CN113046318B - Culture medium and method for inducing pluripotent stem cells to differentiate into hematopoietic precursor cells - Google Patents

Abstract

The present invention relates to a culture medium and a method for inducing differentiation of pluripotent stem cells into hematopoietic precursor cells. Specifically, the method comprises 1) differentiating human pluripotent stem cells to generate hematopoietic endothelial precursor cells, and preparing to obtain the hematopoietic endothelial precursor cells; 2) Hematopoietic progenitor cells are prepared by a method of promoting differentiation of hematopoietic endothelial progenitor cells into hematopoietic progenitor cells. The culture medium is adopted in the preparation process and cultured in a random rotation mode, so that excellent effective efficiency is obtained, and the components of the culture medium are clear.

Description

Translated fromChinese

一种诱导多能干细胞向造血前体细胞分化的培养基以及方法A medium and method for inducing differentiation of pluripotent stem cells into hematopoietic precursor cells

技术领域technical field

本发明属于生物技术领域，涉及一种诱导多能干细胞向造血前体细胞分化的方法。The invention belongs to the field of biotechnology, and relates to a method for inducing differentiation of pluripotent stem cells to hematopoietic precursor cells.

背景技术Background technique

多能性干细胞，包括胚胎来源的胚胎干细胞和体外重编程诱导来源的诱导型多能干细胞。多能性干细胞能够在体外具有长期培养保持自我更新能力，并且具有多向分化的潜能，包括分化为几乎所有功能性的血液细胞。造血前体细胞是具有自我更新能力并能分化为各种血细胞的前体细胞，最终生成各种血细胞成分，包括红细胞、白细胞和血小板，它们也可以分化成各种其他细胞。在临床中，造血干/祖细胞以及各种成熟的血液细胞可服务于临床上的造血干细胞移植和血细胞输注治疗的重大战略需求。而如何获得足够多的造血干细胞一直以来是困扰研究人员和医务人员的难题。自2001年，Kaufman等首次实现将人胚胎干细胞分化为CD34+的造血干/祖细胞后[Kaufman D,Hanson E,Lewis etal.Hematopoietic colony-forming cells derived from human embryonic stemcells.Proc Natl Acad USA.2001；98(19):10716-21]，人们相继开展了人类多能干细胞体外分化为造血干细胞及相关的研究。虽然在这个领域已经有了一定的进展，从目前临床应用的角度来看，人多能干细胞体外造血分化的研究仍然面临着非常大挑战，例如人多能干细胞诱导分化而来的生血内皮前体细胞和造血干细胞和功能细胞数量不足以满足一次输入的需求；人多能干细胞诱导的分化而来的造血干细胞不具备体内移植的能力；体外培养的条件包含了血清、饲养层细胞等外源性物质，这些问题会大大的限制了目前采用人多能干细胞体外造血分化的临床应用。Pluripotent stem cells include embryonic stem cells derived from embryos and induced pluripotent stem cells derived from in vitro reprogramming induction. Pluripotent stem cells can maintain self-renewal ability in long-term culture in vitro, and have the potential of multidirectional differentiation, including differentiation into almost all functional blood cells. Hematopoietic progenitor cells are precursor cells that have the ability to self-renew and differentiate into various blood cells, eventually producing various blood cell components, including red blood cells, white blood cells, and platelets, which can also differentiate into various other cells. In the clinic, hematopoietic stem/progenitor cells and various mature blood cells can serve the important strategic needs of clinical hematopoietic stem cell transplantation and blood cell transfusion therapy. How to obtain enough hematopoietic stem cells has always been a difficult problem for researchers and medical staff. Since 2001, Kaufman et al. first realized the differentiation of human embryonic stem cells into CD34+ hematopoietic stem/progenitor cells [Kaufman D, Hanson E, Lewis et al. Hematopoietic colony-forming cells derived from human embryonic stemcells. Proc Natl Acad USA.2001; 98(19):10716-21], people have carried out in vitro differentiation of human pluripotent stem cells into hematopoietic stem cells and related research. Although some progress has been made in this field, from the perspective of current clinical application, the research on hematopoietic differentiation of human pluripotent stem cells in vitro still faces great challenges, such as the hematopoietic endothelial precursors induced by human pluripotent stem cells The number of cells, hematopoietic stem cells and functional cells is not enough to meet the needs of one input; the differentiated hematopoietic stem cells induced by human pluripotent stem cells do not have the ability to transplant in vivo; the conditions for in vitro culture include exogenous sources such as serum and feeder cells These problems will greatly limit the current clinical application of human pluripotent stem cells in vitro hematopoietic differentiation.

由于目前的这些方法培养条件较为复杂，分化周期相对较长，分化效率和产量较低，可操作性较差。基于以上现有分化技术中存在的问题，亟需寻找一种诱导血管/生血内皮前体细胞生成造血前体细胞的方法。Due to the complex culture conditions of these current methods, the differentiation cycle is relatively long, the differentiation efficiency and yield are low, and the operability is poor. Based on the above problems existing in existing differentiation techniques, it is urgent to find a method for inducing vascular/hematopoietic endothelial precursor cells to generate hematopoietic precursor cells.

发明内容Contents of the invention

本发明的目的是提供一种操作简单、高效的多能干细胞向造血前体诱导分化体系，其包括多能干细胞向生血内皮前体诱导分化体系，以及生血内皮前体细胞向造血前体细胞诱导分化体系。具体地，本发明提供了一种化学成分明确的多能干细胞向造血前体细胞分化的培养基，以及使用该培养基探索了一种利用随机回转仪模拟微重力效应来高效诱导多能干细胞向人造血前体细胞分化的方法；该方法与现有方法相比，具有较高的可操作性与可重复性，为将来大规模生产造血前体细胞用于生物医学和临床治疗奠定技术基础。The purpose of the present invention is to provide a simple and efficient system for inducing differentiation of pluripotent stem cells to hematopoietic precursors, which includes a system for inducing differentiation of pluripotent stem cells to hematopoietic endothelial precursors and a system for inducing hematopoietic endothelial precursors to hematopoietic precursors differentiation system. Specifically, the present invention provides a chemically defined medium for the differentiation of pluripotent stem cells into hematopoietic precursor cells, and uses the medium to explore a way to efficiently induce pluripotent stem cells to differentiate into hematopoietic precursor cells by using a random gyroscope to simulate microgravity effects. A method for differentiation of human hematopoietic precursor cells; compared with existing methods, this method has higher operability and repeatability, and lays a technical foundation for the large-scale production of hematopoietic precursor cells for biomedicine and clinical treatment in the future.

第一方面，本发明提供一种中胚层细胞诱导分化培养基，其包括基础分化培养基和骨形成蛋白4(BMP4)、活化素A(Activin A)以及GSK-3α/β抑制剂(CHIR99021)；In a first aspect, the present invention provides a medium for inducing differentiation of mesoderm cells, which includes basal differentiation medium and bone morphogenic protein 4 (BMP4), activin A (Activin A) and GSK-3α/β inhibitor (CHIR99021) ;

所述基础分化培养基为IF9S完全培养基，包括按1:0.8-1.2的体积比混合的IMDM培养基和F12培养基，以及终浓度为8-12mg/L的聚乙烯醇(PVA)、体积百分比为所述培养基的0.08-0.12％的Lipids 100X添加剂、体积百分比为所述IF9S完全培养基的1.8％-2.2％的ITS-X100X添加剂、终浓度为35-45μl/L的一硫代甘油(αMTG)、终浓度为55-70mg/L的抗坏血酸磷酸酯钠(AA2P)、体积百分比为所述IF9S完全培养基的0.8％-1.2％的谷丙氨酸二肽(GlutaMax^TM)100X添加剂以及体积百分比为所述IF9S完全培养基的0.8％-1.2％的非必需氨基酸添加剂100X(NEAA)。Described basal differentiation medium is the complete medium of IF9S, comprises the IMDM medium and the F12 medium that mix by the volume ratio of 1:0.8-1.2, and the polyvinyl alcohol (PVA) that final concentration is 8-12mg/L, volume The percentage is 0.08-0.12% of the Lipids 100X supplement of the medium, the volume percentage is the 1.8%-2.2% of the ITS-X100X supplement of the IF9S complete medium, and the monothioglycerol with a final concentration of 35-45 μl/L (αMTG), sodium ascorbyl phosphate (AA2P) with a final concentration of 55-70 mg/L, glutalanine dipeptide (GlutaMax^TM ) 100X supplement with a volume percentage of 0.8%-1.2% of the IF9S complete medium, and The volume percentage is 0.8%-1.2% non-essential amino acid supplement 100X (NEAA) of the IF9S complete medium.

优选地，所述IF9S完全培养基，包括按1:1的体积比混合的IMDM培养基和F12培养基，以及终浓度为10mg/L的聚乙烯醇(PVA)、体积百分比为所述培养基的0.1％的Lipids100X添加剂、体积百分比为所述IF9S完全培养基的2％的ITS-X 100X添加剂、终浓度为40μl/L的一硫代甘油(αMTG)、终浓度为64mg/L的抗坏血酸磷酸酯钠(AA2P)、体积百分比为所述IF9S完全培养基的1％的GlutaMax^TM 100X添加剂以及体积百分比为所述IF9S完全培养基的1％的非必需氨基酸添加剂100X(NEAA)。Preferably, the IF9S complete medium includes IMDM medium and F12 medium mixed in a volume ratio of 1:1, and polyvinyl alcohol (PVA) with a final concentration of 10 mg/L, the volume percentage of which is the medium The 0.1% Lipids100X additive, the volume percentage is the 2% ITS-X 100X additive of the IF9S complete medium, the monothioglycerol (αMTG) that the final concentration is 40 μ l/L, the ascorbyl phosphate that the final concentration is 64mg/L Sodium ester (AA2P), GlutaMax^™ 100X Supplement at 1% by volume of the IF9S complete medium, and Non-Essential Amino Acid Supplement 100X (NEAA) at 1% by volume of the IF9S complete medium.

优选地，所述中胚层细胞分化培养基为IF9S完全培养基添加40-60ng/mL的BMP4，15-25μg/mL的Activin A，0.8-1.2μM CHIR99021。Preferably, the mesoderm cell differentiation medium is IF9S complete medium supplemented with 40-60 ng/mL of BMP4, 15-25 μg/mL of Activin A, and 0.8-1.2 μM CHIR99021.

更优选地，所述中胚层细胞分化培养基为IF9S完全培养基添加50ng/mL的BMP4，20μg/mL的Activin A，1μM CHIR99021。More preferably, the mesoderm cell differentiation medium is IF9S complete medium supplemented with 50 ng/mL of BMP4, 20 μg/mL of Activin A, and 1 μM of CHIR99021.

第二方面，本发明提供了一种生血内皮前体细胞诱导分化培养基，其包括基础分化培养基、SB431542、VEGF、bFGF和SCF；In a second aspect, the present invention provides a hematopoietic endothelial precursor cell differentiation medium, which includes basal differentiation medium, SB431542, VEGF, bFGF and SCF;

所述基础分化培养基为IF9S完全培养基，包括按1:0.8-1.2的体积比混合的IMDM培养基和F12培养基，以及终浓度为8-12mg/L的聚乙烯醇(PVA)、体积百分比为所述培养基的0.08-0.12％的Lipids 100X添加剂、体积百分比为所述IF9S完全培养基的1.8％-2.2％的ITS-X100X添加剂、终浓度为35-45μl/L的一硫代甘油(αMTG)、终浓度为55-70mg/L的抗坏血酸磷酸酯钠(AA2P)、体积百分比为所述IF9S完全培养基的0.8％-1.2％的GlutaMax^TM100X添加剂以及体积百分比为所述IF9S完全培养基的0.8％-1.2％的非必需氨基酸添加剂100X(NEAA)。Described basal differentiation medium is the complete medium of IF9S, comprises the IMDM medium and the F12 medium that mix by the volume ratio of 1:0.8-1.2, and the polyvinyl alcohol (PVA) that final concentration is 8-12mg/L, volume The percentage is 0.08-0.12% of the Lipids 100X supplement of the medium, the volume percentage is the 1.8%-2.2% of the ITS-X100X supplement of the IF9S complete medium, and the monothioglycerol with a final concentration of 35-45 μl/L (αMTG), sodium ascorbyl phosphate (AA2P) at a final concentration of 55-70 mg/L, GlutaMax^™ 100X Supplement at a volume percentage of 0.8%-1.2% of the IF9S complete medium, and a volume percentage of the IF9S complete culture 0.8%-1.2% of the base non-essential amino acid additive 100X (NEAA).

优选地，所述IF9S完全培养基，包括按1:1的体积比混合的IMDM培养基和F12培养基，以及终浓度为10mg/L的聚乙烯醇(PVA)、体积百分比为所述培养基的0.1％的Lipids100X添加剂、体积百分比为所述IF9S完全培养基的2％的ITS-X 100X添加剂、终浓度为40μl/L的一硫代甘油(αMTG)、终浓度为64mg/L的抗坏血酸磷酸酯钠(AA2P)、体积百分比为所述IF9S完全培养基的1％的GlutaMax^TM 100X添加剂以及体积百分比为所述IF9S完全培养基的1％的非必需氨基酸添加剂100X(NEAA)。Preferably, the IF9S complete medium includes IMDM medium and F12 medium mixed in a volume ratio of 1:1, and polyvinyl alcohol (PVA) with a final concentration of 10 mg/L, the volume percentage of which is the medium The 0.1% Lipids100X additive, the volume percentage is the 2% ITS-X 100X additive of the IF9S complete medium, the monothioglycerol (αMTG) that the final concentration is 40 μ l/L, the ascorbyl phosphate that the final concentration is 64mg/L Sodium ester (AA2P), GlutaMax^™ 100X Supplement at 1% by volume of the IF9S complete medium, and Non-Essential Amino Acid Supplement 100X (NEAA) at 1% by volume of the IF9S complete medium.

优选地，所述生血内皮前体分化培养基为IF9S完全培养基添加8-12μM SB431542、40-60ng/mL的VEGF、40-60ng/mL的bFGF和40-60ng/mL的SCF；Preferably, the hematopoietic endothelial precursor differentiation medium is IF9S complete medium supplemented with 8-12 μM SB431542, VEGF of 40-60 ng/mL, bFGF of 40-60 ng/mL and SCF of 40-60 ng/mL;

更优选地，所述生血内皮前体分化培养基为IF9S完全培养基添加10μM SB431542、50ng/mL的VEGF、50ng/mL的bFGF和50ng/mL的SCF。More preferably, the hematopoietic endothelial precursor differentiation medium is IF9S complete medium supplemented with 10 μM SB431542, 50 ng/mL of VEGF, 50 ng/mL of bFGF and 50 ng/mL of SCF.

优选地，在配制IF9S完全培养基前，使用去离子水溶解聚乙烯醇(PVA)，并配成5％浓度的储液。Preferably, before preparing the IF9S complete medium, polyvinyl alcohol (PVA) is dissolved in deionized water and prepared as a stock solution with a concentration of 5%.

优选地，在配制IF9S完全培养基前，使用IMDM培养基将一硫代甘油按2％进行稀释，配成了500X的储液。Preferably, before preparing the IF9S complete medium, use IMDM medium to dilute monothioglycerol by 2%, and prepare a 500X stock solution.

优选地，在配制IF9S完全培养基前，使用F12培养基先将AA2P溶解，并配成5mg/mL的储液。Preferably, before preparing the IF9S complete medium, use the F12 medium to dissolve AA2P first, and prepare a 5 mg/mL stock solution.

其中，所述IMDM培养基和F12基础基为常规培养基，可以购买商业化的培养基，也可以自行配制，在本发明的一个优选的实施例中，所述IMDM培养基和F12培养基均购自Gibco公司。Wherein, the IMDM medium and the F12 basal base are conventional mediums, commercial mediums can be purchased, or self-prepared. In a preferred embodiment of the present invention, both the IMDM medium and the F12 medium are Purchased from Gibco Company.

所述Lipids(100X)为化学成分确定的脂质浓缩物，可用于减少或替代细胞培养基中的胎牛血清。在本发明的一个优选的实施例中，所述Lipids添加剂购自Gibco公司，货号为11905031。The Lipids (100X) are chemically defined lipid concentrates that can be used to reduce or replace fetal bovine serum in cell culture media. In a preferred embodiment of the present invention, the Lipids additive is purchased from Gibco Company, the article number is 11905031.

所述ITS-X(100X)添加剂为胰岛素-转铁蛋白-硒-乙醇胺的基础培养基补充物，用于减少或替代细胞培养基中的胎牛血清。在本发明的一个优选的实施例中，所述ITS-X添加剂购自Gibico公司，货号为51500056。The ITS-X (100X) additive is a basal medium supplement of insulin-transferrin-selenium-ethanolamine, which is used to reduce or replace fetal bovine serum in the cell culture medium. In a preferred embodiment of the present invention, the ITS-X additive is purchased from Gibico Company, the product number is 51500056.

所述GlutaMax^TM(100X)添加剂为谷丙氨酸二肽，是一种细胞培养添加剂，可直接替代细胞培养基中的L-谷氨酰胺。在本发明的一个优选的实施例中，所述GlutaMax添加剂购自Gibico公司，货号为35050-061。The GlutaMax^™ (100X) additive is glutalanine dipeptide, which is a cell culture additive and can directly replace L-glutamine in the cell culture medium. In a preferred embodiment of the present invention, the GlutaMax additive is purchased from Gibico Company, the product number is 35050-061.

所述NEAA(100X)添加剂为非必需氨基酸，是一种细胞培养添加剂。在本发明的一个优选的实施例中，所述NEAA添加剂购自Gibico公司，货号为35050-061。The NEAA (100X) additive is a non-essential amino acid, which is a cell culture additive. In a preferred embodiment of the present invention, the NEAA additive is purchased from Gibico Company, the product number is 35050-061.

第三方面，本发明提供了一种生血内皮前体细胞诱导分化组合培养基，其包括上述中胚层细胞诱导分化培养基和上述生血内皮前体细胞诱导分化培养基。In a third aspect, the present invention provides a combined medium for inducing differentiation of hematopoietic endothelial precursor cells, which includes the above-mentioned medium for inducing differentiation of mesoderm cells and the above-mentioned medium for inducing differentiation of hematopoietic endothelial precursor cells.

优选地，中胚层细胞诱导分化培养基和生血内皮前体细胞诱导分化培养基分开放置或配制。Preferably, the medium for inducing differentiation of mesoderm cells and the medium for inducing differentiation of hematopoietic endothelial precursor cells are placed or prepared separately.

第四方面，本发明提供一种诱导生血内皮前体细胞向造血前体细胞分化的培养基，其包括基础分化培养基和血管内皮生长因子(VEGF)、碱性成纤维细胞生长因子(bFGF)、干细胞因子(SCF)、白介素3(IL-3)、白介素6(IL-6)以及血小板生成素(TPO)；In a fourth aspect, the present invention provides a medium for inducing the differentiation of hematopoietic endothelial precursor cells to hematopoietic precursor cells, which includes basal differentiation medium and vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF) , stem cell factor (SCF), interleukin 3 (IL-3), interleukin 6 (IL-6) and thrombopoietin (TPO);

所述基础分化培养基为IF9S完全培养基，包括按1:0.8-1.2的体积比混合的IMDM基础培养基和F12基础培养基，以及终浓度为:8-12mg/L的聚乙烯醇(PVA)、体积百分比为所述培养基的0.08-0.12％的Lipids(100X)添加剂、体积百分比为所述培养基的1.8％-2.2％的ITS-X(100X)添加剂、终浓度为35-45μl/L的一硫代甘油(αMTG)、终浓度为55-70mg/L的AA2P、体积百分比为所述培养基的:0.8-1.2％的GlutaMax^TM(100X)添加剂以及体积百分比为所述培养基的0.8-1.2％的非必需氨基酸添加剂(NEAA，100X)。Described basal differentiation medium is the complete medium of IF9S, comprises the IMDM basal medium and the F12 basal medium mixed by the volume ratio of 1:0.8-1.2, and final concentration is: polyvinyl alcohol (PVA) of 8-12mg/L ), the volume percentage is the 0.08-0.12% Lipids (100X) additive of the described culture medium, the volume percentage is the 1.8%-2.2% ITS-X (100X) additive of the described culture medium, and the final concentration is 35-45 μ l/ L of monothioglycerol (αMTG), AA2P at a final concentration of 55-70 mg/L, percent by volume of the medium: 0.8-1.2% GlutaMax^™ (100X) supplement and percent by volume of the medium 0.8-1.2% non-essential amino acid supplement (NEAA, 100X).

优选地，所述基础分化培养基为IF9S完全培养基，包括按1:1的体积比混合的IMDM基础培养基和F12基础培养基，以及终浓度为10mg/L的聚乙烯醇(PVA)、体积百分比为所述培养基的0.1％的Lipids(100X)添加剂、体积百分比为所述培养基的2％的ITS-X(100X)添加剂、终浓度为40μl/L的一硫代甘油(αMTG)、终浓度为64mg/L的AA2P、体积百分比为所述培养基的1％的GlutaMax^TM(100X)添加剂以及体积百分比为所述培养基的1％的非必需氨基酸添加剂(NEAA，100X)。Preferably, the basal differentiation medium is IF9S complete medium, including IMDM basal medium and F12 basal medium mixed in a volume ratio of 1:1, and polyvinyl alcohol (PVA) with a final concentration of 10 mg/L, Volume percentage is the 0.1% Lipids (100X) supplement of described medium, the volume percentage is the 2% ITS-X (100X) supplement of described medium, the monothioglycerol (αMTG) that final concentration is 40 μ l/L , AA2P at a final concentration of 64 mg/L, GlutaMax^™ (100X) supplement of 1% by volume of the medium, and non-essential amino acid supplement (NEAA, 100X) of 1% by volume of the medium.

优选地，所述造血前体细胞分化培养基为IF9S完全培养基添加40-60ng/mL的VEGF，15-25ng/mL的bFGF，40-60ng/mL的SCF，8-12ng/mL的IL-3，40-60ng/mL的IL-6以及15-25ng/mL TPO。Preferably, the hematopoietic precursor cell differentiation medium is IF9S complete medium supplemented with 40-60 ng/mL of VEGF, 15-25 ng/mL of bFGF, 40-60 ng/mL of SCF, 8-12 ng/mL of IL- 3. 40-60ng/mL IL-6 and 15-25ng/mL TPO.

优选地，所述造血前体细胞分化培养基为IF9S完全培养基添加50ng/mL的VEGF，20ng/mL的bFGF，50ng/mL的SCF，10ng/mL的IL-3，50ng/mL的IL-6以及20ng/mL TPO。Preferably, the hematopoietic precursor cell differentiation medium is IF9S complete medium supplemented with 50ng/mL of VEGF, 20ng/mL of bFGF, 50ng/mL of SCF, 10ng/mL of IL-3, 50ng/mL of IL- 6 and 20ng/mL TPO.

优选地，在配制IF9S完全培养基前，使用去离子水溶解聚乙烯醇(PVA),并配成5％浓度的储液。Preferably, before preparing the IF9S complete medium, polyvinyl alcohol (PVA) is dissolved in deionized water and prepared as a stock solution with a concentration of 5%.

优选地，在配制IF9S完全培养基前，使用IMDM培养基将一硫代甘油按2％进行稀释，配成了500X的储液。Preferably, before preparing the IF9S complete medium, use IMDM medium to dilute monothioglycerol by 2%, and prepare a 500X stock solution.

优选地，在配制IF9S完全培养基前，使用F12培养基先将AA2P溶解，并配成5mg/mL的储液。Preferably, before preparing the IF9S complete medium, use the F12 medium to dissolve AA2P first, and prepare a 5 mg/mL stock solution.

其中，所述IMDM基础培养基和F12基础培养基为常规培养基，可以购买商业化的培养基，也可以自行配制，在本发明的一个优选的实施例中，所述IMDM和F12培养基均购自Gibco公司。Wherein, the IMDM basal medium and the F12 basal medium are conventional mediums, commercialized mediums can be purchased, or self-prepared. In a preferred embodiment of the present invention, both the IMDM and F12 mediums are Purchased from Gibco Company.

所述Lipids(100X)为化学成分确定的脂质浓缩物，可用于减少或替代细胞培养基中的胎牛血清。在本申请的一个优选的实施例中，所述Lipids添加剂购自Gibco公司，货号为11905031。The Lipids (100X) are chemically defined lipid concentrates that can be used to reduce or replace fetal bovine serum in cell culture media. In a preferred embodiment of the present application, the Lipids additive is purchased from Gibco Company, the product number is 11905031.

所述ITS-X(100X)添加剂为胰岛素-转铁蛋白-硒-乙醇胺的基础培养基补充物，用于减少或替代细胞培养基中的胎牛血清。在本发明的一个优选的实施例中，所述ITS-X添加剂购自Gibico公司，货号为51500056。The ITS-X (100X) additive is a basal medium supplement of insulin-transferrin-selenium-ethanolamine, which is used to reduce or replace fetal bovine serum in the cell culture medium. In a preferred embodiment of the present invention, the ITS-X additive is purchased from Gibico Company, the product number is 51500056.

所述GlutaMax^TM(100X)添加剂为谷丙氨酸二肽，是一种细胞培养添加剂，可直接替代细胞培养基中的L-谷氨酰胺。在本发明的一个优选的实施例中，所述GlutaMax添加剂购自Gibico公司，货号为35050-061。The GlutaMax^™ (100X) additive is glutalanine dipeptide, which is a cell culture additive and can directly replace L-glutamine in the cell culture medium. In a preferred embodiment of the present invention, the GlutaMax additive is purchased from Gibico Company, the product number is 35050-061.

所述NEAA(100X)添加剂为非必需氨基酸，是一种高级细胞培养添加剂。在本发明的一个优选的实施例中，所述NEAA添加剂购自Gibico公司，货号为35050-061。The NEAA (100X) additive is a non-essential amino acid, which is a high-grade cell culture additive. In a preferred embodiment of the present invention, the NEAA additive is purchased from Gibico Company, the product number is 35050-061.

第五方面，本发明提供一种诱导多能干细胞向造血前体细胞分化的培养基组合物，所述培养基组合物中包含：In a fifth aspect, the present invention provides a medium composition for inducing differentiation of pluripotent stem cells into hematopoietic precursor cells, the medium composition comprising:

1)上述中胚层细胞诱导分化培养基；1) The above-mentioned medium for inducing differentiation of mesoderm cells;

2)上述生血内皮前体细胞诱导分化培养基；2) The above-mentioned differentiation medium for inducing hematopoietic endothelial precursor cells;

3)上述诱导生血内皮前体细胞向造血前体细胞分化的培养基。3) The above medium for inducing the differentiation of hematopoietic endothelial precursor cells into hematopoietic precursor cells.

优选地，中胚层细胞诱导分化培养基、生血内皮前体细胞诱导分化培养基分开和诱导生血内皮前体细胞向造血前体细胞分化的培养基放置或配制。Preferably, the medium for inducing differentiation of mesoderm cells, the medium for inducing differentiation of hematopoietic endothelial precursor cells, and the medium for inducing the differentiation of hematopoietic endothelial precursor cells into hematopoietic precursor cells are placed or prepared.

第六方面，本发明提供一种促进中胚层细胞向生血内皮前体细胞转化的方法，所述方法包括在中胚层细胞培养过程中，采用随机三维回转仪将细胞培养容器在旋转条件下进行细胞培养的步骤。In the sixth aspect, the present invention provides a method for promoting the transformation of mesoderm cells into hematopoietic endothelial precursor cells, the method comprising: during the culture process of mesoderm cells, using a random three-dimensional gyroscope to rotate the cell culture container under rotating conditions. Cultivation steps.

优选地，所述旋转条件下进行培养为以随机三维回转仪将细胞培养容器进行随机旋转，更优选地，随机旋转为0.1-10rpm/分钟的转速；更优选地，旋转速度为5rpm/分钟。Preferably, the cultivation under the rotation condition is to randomly rotate the cell culture container with a random three-dimensional gyroscope, more preferably, the random rotation is 0.1-10 rpm/min; more preferably, the rotation speed is 5 rpm/min.

优选地，所述旋转条件下进行培养的培养时间为48-96小时，优选为60-72小时。Preferably, the cultivation time under the rotation condition is 48-96 hours, preferably 60-72 hours.

优选地，所述旋转条件下进行培养的培养基采用上述生血内皮前体诱导分化培养基。Preferably, the above-mentioned medium for inducing differentiation of hematopoietic endothelial precursors is used as the medium for culturing under the rotation condition.

第七方面，本发明还提供了一种从人多能干细胞分化产生生血内皮前体细胞的方法，所述的方法包括如下步骤：In the seventh aspect, the present invention also provides a method for differentiating human pluripotent stem cells into hematopoietic endothelial precursor cells, the method comprising the following steps:

S1)人多能干细胞向中胚层细胞诱导分化培养:S1) Human pluripotent stem cells are induced to differentiate into mesoderm cells:

S11).细胞容器预包被生长因子减少的基底膜基质(Matrigel基质)；S11). The cell container is pre-coated with growth factor-reduced basement membrane matrix (Matrigel matrix);

S12).采用消化酶将人多能干细胞克隆进行消化、离心处理；S12). Using digestive enzymes to digest and centrifuge the human pluripotent stem cell clones;

S13).将消化、离心处理后的人多能干细胞克隆小团块接种于S11)获得预包被生长因子减少Matrigel的细胞容器中，添加TeSR^TM-E8^TM培养基，静止培养24～48小时；S13). Inoculate the digested and centrifuged human pluripotent stem cell clone pellets into the cell container obtained from S11) with pre-coated growth factor-reduced Matrigel, add TeSR^TM -E8^TM medium, and culture statically for 24 to 48 hours ;

S14)将培养基更换成上述中胚层细胞诱导分化培养基，静止培养24～48小时，获得诱导的中胚层细胞；S14) replacing the medium with the above-mentioned mesoderm cell induction differentiation medium, and statically culturing for 24 to 48 hours to obtain induced mesoderm cells;

S2)诱导的中胚层细胞进一步向生血内皮前体细胞分化培养:S2) The induced mesoderm cells are further differentiated into hematopoietic endothelial precursor cells:

S21).将中胚层细胞的细胞培养基更换为上述生血内皮前体细胞诱导分化培养基；S21). The cell culture medium of mesoderm cells is replaced with the above-mentioned medium for inducing differentiation of hematopoietic endothelial precursor cells;

S22).将细胞培养容器安装到随机三维回转仪上，设定旋转的方式为随机旋转，转速调整为0.1-10rpm/分钟的转速；S22). Install the cell culture container on the random three-dimensional gyroscope, set the rotation method as random rotation, and adjust the rotation speed to 0.1-10rpm/min;

S23).将随机三维回转仪放入37℃、5％CO₂孵箱中旋转培养60-96小时。S23). Put the random three-dimensional gyroscope into a 37° C., 5% CO₂ incubator for 60-96 hours to rotate and cultivate.

优选的，S11)中其中所述的生长因子减少的Matirgel基质为Corning公司生产的Growth Factor Reduced Matrigel，货号为354230。Preferably, the growth factor-reduced Matrigel matrix described in S11) is Growth Factor Reduced Matrigel produced by Corning Company, the product number is 354230.

优选地，S13)中接种的密度为50-200个克隆团块/cm²。Preferably, the seeding density in S13) is 50-200 clonal agglomerates/cm² .

再优选地，S13)中所述的接种的密度为100个克隆团块/cm²；。Still preferably, the seeding density described in S13) is 100 clonal agglomerates/cm² ;.

优选地，S21)中所述的生血内皮前体细胞诱导分化培养基加至培养容器的95％-100％；Preferably, the hematopoietic endothelial precursor cell differentiation medium described in S21) is added to 95%-100% of the culture vessel;

优选地，S22)中旋转速度为5rpm/分钟；Preferably, in S22), the rotational speed is 5rpm/min;

优选地，S22)中培养72小时。Preferably, cultured in S22) for 72 hours.

优选地，S12)中，人多能干细胞克隆进行消化前进行扩增培养：Preferably, in S12), human pluripotent stem cell clones are subjected to expansion culture before digestion:

S121)采用胚胎干细胞级别的细胞外基质Matrigel对培养细胞容器进行包被；S121) using embryonic stem cell-level extracellular matrix Matrigel to coat the culture cell container;

S122)将人多能干细胞接种于包被后的细胞容器中并使用TeSR^TM-E8^TM培养基进行培养。S122) Inoculate the human pluripotent stem cells into the coated cell container and use TeSR^™ -E8^™ medium for culturing.

优选地，S122)中每24小时更换一次新鲜培养基，当细胞克隆生长至70～80％密度时，对细胞进行消化传代或之后的诱导分化培养；细胞传代的周期为3-4天。Preferably, in S122), the fresh medium is replaced every 24 hours. When the cell clone grows to a density of 70-80%, the cells are digested and subcultured or subsequently differentiated and cultured; the cycle of cell subculture is 3-4 days.

优选地，所述胚胎干细胞级别的细胞外基质为BioCoat公司生产Matrigel，货号为354277。Preferably, the embryonic stem cell-grade extracellular matrix is Matrigel produced by BioCoat Company, the product number is 354277.

第八方面，本发明还提供了一种促进生血内皮前体细胞向造血前体细胞分化的方法，所述的方法包括在生血内皮前体细胞培养过程中，采用随机回转仪将细胞培养容器在旋转条件下进行培养的步骤。In the eighth aspect, the present invention also provides a method for promoting the differentiation of hematopoietic endothelial precursor cells into hematopoietic precursor cells, the method includes using a random gyrator to place the cell culture container in the The step of culturing under rotation conditions.

优选地，所述旋转条件下进行培养为以随机回转仪将细胞培养容器随机旋转，更优选地，随机旋转内外径均为0.1-10rpm/分钟的转速；更优选地，旋转速度为内外径为5rpm/分钟。Preferably, the cultivation under the rotating condition is to randomly rotate the cell culture container with a random gyroscope, more preferably, the random rotation speed is 0.1-10rpm/min for the inner and outer diameters; more preferably, the rotation speed is 0.1-10rpm/min for the inner and outer diameter 5rpm/min.

优选地，所述旋转条件下进行培养的培养时间为48-72小时，优选为72小时。Preferably, the culture time for culturing under the rotation condition is 48-72 hours, preferably 72 hours.

优选地，所述旋转条件下进行培养的培养基采用上述的诱导血管/生血内皮前体细胞向造血前体细胞分化的培养基。Preferably, the medium for culturing under the rotation condition is the above-mentioned medium for inducing the differentiation of vascular/hematopoietic endothelial precursor cells into hematopoietic precursor cells.

优选地，其包含如下步骤：Preferably, it comprises the steps of:

S3)在原始培养基中培养72小时后，更换细胞培养基为上述的诱导生血内皮前体细胞向造血前体细胞分化的培养基；S3) After culturing in the original medium for 72 hours, replace the cell medium with the above-mentioned medium for inducing the differentiation of hematopoietic endothelial precursor cells into hematopoietic precursor cells;

S4)将细胞培养瓶重新安装到随机回转仪上于继续进行回转培养；S4) Reinstall the cell culture bottle on the random gyrator to continue the rotary culture;

优选地，S3)中所述诱导生血内皮前体细胞向造血前体细胞分化的培养基加至培养容器的95％-100％；Preferably, the medium for inducing the differentiation of hematopoietic endothelial precursor cells into hematopoietic precursor cells described in S3) is added to 95%-100% of the culture container;

优选地，S4)中，细胞培养48-72小时，优选为细胞培养72小时。Preferably, in S4), the cells are cultured for 48-72 hours, preferably the cells are cultured for 72 hours.

优选地，S4)中，细胞培养条件为37℃、5％CO₂。Preferably, in S4), the cell culture conditions are 37°C, 5% CO₂ .

第九方面，本发明还提供了一种从人多能干细胞分化产生造血前体细胞方法，所述方法包括以下步骤：In the ninth aspect, the present invention also provides a method for differentiating human pluripotent stem cells to produce hematopoietic precursor cells, the method comprising the following steps:

1)采用上述从人多能干细胞分化产生生血内皮前体细胞的方法，制备获得生血内皮前体细胞；1) Prepare and obtain hematopoietic endothelial precursor cells by adopting the method for producing hematopoietic endothelial precursor cells from human pluripotent stem cells;

2)采用上述促进生血内皮前体细胞向造血前体细胞分化的方法。2) Using the above-mentioned method for promoting the differentiation of hematopoietic endothelial precursor cells into hematopoietic precursor cells.

第十方面，本发明提供随机三维回转仪在用于促进中胚层细胞向生血内皮前体细胞转化中的用途，或者促进生血内皮前体细胞向造血前体细胞转化的用途。In a tenth aspect, the present invention provides the use of a random three-dimensional gyroscope for promoting the transformation of mesoderm cells into hematopoietic endothelial precursor cells, or the use of promoting the transformation of hematopoietic endothelial precursor cells into hematopoietic precursor cells.

本发明选择了化学成分明确的培养基用于人多能干细胞的培养和分化，本发明还描述了利用一种随机回转器来模拟微重力效应开展多能干细胞向生血内皮前体细胞和造血前体细胞分化技术。总之与现有技术相比，本发明具有以下优点：The present invention selects a culture medium with defined chemical composition for the cultivation and differentiation of human pluripotent stem cells. The present invention also describes the use of a random gyrator to simulate microgravity effects to develop pluripotent stem cells into hematopoietic endothelial precursor cells and prehematopoietic stem cells. Somatic cell differentiation techniques. Compared with the prior art in a word, the present invention has the following advantages:

本培养体系从多能干细胞培养扩增开始到中内胚层分化，生血内皮前体分化以及造血前体细胞都是在无血清、无饲养层且化学成分明确的条件下培养。而目前报道的人多能干细胞向造血前体/干细胞体外分化的方法主要包括需要和小鼠骨髓基质细胞(OP9细胞系)或者小鼠主动脉性腺中肾基质细胞(mAGM细胞系)共培养的体系，以及采用拟胚体(Embryoid Body，EB)分化的体系。共培养体系由于含有动物源性物质，增加了实验的不稳定性和安全性，加大了实验步骤的繁琐；而EB分化体系，其分化效率低。本发明使用的化学成分明确的培养体系和随机三维回转模拟微重力的培养方法，可在短时间内获得大量的多能干细胞来源的生血内皮前体细胞以及造血前体细胞，并且分化效率很高，分化效率较静止培养提高2倍以上。这种低成本、安全可控的无血清、无饲养层细胞的随机三维回转培养策略可为将来大规模生产用于临床治疗的生血内皮前体细胞或造血前体细胞奠定了基础。In this culture system, from the expansion of pluripotent stem cells to mesendoderm differentiation, hematopoietic endothelial precursor differentiation and hematopoietic precursor cells are all cultured under the conditions of no serum, no feeder layer and clear chemical composition. The currently reported methods for in vitro differentiation of human pluripotent stem cells into hematopoietic precursor/stem cells mainly include co-cultivation with mouse bone marrow stromal cells (OP9 cell line) or mouse aortic mesenchymal stromal cells (mAGM cell line). system, and a system using embryoid body (Embryoid Body, EB) differentiation. The co-culture system contains animal-derived substances, which increases the instability and safety of the experiment, and increases the complexity of the experimental steps; while the EB differentiation system has low differentiation efficiency. The culture system with defined chemical composition and the culture method of random three-dimensional rotation simulating microgravity used in the present invention can obtain a large number of hematopoietic endothelial precursor cells and hematopoietic precursor cells derived from pluripotent stem cells in a short time, and the differentiation efficiency is very high , The differentiation efficiency is more than 2 times higher than that of static culture. This low-cost, safe and controllable random three-dimensional rotation culture strategy of serum-free and feeder-free cells can lay the foundation for the large-scale production of hematopoietic endothelial precursor cells or hematopoietic precursor cells for clinical treatment in the future.

附图说明Description of drawings

图1是采用本发明方法从人多能干细胞诱导来源的生血内皮前体细胞明场图片。Fig. 1 is a bright field picture of hematopoietic endothelial precursor cells derived from human pluripotent stem cells induced by the method of the present invention.

图2是采用本发明方法从人多能干细胞诱导来源的生血内皮前体细胞进行生血内皮前体细胞标志分子CD31和CD34的荧光染色图片。Fig. 2 is a picture of fluorescent staining of hematopoietic endothelial precursor cell marker molecules CD31 and CD34 from hematopoietic endothelial precursor cells induced by the method of the present invention.

图3是采用本发明随机三维回转培养与传统静止培养下生血内皮前体细胞分化进行流式细胞分析，比较分化后细胞表达CD31+CD34+的效率。其中A图为对照组，B图为随机三维回转组。Fig. 3 is a flow cytometric analysis of hematopoietic endothelial precursor cells differentiated under random three-dimensional rotary culture of the present invention and traditional static culture, and compares the efficiency of expressing CD31+CD34+ cells after differentiation. Among them, picture A is the control group, and picture B is the random three-dimensional rotation group.

图4是采用本发明方法从人多能干细胞诱导来源的造血前体细胞明场图片。Fig. 4 is a bright field picture of hematopoietic precursor cells derived from human pluripotent stem cells induced by the method of the present invention.

图5是采用本发明方法从人多能干细胞诱导来源的造血前体细胞进行造血前体细胞标志分子CD34和CD43的荧光染色图片。Fig. 5 is a picture of fluorescent staining of hematopoietic precursor cell marker molecules CD34 and CD43 from hematopoietic precursor cells induced from human pluripotent stem cells by the method of the present invention.

图6是采用本发明随机回转培养与传统静止培养下造血前体细胞分化进行流式细胞分析，比较分化后细胞表达CD34+CD43+的效率。其中A图为对照组，B图为随机回转组。Fig. 6 is a flow cytometric analysis of the differentiation of hematopoietic precursor cells under the random rotation culture of the present invention and the traditional static culture, and compares the efficiency of expressing CD34+CD43+ cells after differentiation. Among them, picture A is the control group, and picture B is the random rotation group.

具体实施方式Detailed ways

现结合实施例，对本发明做进一步阐述，但本发明的实施并不仅限于此。Now in conjunction with the examples, the present invention will be further described, but the implementation of the present invention is not limited thereto.

除非特别指明，以下实施例中所用的试剂均为分析纯级试剂，且可从正规渠道商购获得。Unless otherwise specified, the reagents used in the following examples are of analytical grade and commercially available from formal channels.

除非特别说明，本发明使用的人多能干细胞为人胚胎干细胞系H1。Unless otherwise specified, the human pluripotent stem cells used in the present invention are human embryonic stem cell line H1.

除非特别指明，以下实施例中所用的试剂均为分析纯级试剂，且可从正规渠道商购获得。Unless otherwise specified, the reagents used in the following examples are of analytical grade and commercially available from formal channels.

实施例1：无血清培养体系培养基的制备Embodiment 1: Preparation of serum-free culture system medium

(1)IF9S培养基的配制：(1) Preparation of IF9S medium:

本申请使用IMDM培养基、F12培养基、聚乙烯醇(PVA)、Lipids(100X)、ITS-X(100X)、一硫代甘油(αMTG)、AA2P、GlutaMax^TM(100X)、非必需氨基酸(NEAA，100X)来配制IF9S培养基。This application uses IMDM medium, F12 medium, polyvinyl alcohol (PVA), Lipids (100X), ITS-X (100X), monothioglycerol (αMTG), AA2P, GlutaMax^TM (100X), non-essential amino acids ( NEAA, 100X) to prepare IF9S medium.

其中，培养基的各组分分别购自：Wherein, each component of culture medium was purchased from:

IMDM培养基购自Gibco公司，货号为21056-023；IMDM medium was purchased from Gibco Company, the article number is 21056-023;

F12培养基购自Gibco公司，货号为31765-027；F12 medium was purchased from Gibco, the product number is 31765-027;

聚乙烯醇(PVA)购自Sigma公司，货号为P8136；Polyvinyl alcohol (PVA) is purchased from Sigma company, and article number is P8136;

Lipids(100X)购自Gibco公司，货号为11905031；Lipids (100X) is purchased from Gibco company, and article number is 11905031;

ITS-X(100X)购自Gibco公司，货号为51500-056；ITS-X (100X) was purchased from Gibco Company, the article number is 51500-056;

一硫代甘油(αMTG)购自Sigma公司，货号为M6145；Monothioglycerol (αMTG) was purchased from Sigma Company, the product number is M6145;

AA2P购自Sigma公司，货号为A8960；AA2P was purchased from Sigma Company, the article number is A8960;

GlutaMax^TM(100X)购自Gibco公司，货号为35050-061；GlutaMax^™ (100X) was purchased from Gibco Company, the article number is 35050-061;

非必需氨基酸(NEAA，100X)购自Gibco公司，货号为11140-035；Non-essential amino acids (NEAA, 100X) were purchased from Gibco, the product number is 11140-035;

配制所述的培养基。其中聚乙烯醇(PVA)为粉末，使用前需用去离子水溶解并配成5％浓度的储液。Prepare the culture medium. Wherein polyvinyl alcohol (PVA) is powder, needs to dissolve with deionized water and be made into the stock solution of 5% concentration before use.

具体地，所述IF9S培养基通过以下方法配制：Specifically, the IF9S medium is prepared by the following method:

首先将向IMDM培养基和F12培养基培养基按1:1混合，搅拌均匀后，在混合后的培养基中添加以下试剂，各试剂的最终浓度为：10mg/L的聚乙烯醇(PVA)、体积百分比为0.1％的Lipids(100X)添加剂、体积百分比为2％的ITS-X(100X)添加剂、40μl/L的一硫代甘油(αMTG)、64mg/L的AA2P、体积百分比为1％的GlutaMax^TM(100X)添加剂以及体积百分比为1％的非必需氨基酸添加剂(NEAA，100X)。First, mix the IMDM medium and the F12 medium medium by 1:1, and after stirring evenly, add the following reagents to the mixed medium, the final concentration of each reagent is: polyvinyl alcohol (PVA) of 10mg/L , 0.1% by volume of Lipids (100X) additive, 2% by volume of ITS-X (100X) additive, 40 μl/L of monothioglycerol (αMTG), 64 mg/L of AA2P, 1% by volume GlutaMax^TM (100X) supplement and 1% by volume non-essential amino acid supplement (NEAA, 100X).

各种试剂在添加无严格的顺序，在常温操作即可，配制的过程无需加热，但培养基配制后需用0.22μm的滤器进行无菌过滤，放置4℃冰箱保存。There is no strict order for the addition of various reagents, and it can be operated at room temperature. The preparation process does not require heating, but after the preparation of the medium, it needs to be sterile filtered with a 0.22 μm filter and stored in a 4°C refrigerator.

(2)中胚层细胞分化培养基配制(2) Preparation of mesoderm cell differentiation medium

所述中胚层细胞分化培养基为IF9S培养基添加50ng/mL的BMP4，20μg/mL的Activin A，1μM CHIR99021。The mesoderm cell differentiation medium is IF9S medium supplemented with 50 ng/mL of BMP4, 20 μg/mL of Activin A, and 1 μM of CHIR99021.

各种因子在添加无严格的顺序，整个过程需在超净工作台中无菌条件下进行。The various factors are added in no strict order, and the whole process needs to be carried out under sterile conditions in an ultra-clean workbench.

(3)生血内皮前体分化培养基配制(3) Preparation of Hematopoietic Endothelial Precursor Differentiation Medium

所述生血内皮前体分化培养基为IF9S培养基添加10μM的SB431542，50ng/mL的VEGF，50ng/mL的bFGF，50ng/mL的SCF。The hematopoietic endothelial precursor differentiation medium is IF9S medium supplemented with 10 μM SB431542, 50 ng/mL VEGF, 50 ng/mL bFGF, and 50 ng/mL SCF.

各种因子在添加无严格的顺序，整个过程需在超净工作台中无菌条件下进行。The various factors are added in no strict order, and the whole process needs to be carried out under sterile conditions in an ultra-clean workbench.

(4)造血前体细胞分化培养基配制(4) Preparation of Hematopoietic Precursor Cell Differentiation Medium

所述造血前体/干细胞分化培养基为IF9S完全培养基添加50ng/mL的VEGF，20ng/mL的bFGF，50ng/mL的SCF，10ng/mL的IL-3，50ng/mL的IL-6以及20ng/mL TPO。The hematopoietic precursor/stem cell differentiation medium is IF9S complete medium supplemented with 50ng/mL of VEGF, 20ng/mL of bFGF, 50ng/mL of SCF, 10ng/mL of IL-3, 50ng/mL of IL-6 and 20ng/mL TPO.

各种因子在添加无严格的顺序，整个过程需在超净工作台中无菌条件下进行。The various factors are added in no strict order, and the whole process needs to be carried out under sterile conditions in an ultra-clean workbench.

实施例2：人多能干细胞来源的生血内皮前体细胞的制备Example 2: Preparation of hematopoietic endothelial precursor cells derived from human pluripotent stem cells

本实施例采用的人多能干细胞是人胚胎干细胞系(H1)(来源于美国威斯康辛大学)。The human pluripotent stem cells used in this example are human embryonic stem cell lines (H1) (derived from the University of Wisconsin, USA).

a.首先采用胚胎干细胞级别的细胞外基质Matrigel对培养细胞的皿进行包被，再将人胚胎干细胞接种于包被后的皿中并使用化学成分明确的商品化培养基TeSR^TM-E8^TM进行培养，每24小时更换一次新鲜培养基。人胚胎干细胞以克隆的形式生长，当细胞克隆生长至70～80％密度时，需要对细胞进行消化传代或之后的诱导分化培养。细胞传代的周期一般为3-4天。a. Firstly, the dish of cultured cells was coated with Matrigel, an extracellular matrix of embryonic stem cell level, and then the human embryonic stem cells were inoculated in the coated dish, and the chemically defined commercial medium TeSR^TM -E8^TM was used for further development. Cultures were replaced with fresh medium every 24 hours. Human embryonic stem cells grow in the form of clones, and when the cell clone grows to a density of 70-80%, the cells need to be digested and passaged or subsequently induced and differentiated. The cycle of cell subculture is generally 3-4 days.

b.采用ReLeSRTM消化酶将上述a.中生长后的人胚胎干细胞克隆进行消化处理，37℃消化2～3分钟。消化2分钟后镜下观察细胞消化情况，待细胞克隆边缘出现“卷边”时用TeSR^TM-E8^TM培养基终止消化，避免消化过度；b. Digest the human embryonic stem cell clone grown in the above a. with ReLeSRTM digestive enzyme, and digest at 37° C. for 2 to 3 minutes. After 2 minutes of digestion, observe the digestion of the cells under the microscope, and stop the digestion with TeSR^TM -E8^TM medium when the edge of the cell clone appears "curled" to avoid excessive digestion;

c.将消化、离心处理后的人胚胎干细胞克隆小团块接种于上述预包被生长因子减少Matrigel的培养瓶中，接种的密度为50-200个克隆团块/cm²。添加适量的TeSR^TM-E8^TM培养基，在37℃、5％CO₂孵箱中静置培养24～48小时；培养24～48小时后将培养基更换成中胚层细胞分化培养基；c. Inoculate the digested and centrifuged small clusters of human embryonic stem cell clones into the culture flask pre-coated with growth factor-reduced Matrigel, at a density of 50-200 clone clusters/cm² . Add an appropriate amount of TeSR^TM -E8^TM medium, and culture it statically in a 37°C, 5% CO₂ incubator for 24 to 48 hours; after 24 to 48 hours of culture, replace the medium with mesoderm cell differentiation medium;

d.添加中胚层细胞分化培养基后，在37℃、5％CO₂孵箱中静止培养48小时；d. After adding mesoderm cell differentiation medium, culture it statically for 48 hours at 37°C and 5% CO in_an incubator;

e.细胞在中胚层细胞诱导培养基中培养48小时后，将培养瓶中的培养基吸弃更换为新鲜的生血内皮前体细胞诱导培养基，将培养瓶灌满培养基；e. After the cells are cultured in the mesoderm cell induction medium for 48 hours, the medium in the culture bottle is sucked out and replaced with fresh hematopoietic endothelial precursor cell induction medium, and the culture bottle is filled with the medium;

f.将细胞培养瓶封口，然后安装到随机三维回转仪上，设定旋转的方式为随机旋转模式，转速调整为0.1-10rpm/分钟的转速；f. Seal the cell culture bottle, then install it on the random three-dimensional gyroscope, set the rotation mode as random rotation mode, and adjust the speed to 0.1-10rpm/min;

g.将随机三维回转移放入37℃、5％CO₂孵箱中旋转培养72小时，培养72小时候后可见细胞组成的血管内皮样网格状形态，此时即为血管/生血内皮前体细胞；生血内皮前体细胞的形态见图1。g. Put the random three-dimensional back transfer in a 37°C, 5% CO₂ incubator and rotate it for 72 hours. After 72 hours of culture, the vascular endothelial-like grid-like morphology composed of cells can be seen, which is the precursor of blood vessels/hematogenesis endothelium. cells; the morphology of hemogenic endothelial precursor cells is shown in Figure 1.

实施例3：生血内皮前体细胞的检测Example 3: Detection of hematopoietic endothelial precursor cells

收集实施例2中产生的血管/生血内皮前体细胞进行细胞免疫荧光染色鉴定。The vascular/hematopoietic endothelial precursor cells produced in Example 2 were collected for cell immunofluorescence staining identification.

具体地：specifically:

a.吸弃培养瓶中的生血内皮前体细胞诱导培养基，用PBS洗涤细胞2遍，以去除死细胞；a. Discard the hematopoietic endothelial precursor cell induction medium in the culture flask, and wash the cells twice with PBS to remove dead cells;

b.加入4％的多聚甲醛进行固定，室温静置固定20分钟；b. Add 4% paraformaldehyde for fixation, and let it stand at room temperature for 20 minutes;

c.吸弃4％多聚甲醛，加入PBS洗涤2-3次；c. Discard 4% paraformaldehyde, add PBS to wash 2-3 times;

d.加入5％的驴血清进行封闭，室温封闭1小时；d. Add 5% donkey serum for blocking, and block for 1 hour at room temperature;

e.加入荧光标记小鼠抗人PE-CD31抗体(货号：560983,BD公司)，荧光标记小鼠抗人FITC-CD34的抗体(货号：560942,BD公司)以及0.1μg/mL的Hoechst33342，抗体稀释倍数按照说明数建议用封闭液进行稀释，室温孵育1小时或4℃孵育过夜；e. Add fluorescently labeled mouse anti-human PE-CD31 antibody (Cat. No.: 560983, BD Company), fluorescently labeled mouse anti-human FITC-CD34 antibody (Cat. No.: 560942, BD Company) and 0.1 μg/mL Hoechst33342, antibody The dilution factor is recommended to dilute with blocking solution according to the instructions, and incubate at room temperature for 1 hour or overnight at 4°C;

f.吸弃含抗体的染色液体，加入适量的PBS进行洗涤细胞，洗涤3次，每次5分钟。f. Discard the staining solution containing the antibody, add an appropriate amount of PBS to wash the cells, wash 3 times, 5 minutes each time.

g.采用共聚焦荧光显微镜对细胞进行CD31和CD34蛋白的检测，Hoechst33342为标记细胞核，注意操作的时候避光，防止荧光淬灭。图2结果显示的为染色鉴定的结果。g. Use a confocal fluorescence microscope to detect the CD31 and CD34 proteins on the cells. Hoechst33342 is used to mark the nucleus. Pay attention to avoid light when operating to prevent fluorescence quenching. Figure 2 shows the results of staining identification.

CD31和CD34是血管/生血内皮前体细胞重要的标志物，荧光颜色结果可以证实通过本发明的方法可以将中胚层细胞进一步诱导为CD31和CD34阳性的细胞，即生血内皮前体细胞。CD31 and CD34 are important markers of blood vessel/hematopoietic endothelial precursor cells, and the fluorescent color results can confirm that mesoderm cells can be further induced into CD31 and CD34 positive cells, ie hematopoietic endothelial precursor cells, by the method of the present invention.

实施例4：生血内皮前体细胞的效率分析Example 4: Efficiency Analysis of Hematopoietic Endothelial Precursor Cells

收集实施例2中产生的产生的生血内皮前体细胞进行流式细胞术分析鉴定。The hematopoietic endothelial precursor cells produced in Example 2 were collected for flow cytometry analysis and identification.

具体地：specifically:

a.细胞消化：采用Accutase酶(货号：A1110501，Gibco公司)将实施例2中所述细胞消化为单细胞，用IF9S培养基终止消化。a. Cell digestion: the cells described in Example 2 were digested into single cells using Accutase enzyme (product number: A1110501, Gibco Company), and the digestion was terminated with IF9S medium.

b.细胞收集：将细胞移入离心管中，1000转/分钟，离心5分钟富集细胞。b. Cell collection: transfer the cells into a centrifuge tube, centrifuge at 1000 rpm for 5 minutes to enrich the cells.

c.抗体孵育标记：离心后吸弃上清，用0.2％BSA重悬细胞，根据实验的具体情况设置细胞的分组及抗体的标记，其中生血内皮前体细胞鉴定标记抗体为小鼠抗人PE-CD31抗体(货号：560983,BD公司)和小鼠抗人FITC-CD34的抗体(货号：560942,BD公司)；造血前体/干细胞鉴定的标志分子抗体分别为小鼠抗人FITC-CD34的抗体(货号：560942,BD公司)以及小鼠抗人APC-CD43抗体(货号：560198,BD公司)。抗体的稀释比例一般为1:200，水平摇床慢摇室温标记抗体30分钟。c. Antibody incubation labeling: after centrifugation, the supernatant was discarded, and the cells were resuspended with 0.2% BSA. Cell grouping and antibody labeling were set according to the specific conditions of the experiment. The antibody for identification of hematopoietic endothelial precursor cells was mouse anti-human PE - CD31 antibody (Cat. No.: 560983, BD Company) and mouse anti-human FITC-CD34 antibody (Cat. No.: 560942, BD Company); the marker molecule antibody for identification of hematopoietic precursor/stem cell is the mouse anti-human FITC-CD34 antibody Antibody (Product No.: 560942, BD Company) and mouse anti-human APC-CD43 antibody (Product No.: 560198, BD Company). The dilution ratio of the antibody is generally 1:200, and the antibody is labeled at room temperature for 30 minutes by slow shaking on a horizontal shaker.

d.细胞洗涤：加入适量PBS，1000转/分钟，离心5分钟富集细胞，吸弃上清。d. Cell washing: add an appropriate amount of PBS, centrifuge at 1000 rpm for 5 minutes to enrich the cells, and discard the supernatant.

e.细胞固定及上机检测：用含1％多聚甲醛PBS重悬细胞。上机检测前用细胞筛(40um)进行过筛处理。将细胞悬液移入流式管准备进行流式分析。流式细胞仪为FACSCalibur分析型流式细胞仪(BD公司)，测试结束后，用FlowJo 10.0.7软件进行数据分析。e. Cell fixation and detection on the machine: resuspend the cells in PBS containing 1% paraformaldehyde. Sieve with a cell sieve (40um) before testing on the machine. Transfer the cell suspension into flow tubes to prepare for flow analysis. The flow cytometer is a FACSCalibur analytical flow cytometer (BD Company). After the test, FlowJo 10.0.7 software is used for data analysis.

图3显示为对照组和随机三维回转组中生血内皮前体细胞流式细胞分析结果。Figure 3 shows the flow cytometric analysis results of hematopoietic endothelial precursor cells in the control group and the random three-dimensional rotation group.

其中随机三维回转组细胞制备方法为实施例2的方法，对照组细胞的制备方法与实施例2基本相同，区别仅在于步骤e后直接进行在37℃、5％CO₂孵箱中静置培养72小时，得到对照组细胞。The method for preparing the cells in the random three-dimensional rotation group is the method in Example 2, and the method for preparing the cells in the control group is basically the same as in Example 2, the only difference being that after step e, they are directly cultured in a 37°C, 5%_CO2 incubator After 72 hours, cells in the control group were obtained.

通过图3结果可知，对照组仅采用本发明的培养基实现了将37.6％的中胚层细胞诱导为生血内皮前体细胞。而通过结合本发明的培养基以及随机三维回转，实现了71.6％的的中胚层细胞诱导为生血内皮前体细胞。本发明的方法证实了随机旋转可以增加向生血内皮前体细胞的诱导效率，而进一步配合本发明的培养基实现了高效诱导生血内皮前体细胞。It can be seen from the results in Fig. 3 that in the control group, 37.6% of mesoderm cells can be induced into hematopoietic endothelial precursor cells only by using the culture medium of the present invention. However, by combining the medium of the present invention and random three-dimensional rotation, 71.6% of mesoderm cells can be induced into hematopoietic endothelial precursor cells. The method of the present invention proves that random rotation can increase the induction efficiency of hematopoietic endothelial precursor cells, and further cooperate with the medium of the present invention to achieve high-efficiency induction of hematopoietic endothelial precursor cells.

实施例5：人多能干细胞来源的造血前体细胞的制备Example 5: Preparation of hematopoietic precursor cells derived from human pluripotent stem cells

具体地：specifically:

a.弃去培养瓶中生血内皮前体细胞原培养基，更换为实施例1获得的造血前体细胞诱导培养基；培养瓶中应将培养基灌满；a. Discard the original culture medium of hematopoietic endothelial precursor cells in the culture bottle, and replace it with the hematopoietic precursor cell induction medium obtained in Example 1; the culture bottle should be filled with the culture medium;

b.将细胞培养瓶安装到随机回转仪上，设定回转的方式为随机回转，转速调整为内外径均为5rpm/分钟的转速；b. Install the cell culture bottle on the random gyroscope, set the rotation method as random rotation, and adjust the rotation speed to a rotation speed of 5 rpm/min for both the inner and outer diameters;

c.将随机回转仪置于37℃、5％CO₂孵箱中继续进行回转培养48-72小时；c. Place the random gyrator in a 37°C, 5% CO₂ incubator to continue the rotary culture for 48-72 hours;

d.细胞培养48小时候后即可见培养瓶中出现大量的“卵石样”造血前体细胞，部分造血前体细胞从皿底脱离成为悬浮的造血前体细胞。生成的造血前体细胞见图4，其中A为“卵石样”造血前体细胞，B为部分造血前体细胞从皿底脱离的悬浮造血前体细胞。d. After 48 hours of cell culture, a large number of "pebble-like" hematopoietic precursor cells can be seen in the culture flask, and some hematopoietic precursor cells detach from the bottom of the dish to become suspended hematopoietic precursor cells. The generated hematopoietic precursor cells are shown in Figure 4, where A is a "pebble-like" hematopoietic precursor cell, and B is a suspended hematopoietic precursor cell in which some hematopoietic precursor cells have detached from the bottom of the dish.

实施例6：造血前体细胞的检测Example 6: Detection of hematopoietic precursor cells

收集实施例5中产生的造血前体细胞进行细胞免疫荧光染色鉴定。The hematopoietic precursor cells produced in Example 5 were collected and identified by immunofluorescence staining.

h.吸弃培养瓶中的造血前体细胞诱导培养基，用PBS洗涤细胞2遍，以去除死细胞；h. Discard the hematopoietic precursor cell induction medium in the culture flask, and wash the cells twice with PBS to remove dead cells;

i.加入4％的多聚甲醛进行固定，室温静置固定20分钟；i. Add 4% paraformaldehyde for fixation, and let it stand at room temperature for 20 minutes;

j.吸弃4％多聚甲醛，加入PBS洗涤2-3次；j. Discard 4% paraformaldehyde, add PBS to wash 2-3 times;

k.加入5％的驴血清进行封闭，室温封闭1小时；k. Add 5% donkey serum for blocking, and block for 1 hour at room temperature;

l.加入荧光标记小鼠抗人FITC-CD34的抗体(货号：560942,BD公司)，荧光标记小鼠抗人APC-CD43抗体(货号：560198,BD公司)以及0.1μg/mL的Hoechst33342，抗体稀释倍数按照说明数建议用封闭液进行稀释，室温孵育1小时或4℃孵育过夜；l. Add fluorescently labeled mouse anti-human FITC-CD34 antibody (Product No.: 560942, BD Company), fluorescently labeled mouse anti-human APC-CD43 antibody (Product No.: 560198, BD Company) and 0.1 μg/mL Hoechst33342, antibody The dilution factor is recommended to dilute with blocking solution according to the instructions, and incubate at room temperature for 1 hour or overnight at 4°C;

m.吸弃含抗体的染色液体，加入适量的PBS进行洗涤细胞，洗涤3次，每次5分钟。m. Discard the staining solution containing the antibody, add an appropriate amount of PBS to wash the cells, wash 3 times, 5 minutes each time.

n.采用共聚焦荧光显微镜对细胞进行CD34和CD43蛋白的检测，Hoechst33342为标记细胞核，注意操作的时候避光，防止荧光淬灭。图5结果显示的为染色鉴定的结果。n. Use a confocal fluorescence microscope to detect the CD34 and CD43 proteins on the cells. Hoechst33342 is used to mark the nucleus. Pay attention to avoid light when operating to prevent fluorescence quenching. Figure 5 shows the results of staining identification.

CD34和CD43是造血前体细胞的重要的标志物，荧光颜色结果可以证实通过本发明的方法可以将生血内皮前体细胞诱导为CD34和CD43阳性的细胞，即造血前体细胞。CD34 and CD43 are important markers of hematopoietic precursor cells, and the fluorescent color results can confirm that hematopoietic endothelial precursor cells can be induced into CD34 and CD43 positive cells, ie hematopoietic precursor cells, by the method of the present invention.

图2结果显示的为造血前体细胞染色鉴定的结果。Figure 2 shows the results of staining and identification of hematopoietic precursor cells.

实施例7：造血前体细胞的效率分析Example 7: Efficiency Analysis of Hematopoietic Precursor Cells

收集实施例2中产生的造血前体细胞进行流式细胞术分析鉴定。The hematopoietic precursor cells produced in Example 2 were collected for flow cytometry analysis and identification.

具体地：specifically:

f.细胞消化：采用Accutase酶(货号：A1110501，Gibco公司)将实施例2中所述细胞消化为单细胞，分别用IF9S培养基终止消化。f. Cell digestion: the cells described in Example 2 were digested into single cells using Accutase enzyme (product number: A1110501, Gibco Company), and the digestion was terminated with IF9S medium respectively.

g.细胞收集：将细胞移入离心管中，1000转/分钟，离心5分钟富集细胞。g. Cell collection: transfer the cells into a centrifuge tube, centrifuge at 1000 rpm for 5 minutes to enrich the cells.

h.抗体孵育标记：离心后吸弃上清，用0.2％BSA重悬细胞，根据实验的具体情况设置细胞的分组及抗体的标记，其中造血前体细胞鉴定的标志分子抗体分别为小鼠抗人FITC-CD34的抗体(货号：560942,BD公司)以及小鼠抗人APC-CD43抗体(货号：560198,BD公司)。抗体的稀释比例一般为1:200，水平摇床慢摇室温标记抗体30分钟。h. Antibody incubation labeling: After centrifugation, the supernatant was discarded, the cells were resuspended with 0.2% BSA, and the grouping of cells and antibody labeling were set according to the specific conditions of the experiment. The marker molecule antibodies for identification of hematopoietic precursor cells were mouse anti Human FITC-CD34 antibody (Cat. No.: 560942, BD Company) and mouse anti-human APC-CD43 antibody (Cat. No.: 560198, BD Company). The dilution ratio of the antibody is generally 1:200, and the antibody is labeled at room temperature for 30 minutes by slow shaking on a horizontal shaker.

i.细胞洗涤：加入适量PBS，1000转/分钟，离心5分钟富集细胞，吸弃上清。i. Cell washing: add an appropriate amount of PBS, centrifuge at 1000 rpm for 5 minutes to enrich the cells, and discard the supernatant.

j.细胞固定及上机检测：用含1％多聚甲醛PBS重悬细胞。上机检测前用细胞筛(40μm)进行过筛处理。将细胞悬液移入流式管准备进行流式分析。流式细胞仪为FACS Calibur分析型流式细胞仪(BD公司)，测试结束后，用FlowJo 10.0.7软件进行数据分析。j. Cell fixation and on-machine detection: resuspend the cells in PBS containing 1% paraformaldehyde. Sieve with a cell sieve (40 μm) before testing on the machine. Transfer the cell suspension into flow tubes to prepare for flow analysis. The flow cytometer is a FACS Calibur analytical flow cytometer (BD Company). After the test, FlowJo 10.0.7 software is used for data analysis.

图6显示的为对照组和随机回转组中造血前体细胞流式细胞分析结果。其中随机回转组细胞制备方法为实施例2的方法，对照组细胞的制备方法与实施例2基本相同，区别仅在于步骤a后直接进行在37℃、5％CO₂孵箱中静置培养72小时，得到对照组细胞。Figure 6 shows the results of flow cytometric analysis of hematopoietic precursor cells in the control group and the random switch group. The preparation method of the cells in the random rotation group is the method in Example 2, and the preparation method of the cells in the control group is basically the same as that in Example 2, the only difference being that after step a, they are directly cultured in a 37°C, 5% CO2_incubator for 72 Hours, the cells of the control group were obtained.

通过图3结果可知，对照组仅采用本发明的培养基实现了将28.9％的生血内皮前体细胞诱导为造血前体细胞。而通过结合本发明的培养基以及随机回转，实现了45.9％的生血内皮前体细胞诱导为造血前体细胞。本发明的方法证实了随机旋转可以增加向造血前体细胞的诱导效率，而进一步配合本发明的培养基实现了高效诱导。It can be seen from the results in Fig. 3 that the control group only uses the culture medium of the present invention to induce 28.9% of hematopoietic endothelial precursor cells into hematopoietic precursor cells. However, 45.9% of hematopoietic endothelial precursor cells were induced into hematopoietic precursor cells by combining the medium of the present invention and random rotation. The method of the present invention proves that the random rotation can increase the induction efficiency to the hematopoietic precursor cells, and further cooperate with the medium of the present invention to realize high-efficiency induction.

实施例8：其他方法获得的人多能干细胞来源生血内皮前体细胞向造血前体细胞Example 8: Transformation of human pluripotent stem cell-derived hematopoietic endothelial precursor cells to hematopoietic precursor cells obtained by other methods诱导试验induction test

采用已知方法制备(例如参照文献Xu Cao,Gopala K.Yakala,Francijna E.vanden Hil,Amy Cochrane,Christine L.Mummery,Valeria V.Orlova.Differentiation andFunctional Comparison of Monocytes and Macrophages from hiPSCs withPeripheral Blood Derivatives.Stem Cell Reports.2019June 11；12(6):1282–1297中描述的)生血内皮前体细胞，并通过以下方法向造血前体细胞诱导培养。Prepared by known methods (for example, refer to literature Xu Cao, Gopala K.Yakala, Francijna E.vanden Hil, Amy Cochrane, Christine L.Mummery, Valeria V.Orlova.Differentiation and Functional Comparison of Monocytes and Macrophages from hiPSCs withPeripheral Blood Deriv atives. Stem Cell Reports.2019June 11; 12(6):1282-1297 described in) hematopoietic endothelial precursor cells, and induce culture to hematopoietic precursor cells by the following method.

具体地：specifically:

e.弃去培养瓶中生血内皮前体细胞原培养基，更换为实施例1获得的造血前体细胞诱导培养基；培养瓶中应将培养基灌满；e. Discard the original culture medium of hematopoietic endothelial precursor cells in the culture bottle, and replace it with the hematopoietic precursor cell induction medium obtained in Example 1; the culture bottle should be filled with the culture medium;

f.将细胞培养瓶安装到随机回转仪上，设定回转的方式为随机回转，转速调整为内外径均为5rpm/分钟的转速；f. Install the cell culture bottle on the random gyroscope, set the rotation method as random rotation, and adjust the speed to a speed of 5 rpm/min for both the inner and outer diameters;

g.将随机回转仪置于37℃、5％CO₂孵箱中继续进行回转培养48-72小时；g. Place the random gyrator in a 37°C, 5% CO₂ incubator to continue the rotary culture for 48-72 hours;

h.细胞培养48小时候后即可见培养瓶中出现大量的“卵石样”造血前体细胞，部分造血前体细胞从皿底脱离成为悬浮的造血前体细胞。h. After 48 hours of cell culture, a large number of "pebble-like" hematopoietic precursor cells can be seen in the culture flask, and some hematopoietic precursor cells detach from the bottom of the dish to become suspended hematopoietic precursor cells.

说明本发明的从生血内皮前体细胞向造血前体细胞的培养也可以采用本发明的培养基以及旋转培养方法诱导。It shows that the culture from hematopoietic endothelial precursor cells to hematopoietic precursor cells of the present invention can also be induced by the medium and the rotary culture method of the present invention.

Claims (11)

Translated fromChinese

1.一种从人多能干细胞向造血前体细胞分化的方法，其特征在于，所述的方法包括如下步骤：1. A method of differentiation from human pluripotent stem cells to hematopoietic precursor cells, characterized in that, the method comprises the steps of:S1)人多能干细胞向中胚层细胞诱导分化培养:S1) Human pluripotent stem cells are induced to differentiate into mesoderm cells:S11).细胞容器预包被生长因子减少的基底膜基质；S11). Cell containers are pre-coated with growth factor-reduced basement membrane matrix;S12).采用消化酶将人多能干细胞克隆进行消化、离心处理；S12). Using digestive enzymes to digest and centrifuge the human pluripotent stem cell clones;S13).将消化、离心处理后的人多能干细胞克隆小团块接种于S11)获得预包被生长因子减少Matrigel的细胞容器中，添加TeSR^TM-E8^TM培养基，静止培养24～48小时；S13). Inoculate the digested and centrifuged human pluripotent stem cell clone pellets into the cell container obtained from S11) with pre-coated growth factor-reduced Matrigel, add TeSR^TM -E8^TM medium, and culture statically for 24 to 48 hours ;S14)将培养基更换成中胚层细胞诱导分化培养基，静止培养24～48小时，获得诱导的中胚层细胞；S14) replacing the medium with a mesoderm cell induction differentiation medium, and statically culturing for 24 to 48 hours to obtain induced mesoderm cells;S2)诱导的中胚层细胞进一步向生血内皮前体细胞分化培养:S2) The induced mesoderm cells are further differentiated into hematopoietic endothelial precursor cells:S21).将中胚层细胞的细胞培养基更换为生血内皮前体细胞诱导分化培养基；S21). The cell culture medium of the mesoderm cells is replaced with a differentiation medium for induction of hematopoietic endothelial precursor cells;S22).将细胞培养容器安装到随机三维回转仪上，设定旋转的方式为随机旋转，转速调整为0.1-10rpm/分钟的转速；S22). Install the cell culture container on the random three-dimensional gyroscope, set the rotation method as random rotation, and adjust the rotation speed to 0.1-10rpm/min;S23).将随机三维回转仪放入37℃、5％CO₂孵箱中旋转培养60-96小时，获得生血内皮前体细胞；S23). Put the random three-dimensional gyroscope into a 37°C, 5% CO₂ incubator and rotate it for 60-96 hours to obtain hematopoietic endothelial precursor cells;S3)在原始培养基中培养72小时后，更换细胞培养基为诱导生血内皮前体细胞向造血前体细胞分化的培养基；S3) After culturing in the original medium for 72 hours, replace the cell medium with a medium for inducing the differentiation of hematopoietic endothelial precursor cells into hematopoietic precursor cells;S4)将细胞培养瓶重新安装到随机三维回转仪上继续进行三维回转培养，三维回转培养旋转的方式为随机旋转，转速为0.1-10rpm/分钟；S4) reinstall the cell culture bottle on the random three-dimensional gyroscope to continue the three-dimensional rotary culture, the rotation method of the three-dimensional rotary culture is random rotation, and the rotation speed is 0.1-10rpm/min;所述中胚层细胞诱导分化培养基为IF9S完全培养基添加40-60ng/mL的BMP4，15-25μg/mL的Activin A，0.8-1.2μM CHIR99021；The medium for inducing differentiation of mesoderm cells is IF9S complete medium supplemented with 40-60 ng/mL of BMP4, 15-25 μg/mL of Activin A, and 0.8-1.2 μM CHIR99021;所述生血内皮前体细胞诱导分化培养基为IF9S完全培养基添加8-12μM SB431542、40-60ng/mL的VEGF、40-60ng/mL的bFGF和40-60ng/mL的SCF；The hematopoietic endothelial precursor cell induction differentiation medium is IF9S complete medium supplemented with 8-12 μM SB431542, VEGF of 40-60 ng/mL, bFGF of 40-60 ng/mL and SCF of 40-60 ng/mL;所述诱导生血内皮前体细胞向造血前体细胞分化的培养基为IF9S完全培养基添加40-60ng/mL的VEGF，15-25ng/mL的bFGF，40-60ng/mL的SCF，8-12ng/mL的IL-3，40-60ng/mL的IL-6以及15-25ng/mL TPO；The medium for inducing the differentiation of hematopoietic endothelial precursor cells into hematopoietic precursor cells is IF9S complete medium supplemented with 40-60ng/mL of VEGF, 15-25ng/mL of bFGF, 40-60ng/mL of SCF, 8-12ng /mL IL-3, 40-60ng/mL IL-6 and 15-25ng/mL TPO;所述IF9S完全培养基，包括按1:0.8-1.2的体积比混合的IMDM培养基和F12培养基，以及终浓度为8-12mg/L的聚乙烯醇(PVA)、体积百分比为所述培养基的0.08-0.12％的Lipids100X添加剂、体积百分比为所述IF9S完全培养基的1.8％-2.2％的ITS-X 100X添加剂、终浓度为35-45μl/L的一硫代甘油(αMTG)、终浓度为55-70mg/L的抗坏血酸磷酸酯钠(AA2P)、体积百分比为所述IF9S完全培养基的0.8％-1.2％的GlutaMax^TM 100X添加剂以及体积百分比为所述IF9S完全培养基的0.8％-1.2％的非必需氨基酸添加剂100X(NEAA)。The IF9S complete culture medium comprises the IMDM medium and the F12 medium mixed in a volume ratio of 1:0.8-1.2, and polyvinyl alcohol (PVA) whose final concentration is 8-12mg/L, and the volume percentage is the culture medium 0.08-0.12% Lipids 100X supplement of base, 1.8%-2.2% ITS-X 100X supplement of the IF9S complete medium by volume percentage, monothioglycerol (αMTG) with a final concentration of 35-45 μl/L, final Sodium Ascorbyl Phosphate (AA2P) at a concentration of 55-70 mg/L, GlutaMax^™ 100X Supplement at a volume percentage of 0.8%-1.2% of the IF9S complete medium and a volume percentage of 0.8-1.2% of the IF9S complete medium 1.2% Non-Essential Amino Acid Supplement 100X (NEAA).2.根据权利要求1所述的方法，其特征在于，S22)中旋转速度为5rpm/分钟。2. The method according to claim 1, characterized in that, in S22), the rotational speed is 5 rpm/min.3.根据权利要求2所述的方法，其特征在于，S22)中培养72小时。3. The method according to claim 2, characterized in that, culturing for 72 hours in S22).4.一种促进生血内皮前体细胞向造血前体细胞分化的方法，其特征在于，所述的方法包括如下步骤，在生血内皮前体细胞向造血前体细胞分化培养过程中，采用随机三维回转仪将细胞培养容器在旋转条件下进行培养；4. A method for promoting the differentiation of hematopoietic endothelial precursor cells to hematopoietic precursor cells, characterized in that, the method comprises the following steps, during the differentiation culture process of hematopoietic endothelial precursor cells to hematopoietic precursor cells, using random three-dimensional The gyroscope cultures the cell culture vessel under rotating conditions;所述旋转条件下进行培养的培养基采用诱导生血内皮前体细胞向造血前体细胞分化的培养基；The medium for culturing under the rotation condition is a medium that induces the differentiation of hematopoietic endothelial precursor cells into hematopoietic precursor cells;三维回转仪旋转的方式为随机旋转，转速为0.1-10rpm/分钟；The rotation method of the three-dimensional gyroscope is random rotation, and the rotation speed is 0.1-10rpm/min;所述诱导生血内皮前体细胞向造血前体细胞分化培养基为IF9S完全培养基添加40-60ng/mL的VEGF，15-25ng/mL的bFGF，40-60ng/mL的SCF，8-12ng/mL的IL-3，40-60ng/mL的IL-6以及15-25ng/mL TPO；The induction of hematopoietic endothelial precursor cells to hematopoietic precursor cell differentiation medium is IF9S complete medium with 40-60 ng/mL of VEGF, 15-25 ng/mL of bFGF, 40-60 ng/mL of SCF, 8-12 ng/mL mL of IL-3, 40-60ng/mL of IL-6 and 15-25ng/mL of TPO;所述IF9S完全培养基包括按1:0.8-1.2的体积比混合的IMDM基础培养基和F12基础培养基，以及终浓度为:8-12mg/L的聚乙烯醇(PVA)、体积百分比为所述培养基的0.08-0.12％的Lipids 100X添加剂、体积百分比为所述培养基的1.8％-2.2％的ITS-X 100X添加剂、终浓度为35-45μl/L的一硫代甘油(αMTG)、终浓度为55-70mg/L的AA2P、体积百分比为所述培养基的:0.8-1.2％的GlutaMax^TM 100X添加剂以及体积百分比为所述培养基的0.8-1.2％的非必需氨基酸添加剂100X(NEAA)。The IF9S complete medium comprises the IMDM basal medium and the F12 basal medium mixed in a volume ratio of 1:0.8-1.2, and the final concentration is: polyvinyl alcohol (PVA) of 8-12mg/L, the volume percent is all 0.08-0.12% of the Lipids 100X supplement of the medium, the volume percentage of 1.8%-2.2% of the ITS-X 100X supplement of the medium, the monothioglycerol (αMTG) of 35-45 μl/L in the final concentration, AA2P at a final concentration of 55-70 mg/L, GlutaMax^™ 100X Supplement at 0.8-1.2% by volume of the medium, and Non-Essential Amino Acid Supplement 100X (NEAA) at 0.8-1.2% by volume of the medium ).5.权利要求4所述的方法，其特征在于，在生血内皮前体细胞向造血前体细胞分化培养之前对生血内皮前体细胞培养72小时。5. The method according to claim 4, wherein the hematopoietic endothelial precursor cells are cultured for 72 hours before the hematopoietic endothelial precursor cells are differentiated into hematopoietic precursor cells.6.根据权利要求4所述的方法，其特征在于，所述随机旋转内外径均为5rpm/分钟的转速。6. The method according to claim 4, characterized in that, both the inner and outer diameters of the random rotation rotate at a speed of 5 rpm/min.7.根据权利要求4所述的方法，其特征在于，所述旋转条件下进行培养的培养时间为48-72小时。7. The method according to claim 4, characterized in that, the cultivation time for culturing under the rotation condition is 48-72 hours.8.根据权利要求4所述的方法，其特征在于，所述诱导生血内皮前体细胞向造血前体细胞分化的培养基加至培养容器的95％-100％。8. The method according to claim 4, characterized in that the medium for inducing the differentiation of hematopoietic endothelial precursor cells into hematopoietic precursor cells is added to 95%-100% of the culture container.9.根据权利要求4所述的方法，其特征在于，细胞培养条件为37℃、5％CO₂。9 . The method according to claim 4 , wherein the cell culture conditions are 37° C., 5% CO₂ .10.随机三维回转仪在用于促进中胚层细胞向生血内皮前体细胞转化中的用途；10. The use of a random three-dimensional gyroscope for promoting the transformation of mesoderm cells into hematopoietic endothelial precursor cells;将中胚层细胞在随机三维回转仪中培养，培养基为生血内皮前体细胞诱导分化培养基；The mesoderm cells are cultured in a random three-dimensional gyroscope, and the medium is a hematopoietic endothelial precursor cell-induced differentiation medium;所述生血内皮前体细胞诱导分化培养基为IF9S完全培养基添加8-12μM SB431542、40-60ng/mL的VEGF、40-60ng/mL的bFGF和40-60ng/mL的SCF；The hematopoietic endothelial precursor cell induction differentiation medium is IF9S complete medium supplemented with 8-12 μM SB431542, VEGF of 40-60 ng/mL, bFGF of 40-60 ng/mL and SCF of 40-60 ng/mL;所述IF9S完全培养基，包括按1:0.8-1.2的体积比混合的IMDM培养基和F12培养基，以及终浓度为8-12mg/L的聚乙烯醇(PVA)、体积百分比为所述培养基的0.08-0.12％的Lipids100X添加剂、体积百分比为所述IF9S完全培养基的1.8％-2.2％的ITS-X 100X添加剂、终浓度为35-45μl/L的一硫代甘油(αMTG)、终浓度为55-70mg/L的抗坏血酸磷酸酯钠(AA2P)、体积百分比为所述IF9S完全培养基的0.8％-1.2％的GlutaMax^TM 100X添加剂以及体积百分比为所述IF9S完全培养基的0.8％-1.2％的非必需氨基酸添加剂100X(NEAA)；The IF9S complete culture medium comprises the IMDM medium and the F12 medium mixed in a volume ratio of 1:0.8-1.2, and polyvinyl alcohol (PVA) whose final concentration is 8-12mg/L, and the volume percentage is the culture medium 0.08-0.12% Lipids 100X supplement of base, 1.8%-2.2% ITS-X 100X supplement of the IF9S complete medium by volume percentage, monothioglycerol (αMTG) with a final concentration of 35-45 μl/L, final Sodium Ascorbyl Phosphate (AA2P) at a concentration of 55-70 mg/L, GlutaMax^™ 100X Supplement at a volume percentage of 0.8%-1.2% of the IF9S complete medium and a volume percentage of 0.8-1.2% of the IF9S complete medium 1.2% non-essential amino acid supplement 100X (NEAA);随机三维回转仪的旋转方式为随机旋转，转速为0.1-10rpm/分钟。The rotation mode of the random three-dimensional gyroscope is random rotation, and the rotation speed is 0.1-10rpm/min.11.随机三维回转仪在用于促进生血内皮前体细胞向造血前体细胞转化的用途；11. The use of a random three-dimensional gyroscope for promoting the transformation of hematopoietic endothelial precursor cells into hematopoietic precursor cells;将生血内皮前体细胞在随机三维回转仪中培养，所述培养基为诱导生血内皮前体细胞向造血前体细胞分化的培养基，The hematopoietic endothelial precursor cells are cultured in a random three-dimensional gyroscope, and the medium is a medium for inducing the hematopoietic endothelial precursor cells to differentiate into hematopoietic precursor cells,所述诱导生血内皮前体细胞向造血前体细胞分化的培养基为IF9S完全培养基添加40-60ng/mL的VEGF，15-25ng/mL的bFGF，40-60ng/mL的SCF，8-12ng/mL的IL-3，40-60ng/mL的IL-6以及15-25ng/mL TPO；The medium for inducing the differentiation of hematopoietic endothelial precursor cells into hematopoietic precursor cells is IF9S complete medium supplemented with 40-60ng/mL of VEGF, 15-25ng/mL of bFGF, 40-60ng/mL of SCF, 8-12ng /mL IL-3, 40-60ng/mL IL-6 and 15-25ng/mL TPO;所述IF9S完全培养基，包括按1:0.8-1.2的体积比混合的IMDM培养基和F12培养基，以及终浓度为8-12mg/L的聚乙烯醇(PVA)、体积百分比为所述培养基的0.08-0.12％的Lipids100X添加剂、体积百分比为所述IF9S完全培养基的1.8％-2.2％的ITS-X 100X添加剂、终浓度为35-45μl/L的一硫代甘油(αMTG)、终浓度为55-70mg/L的抗坏血酸磷酸酯钠(AA2P)、体积百分比为所述IF9S完全培养基的0.8％-1.2％的GlutaMax^TM 100X添加剂以及体积百分比为所述IF9S完全培养基的0.8％-1.2％的非必需氨基酸添加剂100X(NEAA)；The IF9S complete culture medium comprises the IMDM medium and the F12 medium mixed in a volume ratio of 1:0.8-1.2, and polyvinyl alcohol (PVA) whose final concentration is 8-12mg/L, and the volume percentage is the culture medium 0.08-0.12% Lipids 100X supplement of base, 1.8%-2.2% ITS-X 100X supplement of the IF9S complete medium by volume percentage, monothioglycerol (αMTG) with a final concentration of 35-45 μl/L, final Sodium Ascorbyl Phosphate (AA2P) at a concentration of 55-70 mg/L, GlutaMax^™ 100X Supplement at a volume percentage of 0.8%-1.2% of the IF9S complete medium and a volume percentage of 0.8-1.2% of the IF9S complete medium 1.2% non-essential amino acid supplement 100X (NEAA);随机三维回转仪的旋转方式为随机旋转，转速为0.1-10rpm/分钟。The rotation mode of the random three-dimensional gyroscope is random rotation, and the rotation speed is 0.1-10rpm/min.

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