its substitute, glycine, L-alanine, L-aspartic acid, L-glutamic acid, L-proline, L-serine, penicillin, streptomycin, vitamin C, human interleukin-3 and human macrophage colony stimulating factor;
(e5) a culture solution containing insulin-added B27 additive, L-glutamine or its substitute, glycine, L-alanine, L-aspartic acid, L-glutamic acid, L-proline, L-serine, penicillin, streptomycin, vitamin C, human interleukin-3 and human macrophage colony stimulating factor; the volume percentage of the B27 additive added with insulin in the culture solution VI is 1-2%; the concentration of the L-glutamine or the substitute thereof in the culture solution VI is 1-2 mM; the concentration of the glycine in the culture solution VI is 7.5 mu g/mL; the concentration of the L-alanine in the culture solution VI is 8.9 mu g/mL; the concentration of the L-aspartic acid in the culture solution VI is 13.2 mug/mL; the concentration of the L-aspartic acid in the culture solution VI is 13.30 mu g/mL; the concentration of the L-glutamic acid in the culture solution VI is 14.70 mu g/mL; the concentration of the L-proline in the culture solution VI is 11.50 mu g/mL; the concentration of the L-serine in the culture solution VI is 10.5 mu g/mL; the concentration of the penicillin in the culture solution VI is 50-100U/ml; the concentration of the streptomycin in the culture solution VI is 50-100 mug/ml; the concentration of the vitamin C in the culture solution VI is 25-50 ng/ml; the concentration of the human interleukin-3 in the culture solution VI is 10-20 ng/ml; the concentration of the human macrophage colony stimulating factor in the culture solution VI is 50-100 ng/ml;
(e6) a culture solution containing insulin-added B27 additive, L-glutamine or its substitute, glycine, L-alanine, L-aspartic acid, L-glutamic acid, L-proline, L-serine, penicillin, streptomycin, vitamin C, human interleukin-3 and human macrophage colony stimulating factor; the volume percentage of the B27 additive added with insulin in the culture solution VI is 2 percent; the concentration of the L-glutamine or the substitute thereof in the culture solution VI is 2 mM; the concentration of the glycine in the culture solution VI is 7.5 mu g/mL; the concentration of the L-alanine in the culture solution VI is 8.9 mu g/mL; the concentration of the L-aspartic acid in the culture solution VI is 13.2 mug/mL; the concentration of the L-aspartic acid in the culture solution VI is 13.30 mu g/mL; the concentration of the L-glutamic acid in the culture solution VI is 14.70 mu g/mL; the concentration of the L-proline in the culture solution VI is 11.50 mu g/mL; the concentration of the L-serine in the culture solution VI was 10.5. mu.g/mL. The concentration of the penicillin in the culture solution VI is 100U/ml; the concentration of the streptomycin in the culture solution VI is 100 mug/ml; the concentration of the vitamin C in the culture solution VI is 50 ng/ml; the concentration of the human interleukin-3 in the culture solution VI is 10 ng/ml; the concentration of the human macrophage colony stimulating factor in the culture solution VI is 50 ng/ml.
The culture solution VII is any one of the following (f1) - (f 6):
(f1) a culture solution containing insulin-supplemented B27supplement and human macrophage colony stimulating factor;
(f2) a culture solution containing insulin-supplemented B27supplement and human macrophage colony stimulating factor; the volume percentage of the B27 additive added with insulin in the culture solution VII is 1-2%; the concentration of the human macrophage colony stimulating factor in a culture solution VII is 50-100 ng/ml;
(f3) a culture solution containing insulin-supplemented B27supplement and human macrophage colony stimulating factor; the volume percentage of the B27 additive added with insulin in the culture solution VII is 2%; the concentration of the human macrophage colony stimulating factor in a culture solution VII is 50 ng/ml;
(f4) a culture solution containing insulin-supplemented B27 additive, L-glutamine or a substitute therefor, glycine, L-alanine, L-aspartic acid, L-glutamic acid, L-proline, L-serine, penicillin, streptomycin, vitamin C, and human macrophage colony stimulating factor;
(f5) a culture solution containing insulin-supplemented B27 additive, L-glutamine or a substitute therefor, glycine, L-alanine, L-aspartic acid, L-glutamic acid, L-proline, L-serine, penicillin, streptomycin, vitamin C, and human macrophage colony stimulating factor; the volume percentage of the B27 additive added with insulin in the culture solution VII is 1-2%; the concentration of the L-glutamine or the substitute thereof in the culture solution VII is 1-2 mM; the concentration of the glycine in the culture solution VII is 7.5 mu g/mL; the concentration of the L-alanine in the culture solution VII is 8.9 mu g/mL; the concentration of the L-aspartic acid in the culture solution VII is 13.2 mug/mL; the concentration of the L-aspartic acid in the culture solution VII is 13.30 mu g/mL; the concentration of the L-glutamic acid in the culture solution VII is 14.70 mu g/mL; the concentration of the L-proline in the culture solution VII is 11.50 mu g/mL; the concentration of the L-serine in the culture solution VII is 10.5 mu g/mL; the concentration of the penicillin in a culture solution VII is 50-100U/ml; the concentration of the streptomycin in the culture solution VII is 50-100 mug/ml; the concentration of the vitamin C in the culture solution VII is 25-50 ng/ml; the concentration of the human macrophage colony stimulating factor in a culture solution VII is 50-100 ng/ml;
(f6) a culture solution containing insulin-supplemented B27 additive, L-glutamine or a substitute therefor, glycine, L-alanine, L-aspartic acid, L-glutamic acid, L-proline, L-serine, penicillin, streptomycin, vitamin C, and human macrophage colony stimulating factor; the volume percentage of the B27 additive added with insulin in the culture solution VII is 2%; the concentration of the L-glutamine or the substitute thereof in the culture solution VII is 2 mM; the concentration of the glycine in the culture solution VII is 7.5 mu g/mL; the concentration of the L-alanine in the culture solution VII is 8.9 mu g/mL; the concentration of the L-aspartic acid in the culture solution VII is 13.2 mug/mL; the concentration of the L-aspartic acid in the culture solution VII is 13.30 mu g/mL; the concentration of the L-glutamic acid in the culture solution VII is 14.70 mu g/mL; the concentration of the L-proline in the culture solution VII is 11.50 mu g/mL; the concentration of the L-serine in the culture solution VII is 10.5 mu g/mL; the concentration of the penicillin in a culture solution VII is 100U/ml; the concentration of the streptomycin in the culture solution VII is 100 mug/ml; the concentration of the vitamin C in the culture solution VII is 50 ng/ml; the concentration of the human macrophage colony stimulating factor in the culture solution VII is 50 ng/ml.
The culture solution II comprises the following components: insulin-free B27 additive, human bone morphogenetic protein 4 and cell basal medium.
The culture solution II comprises the following components: insulin-free B27 additive, L-glutamine or its substitute, glycine, L-alanine, L-aspartic acid, L-glutamic acid, L-proline, L-serine, penicillin, streptomycin, vitamin C, human bone morphogenetic protein 4 and cell basal medium.
The composition of the culture solution III is as follows: GSK3 inhibitor and culture solution II.
The composition of the culture solution IV is as follows: b27 additive containing insulin, human vascular endothelial growth factor VEGF-165, human fibroblast growth factor and cell basic culture solution.
The composition of the culture solution IV is as follows: b27 additive added with insulin, L-glutamine or its substitute, glycine, L-alanine, L-aspartic acid, L-glutamic acid, L-proline, L-serine, penicillin, streptomycin, vitamin C, human vascular endothelial growth factor VEGF-165, human fibroblast growth factor and cell basic culture solution.
The composition of the culture solution V is as follows: TGF beta inhibitor and culture solution IV.
The composition of the culture solution VI is as follows: b27 additive containing insulin, human interleukin-3, human macrophage colony stimulating factor and cell basic culture solution.
The composition of the culture solution VI is as follows: b27 additive containing insulin, L-glutamine or its substitute, glycine, L-alanine, L-aspartic acid, L-glutamic acid, L-proline, L-serine, penicillin, streptomycin, vitamin C, human interleukin-3, human macrophage colony stimulating factor and cell basic culture solution.
The composition of the culture solution VII is as follows: b27 additive containing insulin, human macrophage colony stimulating factor and cell basic culture solution.
The composition of the culture solution VII is as follows: b27 additive containing insulin, L-glutamine or its substitute, glycine, L-alanine, L-aspartic acid, L-glutamic acid, L-proline, L-serine, penicillin, streptomycin, vitamin C, human macrophage colony stimulating factor and cell basic culture solution.
The cell basic culture solution may be RPMI1640 basic culture solution.
Further, the air conditioner is provided with a fan,the L-glutamine or its substitute is GlutaMAX, specifically "GlutaMAXTMSupplement "(e.g., Gibco #35050061, or other products of the same composition). The "GlutaMAXTMThe Supplement' is composed of L-allyl-L-glutamine as a substitute for L-glutamine, and has a concentration of 200mM and a solvent of 0.85% NaCl solution.
The protein sequence of any one of the human bone morphogenetic proteins 4(BMP4) is shown in SEQ ID No. 1.
The protein sequence of any of the angiogenesis factors (VEGF-165) is shown in SEQ ID No. 2.
The protein sequence of any one of the human fibroblast growth factors (bFGF) is shown in SEQ ID No. 3.
The protein sequence of any one of the above human interleukin-3 (IL3) is shown in SEQ ID No. 4.
The protein sequence of any one of the human macrophage colony stimulating factor (M-CSF) is shown in SEQ ID No. 5.
In a second aspect, the invention claims a kit for differentiating hematopoietic stem cells into macrophages.
The claimed kit for differentiating hematopoietic stem cells into macrophages comprises culture solution VI and culture solution VII as described above.
In the first aspect, the GSK3 inhibitor may be any one of the following (g1) - (g 4):
(g1)CHIR-99021；
(g2)B216763；
(g3)BIO；
(g4)TWS119。
in the first and second aspects, the TGF β inhibitor may be SB 431542.
In the first aspect, the kit may further comprise a culture solution i; the culture solution I may be any one of the following (h1) to (h 6):
(h1) a stem cell culture fluid comprising a ROCK inhibitor;
(h2) a stem cell culture fluid containing 5-10 μ M ROCK inhibitor;
(h3) a stem cell culture broth containing 10 μ M ROCK inhibitor;
(h4) stem cell culture broth containing Y27632;
(h5) a stem cell culture fluid containing 5-10 μ M Y27632;
(h6) stem cell culture broth containing 10. mu. M Y27632.
The culture solution I comprises the following components: ROCK inhibitors and stem cell culture fluids.
The culture solution I comprises the following components: y27632 inhibitor and stem cell culture fluid.
Wherein, the stem cell culture solution can be TeSR-E8 culture solution.
In a third aspect, the invention claims a method of differentiating pluripotent stem cells into macrophages.
The method for differentiating pluripotent stem cells into macrophages as claimed by the invention can comprise the following steps:
(2) inoculating pluripotent stem cells into the culture solution II, and culturing for 0.5-1.5 days (e.g., 1 day);
(3) replacing the culture solution of the step (2) with the culture solution III, and culturing for 1.5-2.5 days (such as 2 days);
(4) transferring the cells of step (3) to the culture solution IV and culturing for 1.5-2.5 days (such as 2 days);
(5) replacing the culture solution of step (4) with culture solution V, and culturing for 2-4 days (such as 3 days);
(6) transferring the cells obtained in step (5) into the culture solution VI and culturing for 4-6 days (such as 5 days);
(7) the cells obtained in step (6) are transferred to culture broth VII as described above and cultured for 2-4 days (e.g., 3 days).
Further, step (1) can be included before step (2): pluripotent stem cells were inoculated into the culture medium I described above and cultured.
Further, the step (1) may be: (a) inoculating pluripotent stem cells into the culture solution I described above and culturing for 0.5-1.5 days (e.g., 1 day); (b) replacing the culture medium of step (a) with the stem cell culture medium described above for 0.5-1.5 days (e.g., 1 day).
In a fourth aspect, the invention claims a method of differentiating hematopoietic stem cells into macrophages.
The method for differentiating hematopoietic stem cells into macrophages as claimed in the present invention may comprise the steps of:
(6) inoculating hematopoietic stem cells into the culture solution VI and culturing for 4-6 days (such as 5 days);
(7) the cells obtained in step (6) are transferred to culture broth VII as described above and cultured for 2-4 days (e.g., 3 days).
In the third and fourth aspects, the conditions of any of the cultures are 37 ℃ and 5% CO2. Any culture can be carried out by adopting a Marigel coated culture dish, specifically coating for 2h at 37 ℃.
In a fifth aspect, the invention claims the use of a kit as described hereinbefore for the preparation of macrophages.
Further, the application is to prepare the macrophage by taking the pluripotent stem cell (corresponding to the kit of the first aspect) or the hematopoietic stem cell (corresponding to the kit of the second aspect) as a starting cell.
In the present invention, the pluripotent stem cells are specifically human pluripotent stem cells; the hematopoietic stem cells are specifically human hematopoietic stem cells.
Any one of the human pluripotent stem cells is a human embryonic stem cell line or a human induced pluripotent stem cell.
The human embryonic stem cell line is a commercial human embryonic stem cell line, such as human embryonic stem cell line H1. The human embryonic stem cell line H1 can be specifically from the U.S. WiCell cell Bank, with the following numbering: WA 01. The human induced pluripotent stem cell is CD 34-iPSC. The induced pluripotent stem cell CD34-iPSC is obtained by inducing human cord blood hematopoietic stem cells (CD34 positive cells) to reprogram by using a Sendai virus reprogramming kit (Invitrogen, goods number: A16517).
The invention develops a culture solution additive capable of greatly enhancing the differentiation efficiency of human pluripotent stem cells to macrophages, and the additive does not contain animal-derived components, has determined chemical components and is favorable for serving as a clinical-grade stem cell differentiation culture system. The invention also provides a novel method for differentiating the human pluripotent stem cells into the macrophages, which can obviously improve the differentiation efficiency and reduce the differentiation cost compared with the existing method.
The preparation method provided by the invention can be used for obtaining macrophages, hematopoietic stem cells are obtained by differentiation firstly by adopting a monolayer cell culture method, micromolecules for regulating WNT and TGF beta signal paths are added in stages, and a differentiation method for removing insulin in stages is adopted. The method has the advantages of definite chemical components, no animal-derived components, greatly improved safety of cell preparation, short time consumption, high differentiation efficiency, and low cost. The preparation method provided by the invention can be used for producing human macrophages in a large scale, has stable quality and high safety, and provides a large amount of cell sources for tissue engineering, drug research and development and cell therapy.
In a word, the invention adopts a staged induction method based on monolayer cell differentiation, can obtain a large number of macrophages from the differentiation of human pluripotent stem cells in about 16 days, greatly shortens the differentiation period, improves the differentiation efficiency, has determined chemical components, and is beneficial to the future application in clinical cell replacement therapy.
Drawings
FIG. 1 is a morphological diagram of human pluripotent stem cells.
FIG. 2 is a graph of the morphological changes of H1 during differentiation into macrophages.
FIG. 3 is a graph showing the morphological changes of CD34-iPSC during the differentiation process to macrophages.
FIG. 4 shows the result of the detection of the surface marker of hematopoietic progenitor cells by a flow cytometer.
FIG. 5 shows the results of the detection of macrophage surface marker by the flow cytometer.
FIG. 6 shows immunofluorescence results of macrophage surface marker detection.
FIG. 7 shows the results of Giemsa staining of macrophages.
FIG. 8 shows the result of macrophage phagocytosis assay.
FIG. 9 shows the result of LPS stimulation of macrophages.
Detailed Description
The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.
Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
Human embryonic stem cell line H1 (abbreviated as H1 cell): U.S. WiCell cell bank, No.: WA 01.
Induced pluripotent stem cells CD34-iPSC (CD34-iPSC cells for short): obtained by inducing reprogramming of human cord blood hematopoietic stem cells (CD34 positive cells) by using a Sendai virus reprogramming kit (Invitrogen, Cat: A16517), and the specific method is operated according to the kit instruction.
DMEM culture solution: gibco Corp., Cat number: 11965092.
RPMI1640 basal medium: ThermoFisher company, Cat No.: 11875093.
TeSR-E8 culture solution: stemcel corporation, cat # stock: 05990.
the E8-Y culture solution is TeSR-E8 culture solution containing 5-10 μ M ROCK inhibitor Y27632; in the examples of the present invention, the concentration of ROCK inhibitor Y27632 in E8-Y broth was 10. mu.M.
The M1 culture solution is RPMI1640 culture solution containing 1-2% (v/v) insulin-free B27 additive (B27minus insulin), 0.5-1% (v/v) Glutamax (L-alkyl-L-glutamine final concentration of 1-2mM), 0.5-1% (v/v) non-essential amino acid (NEAA), 50-100U/ml penicillin, 50-100. mu.g/ml streptomycin, 25-50ng/ml vitamin C and 5-10ng/ml human osteogenic protein 4(BMP 4). In the embodiment of the invention, the concentration of each component in the M1 culture solution is as follows: 2% (v/v) insulin-free B27 additive (B27minus insulin), 1% (v/v) Glutamax (L-allyl-L-glutamine final concentration of 2mM), 1% (v/v) non-essential amino acid (NEAA), 100U/ml penicillin, 100. mu.g/ml streptomycin, 50ng/ml vitamin C, 5ng/ml human osteogenic protein 4(BMP 4).
The M2 culture solution is RPMI1640 culture solution containing 1-2% (v/v) B27 additive (B27supplement), 0.5-1% (v/v) Glutamax (L-allyl-L-glutamine final concentration is 1-2mM), 0.5-1% (v/v) non-essential amino acid (NEAA), 50-100U/ml penicillin, 50-100 μ g/ml streptomycin, 25-50ng/ml vitamin C, 25-50ng/ml human vascular endothelial growth factor (VEGF-165) and 5-10ng/ml human fibroblast growth factor (bFGF). In the embodiment of the invention, the concentration of each component in the M2 culture solution is as follows: 2% (v/v) B27 additive (B27supplement), 1% (v/v) Glutamax (L-alkyl-L-glutamine final concentration of 2mM), 1% (v/v) non-essential amino acid (NEAA), 100U/ml penicillin, 100. mu.g/ml streptomycin, 50ng/ml vitamin C, 50ng/ml human vascular endothelial growth factor (VEGF-165), 10ng/ml human fibroblast growth factor (bFGF).
The M3 culture solution is RPMI1640 culture solution containing 1-2% (v/v) B27 additive (B27supplement) 0.5-1% (v/v) Glutamax (L-allyl-L-glutamine final concentration is 1-2mM), 0.5-1% (v/v) non-essential amino acid (NEAA), 50-100U/ml penicillin, 50-100ug/ml streptomycin, 25-50ng/ml vitamin C, 10-20ng/ml human interleukin-3 (IL3) and 50-100ng/ml human macrophage colony stimulating factor (M-CSF). In the embodiment of the invention, the concentration of each component in the M3 culture solution is as follows: 2% (v/v) B27 additive (B27supplement), 1% (v/v) Glutamax (L-alkyl-L-glutamine final concentration of 2mM), 1% (v/v) non-essential amino acid (NEAA), 100U/ml penicillin, 100ug/ml streptomycin, 50ng/ml vitamin C, 10ng/ml human interleukin-3 (IL3) and 50ng/ml human macrophage colony stimulating factor (M-CSF)
Non-essential amino acids (NEAA, 100 ×): gibco Corp., Cat number: 11140050, respectively; in the embodiment of the invention, the concentration of each amino acid in the M1 culture solution or the M2 culture solution is as follows: 7.5 mu g/mL of glycine, 8.9 mu g/mL of L-alanine, 13.20 mu g/mL of L-aspartic acid, 13.30 mu g/mL of L-aspartic acid, 14.70 mu g/mL of L-glutamic acid, 11.50 mu g/mL of L-proline and 10.50 mu g/mL of L-serine.
Insulin-containing B27 additive (B27 supplement): gibco Corp., Cat number: 17504-044.
Insulin-free B27 additive (B27minus insulin): gibco Corp., Cat number: A1895601.
glutamax: gibco Corp., Cat number: 35050061.
vitamin C: sigma Aldrich, cat # s: A4403.
ROCK inhibitor Y27632: TargetMol corporation, cat #: t1870. The structural formula of Y27632 is as follows:
human bone morphogenic protein 4(BMP 4): r & D BioSystems, Cat number: 314-BP; the protein sequence is shown in SEQ ID No. 1.
Angiogenic factor (VEGF-165): SinoBiological, Inc., having a Cat number of: 11066-HNAH; the protein sequence is shown in SEQ ID No. 2.
Human fibroblast growth factor (bFGF): SinoBiological, Inc., having a Cat number of: 10014-HNAE; the protein sequence is shown in SEQ ID No. 3.
Human interleukin-3 (IL 3): peprotech company, cat #: 200-03-10; the protein sequence is shown in SEQ ID No. 4.
Human macrophage colony stimulating factor (M-CSF): peprotech company, cat #: 300-25-10; the protein sequence is shown in SEQ ID No. 5.
GSK3 inhibitor CHIR-99021: the Tocris Biosciences company has the following product number: 4423/10. The structural formula of CHIR-99021 is as follows:
TGF β inhibitor SB 431542: selleck, Inc., cat #: s1067. The structural formula of SB431542 is as follows:
cell digest Accutase: merk Millipore company, cat #: SF 006.
0.25% Trypsin: gibco, Cat # is: 25200056.
matrigel: BD Biosciences, Cat #: 356231.
PE-labeled CD43 antibody: eBioscience, inc, cat #: 12-0439-42.
APC-labeled CD34 antibody: miltenyi Inc., cat # is: 555824.
FITC-labeled CD45 antibody: miltenyi Inc., cat # is: 130-113-679.
FITC-labeled CD14 antibody: biolegend, cat #: 301804.
APC-labeled CD11b antibody: biolegend, cat #: 301309.
PE-labeled CD163 antibody: biolegend, cat #: 326505.
FITC-labeled mouse anti-human IgG antibody: eBioscience, inc, cat #: 11-4714-42.
PE-labeled mouse anti-human IgG antibody: eBioscience, inc, cat #: 12-4714-42.
APC-labeled mouse anti-human IgG antibody: eBioscience, inc, cat #: 17-4714-42.
Anti-human CD163 antibodies: biolegend, cat #: 333602
Anti-human CD14 antibody: biolegend, cat #: 367101
DyLight 488-labeled secondary antibody: thermo corporation, cat #: and R37120.
Nuclear dye DAPI: Sigma-Aldrich, Cat #: d9542
Lipopolysaccharide LPS: Sigma-Aldrich, Cat #: l2880-10 MG.
Giemsa dye liquor: Sigma-Aldrich, Cat #: G4507-5G.
RNA extraction solution: invitrogen corporation: the goods number is: 15596026
5 × All-In-One RT MasterMix reverse transcription kit:company, goods number: g490
qPCR master mix fluorescent quantitative PCR kit: promega corporation, cat #: a6001
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