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CN112980770B - Induced Human Pluripotent Stem Cell Directed Endothelial Differentiation Method - Google Patents

Induced Human Pluripotent Stem Cell Directed Endothelial Differentiation Method Download PDF

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CN112980770B
CN112980770B CN202110235774.2A CN202110235774A CN112980770B CN 112980770 B CN112980770 B CN 112980770B CN 202110235774 A CN202110235774 A CN 202110235774A CN 112980770 B CN112980770 B CN 112980770B
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董念国
谢明辉
程敏
乔韡华
曹红
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Abstract

The invention relates to a method for inducing directional endothelial differentiation of human pluripotent stem cells, which specifically comprises the following steps: 1. when the human induced pluripotent stem cells reach more than 80% fusion, using a differentiation culture medium containing 10-50ng/ml WNT3a to induce and differentiate the digested stem cells; 2. continuing to induce differentiation of the cells using a differentiation medium containing 10-50ng/ml WNT3a and 10-30ng/ml BMP 2; 3. continuously inducing and differentiating the cells after inducing and differentiating in the step 2 into mesodermal cells by using a differentiation medium containing 10-30ng/ml of BMP 2; 4. the cells were further induced to differentiate into endothelial cells using a medium containing 10-15ng/ml FGF 8. By improving the culture medium and the related cytokines, the experimental scheme can have higher endothelial induction efficiency.

Description

诱导人类多能干细胞定向内皮分化方法Induced Human Pluripotent Stem Cell Directed Endothelial Differentiation Method

技术领域technical field

本发明涉及诱导人类多能干细胞的定向分化领域,具体涉及一种诱导人类多能干细胞定向内皮分化方法。The invention relates to the field of directional differentiation of induced human pluripotent stem cells, in particular to a method for inducing directional endothelial differentiation of human pluripotent stem cells.

背景技术Background technique

心血管疾病(CVD)通常由内皮功能障碍引发,是世界上主要的死亡原因之一。由于缺乏分离足够数量的功能正常的自体内皮细胞(ECs)移植的方法,以及我们对内源性ECs在各种心血管疾病发生过程中如何功能失调的了解有限,直接通过内皮替代治疗CVD受到阻碍。人类诱导多能干细胞(HiPSCs)的创新,即从体细胞重新编程为类似胚胎干细胞(ESC)的多潜能状态的细胞,可以产生大量针对患者的EC,可用于移植、药物筛选或研究,以探索某些疾病状态下内皮功能障碍的机制。然而,目前大多数的内皮分化方案分化效率低下,分化的细胞异质性较大,一定程度上限制了HiPSCs来源的内皮细胞的应用。Cardiovascular disease (CVD), often caused by endothelial dysfunction, is one of the leading causes of death worldwide. Direct endothelial replacement therapy for CVD is hampered by the lack of methods to isolate sufficient numbers of functioning autologous endothelial cells (ECs) for transplantation, and our limited understanding of how endogenous ECs are dysfunctional in the development of various cardiovascular diseases . Innovations in human induced pluripotent stem cells (HiPSCs), cells reprogrammed from somatic cells to a pluripotent state similar to embryonic stem cells (ESCs), can generate large numbers of patient-specific ECs that can be used for transplantation, drug screening or research to explore Mechanisms of endothelial dysfunction in certain disease states. However, most of the current endothelial differentiation protocols have low differentiation efficiency and great heterogeneity of differentiated cells, which limits the application of HiPSCs-derived endothelial cells to a certain extent.

发明内容SUMMARY OF THE INVENTION

本发明所要解决的技术问题是针对上述问题,提供一种诱导人类多能干细胞定向内皮分化方法,本发明解决上述技术问题的技术方案如下:The technical problem to be solved by the present invention is to provide a method for inducing directional endothelial differentiation of human pluripotent stem cells in view of the above-mentioned problems. The technical solution of the present invention to solve the above-mentioned technical problems is as follows:

诱导人类多能干细胞定向内皮分化方法,包括以下步骤:A method for inducing human pluripotent stem cells directed endothelial differentiation, including the following steps:

步骤1、第0-1天:人类诱导多能干细胞达到80%融合以上时,使用含有10-50ng/mlWNT3a和1μmol/L SU5402的RPMI/B27分化培养基,对消化后的人类诱导多能干细胞进行诱导分化;Step 1. Days 0-1: When human induced pluripotent stem cells reach 80% confluence, use RPMI/B27 differentiation medium containing 10-50ng/ml WNT3a and 1μmol/L SU5402 to treat the digested human induced pluripotent stem cells. to induce differentiation;

步骤2、第1-2天:使用含有10-50ng/ml WNT3a、10-30ng/ml BMP2和1μmol/LSU5402的RPMI/B27分化培养基,对步骤1诱导分化后的细胞继续诱导分化;Step 2, Day 1-2: Use RPMI/B27 differentiation medium containing 10-50ng/ml WNT3a, 10-30ng/ml BMP2 and 1μmol/LSU5402 to continue to induce differentiation of cells after induction in step 1;

步骤3、第2-3天:使用含有10-30ng/ml BMP2和1μmol/L SU5402的RPMI/B27分化培养基,对步骤2诱导分化后的细胞继续诱导分化为中胚层细胞;Step 3. Days 2-3: Use RPMI/B27 differentiation medium containing 10-30ng/ml BMP2 and 1μmol/L SU5402, and continue to induce differentiation into mesoderm cells after induction and differentiation in Step 2;

步骤4、第4-9天:使用含有30-50ng/ml VEGF、10-15ng/ml FGF8和10-15ng/mlbFGF的EGM-2/B27培养基,对步骤3诱导分化后的细胞继续诱导分化为内皮细胞。Step 4. Days 4-9: Use EGM-2/B27 medium containing 30-50ng/ml VEGF, 10-15ng/ml FGF8 and 10-15ng/ml bFGF, continue to induce differentiation of cells after induction in step 3 for endothelial cells.

进一步的,所述步骤2和步骤3中分化培养基中WNT3a的浓度为10ng/ml,BMP2的浓度为10ng/ml。Further, in the steps 2 and 3, the concentration of WNT3a in the differentiation medium is 10 ng/ml, and the concentration of BMP2 is 10 ng/ml.

进一步的,所述步骤4的培养基中FGF8的浓度为10ng/ml。Further, the concentration of FGF8 in the medium of step 4 is 10ng/ml.

本发明的有益效果为:通过改良培养基及相关细胞因子,本实验方案可以具备更高的内皮诱导效率。The beneficial effects of the present invention are: by improving the culture medium and related cytokines, the experimental scheme can have higher endothelial induction efficiency.

附图说明Description of drawings

图1为中胚层标志物CD15的流式细胞图;Figure 1 is a flow cytometry diagram of mesoderm marker CD15;

图2为内皮标志物CD31、CD144双标流式细胞图;Figure 2 is a flow cytometry chart of double-labeled endothelial markers CD31 and CD144;

图3为内皮标志物单标CD144流式细胞图;Figure 3 is a flow cytometry diagram of endothelial marker CD144;

图4为内皮标志物单标CD31流式细胞图。Figure 4 is a flow cytometry diagram of the endothelial marker single-labeled CD31.

具体实施方式Detailed ways

以下结合附图对本发明的原理和特征进行描述,所举实例只用于解释本发明,并非用于限定本发明的范围。The principles and features of the present invention will be described below with reference to the accompanying drawings. The examples are only used to explain the present invention, but not to limit the scope of the present invention.

诱导人类多能干细胞定向内皮分化方法,包括以下步骤:A method for inducing human pluripotent stem cells directed endothelial differentiation, including the following steps:

HiPSCs的维持Maintenance of HiPSCs

HiPSCs常规培养在含有mTeSR1培养基的Matrigel上。每隔3-5天用Versen3-5传代一次,在35mm培养板上细胞需要80%融合才能开始分化。HiPSCs were routinely grown on Matrigel containing mTeSR1 medium. Passage with Versen3-5 every 3-5 days, cells need to be 80% confluent on 35mm culture plates to start differentiation.

第0天:Day 0:

铺胶:Matrigel在冰上解冻,以无生长因子的DMEM培养基1:100稀释后,平铺在60mm的培养板上,37℃孵育0.5h。吸除培养基,用DPBS(不含Ca2+和Mg2+)清洗一次,加入4.5ml预热的mTeSR1+ROCK抑制剂Y27632(终浓度10μM)。Spreading: Matrigel was thawed on ice, diluted 1:100 with growth factor-free DMEM medium, spread on a 60mm culture plate, and incubated at 37°C for 0.5h. The medium was aspirated, washed once with DPBS (without Ca2+ and Mg2+), and 4.5 ml of pre-warmed mTeSR1+ROCK inhibitor Y27632 (final concentration 10 μM) was added.

消化种板:将mTeSR1生长培养基从80%汇合的35mm细胞培养板中吸除,用2ml预热的DPBS清洗。加入2毫升预热的TriplE,37℃孵育5-10分钟,显微镜下检查细胞脱落情况。在上述加入1-2ml预热的mTeSR1,将细胞转移到15毫升的离心管中。在1000rpm(210g)下离心5min后,吸除上清液2ml预热的mTeSR1培养基重新悬浮细胞后进行细胞计数。以每平方厘米20000-30000个细胞接种于在铺胶的培养皿上。在37℃、5%二氧化碳中孵育细胞过夜。Digest seed plate: Aspirate mTeSR1 growth medium from 80% confluent 35 mm cell culture plate and wash with 2 ml of pre-warmed DPBS. Add 2 ml of pre-warmed TriplE, incubate at 37°C for 5-10 minutes, and check for cell detachment under a microscope. Add 1-2 ml of pre-warmed mTeSR1 above and transfer the cells to a 15-ml centrifuge tube. After centrifugation at 1000 rpm (210 g) for 5 min, the supernatant was removed by aspirating 2 ml of pre-warmed mTeSR1 medium and the cells were resuspended for cell counting. 20,000-30,000 cells per square centimeter were plated on gel-coated dishes. Cells were incubated overnight at 37°C, 5% carbon dioxide.

中胚层诱导阶段(第1-3天):Mesoderm induction stage (days 1-3):

中胚层诱导第1天:用预热的RPMI/B27培养基+10ng/ml WNT3a+1μmol/L SU5402(4.5ml/60mm平板/2ml/6孔)更换原培养基。第2天:用预热的含10ng/ml WNT3a+10ng/mlBMP2+1μmol/L SU5402的RPMI/B27培养基(4.5ml/60mm平板/2ml/6孔)更换原培养基。第3天:用含10ng/ml BMP2+1μmol/L SU5402(4.5ml/60mm培养皿/2ml/6孔)的预热RPMI/B27培养基更换原培养基。Mesoderm induction day 1: Replace the original medium with pre-warmed RPMI/B27 medium + 10 ng/ml WNT3a + 1 μmol/L SU5402 (4.5 ml/60 mm plate/2 ml/6 wells). Day 2: Replace the original medium with pre-warmed RPMI/B27 medium (4.5ml/60mm plate/2ml/6 wells) containing 10ng/ml WNT3a+10ng/ml BMP2+1μmol/L SU5402. Day 3: Replace the original medium with pre-warmed RPMI/B27 medium containing 10ng/ml BMP2+1μmol/L SU5402 (4.5ml/60mm dish/2ml/6 well).

内皮诱导阶段(第4-9天):Endothelial induction phase (days 4-9):

第4天Day 4

铺胶:Matrigel在冰上解冻,以无生长因子的DMEM培养基1:100稀释后,平铺在60mm的培养皿上,37℃孵育0.5h。吸除培养基,用DPBS(不含Ca2+和Mg2+)清洗一次,加入4.5ml预热的EGM-2/B27诱导分化培养基+ROCK抑制剂Y27632(终浓度10μM),并加入30ng/mlVEGF+10ng/ml FGF8+10ng/ml bFGF。Laying: Matrigel was thawed on ice, diluted 1:100 with growth factor-free DMEM medium, spread on a 60mm petri dish, and incubated at 37°C for 0.5h. Aspirate the medium, wash once with DPBS (without Ca2+ and Mg2+), add 4.5ml of pre-warmed EGM-2/B27 induction differentiation medium + ROCK inhibitor Y27632 (final concentration 10μM), and add 30ng/ml VEGF+10ng /ml FGF8 + 10ng/ml bFGF.

消化种板:将生长培养基从80%汇合的60mm培养皿中吸除。用2ml预热的DPBS(不含Ca2+和Mg2+)清洗。加入2毫升预热的TriplE,37℃孵育5-10分钟,显微镜下检查细胞脱落情况。加入1-2ml预热的EGM-2培养基。将细胞转移到15毫升的离心管中。在1000rpm(210g)下离心5min后进行细胞计数,以每平方厘米5000个细胞的密度接种在预铺胶的培养皿上。在37℃、5%二氧化碳中孵育细胞过夜。用添加30ng/ml VEGF+10ng/ml FGF8+10ng/ml bFGF的EGM-2/B27培养基(4.5ml/60mm平板/2ml/6孔)更换培养基,每隔一天换一次培养基。Digest seed plate: Aspirate growth medium from 80% confluent 60 mm dishes. Wash with 2 ml of pre-warmed DPBS (without Ca2+ and Mg2+). Add 2 ml of pre-warmed TriplE, incubate at 37°C for 5-10 minutes, and check for cell detachment under a microscope. Add 1-2 ml of pre-warmed EGM-2 medium. Transfer the cells to a 15 mL centrifuge tube. Cells were counted after centrifugation at 1000 rpm (210 g) for 5 min and seeded on pre-gelatinized dishes at a density of 5000 cells per square centimeter. Cells were incubated overnight at 37°C, 5% carbon dioxide. The medium was replaced with EGM-2/B27 medium (4.5ml/60mm plate/2ml/6 wells) supplemented with 30ng/ml VEGF+10ng/ml FGF8+10ng/ml bFGF every other day.

如图1所示为中胚层标志物CD15的流式细胞图;由图1可看出HiPSCs分化为中胚层CD15的分化效率为99.1%(图1的P2区域);Figure 1 shows the flow cytometry of the mesoderm marker CD15; it can be seen from Figure 1 that the differentiation efficiency of HiPSCs into mesoderm CD15 is 99.1% (P2 area in Figure 1);

如图2所示为内皮标志物CD31、CD144双标流式细胞图;由图2可看出在该分化方案诱导下,HiPSCs分化为内皮标志物CD31、CD144双标的分化效率为63.8%(图2的Q2区域);Figure 2 shows the flow cytometry diagram of the endothelial markers CD31 and CD144 double-labeling; it can be seen from Figure 2 that under the induction of the differentiation protocol, the differentiation efficiency of HiPSCs into the endothelial markers CD31 and CD144 double-labeling was 63.8% (Fig. 2's Q2 area);

如图3所示为内皮标志物单标CD144流式细胞图;由图3可看出在该分化方案诱导下,HiPSCs分化为内皮标志物CD144的分化效率为86.2%(图3的P2区域);Figure 3 shows the flow cytometry chart of endothelial marker CD144; it can be seen from Figure 3 that under the induction of this differentiation protocol, the differentiation efficiency of HiPSCs into endothelial marker CD144 is 86.2% (P2 area in Figure 3) ;

如图4所示为内皮标志物单标CD31流式细胞图;由图4可看出在该分化方案诱导下,HiPSCs分化为内皮标志物CD31的分化效率为56.3%(图4的P3区域)。Figure 4 shows the flow cytometry diagram of the endothelial marker CD31; it can be seen from Figure 4 that the differentiation efficiency of HiPSCs into endothelial marker CD31 under the induction of this differentiation protocol is 56.3% (P3 area in Figure 4) .

以上所述仅为本发明的较佳实施例,并不用以限制本发明,凡在本发明的精神和原则之内,所作的任何修改、等同替换、改进等,均应包含在本发明的保护范围之内。The above are only preferred embodiments of the present invention and are not intended to limit the present invention. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection of the present invention. within the range.

Claims (3)

1. The method for inducing the directional endothelial differentiation of the human pluripotent stem cells is characterized by comprising the following steps:
step 1, day 0-1: when the human induced pluripotent stem cells reach more than 80% fusion, using an RPMI/B27 differentiation medium containing 10-50ng/ml WNT3a and 1 mu mol/L SU5402 to induce and differentiate the digested human induced pluripotent stem cells;
step 2, day 1-2: continuing to induce differentiation of the cells after induced differentiation in step 1 by using an RPMI/B27 differentiation medium containing 10-50ng/ml WNT3a, 10-30ng/ml BMP2 and 1. mu. mol/L SU 5402;
step 3, day 2-3: using RPMI/B27 differentiation medium containing 10-30ng/ml BMP2 and 1 mu mol/L SU5402 to continue to induce and differentiate the cells after induced differentiation in the step 2 into mesodermal cells;
and 4, 4-9 days, using EGM-2/B27 culture medium containing 30-50ng/ml VEGF, 10-15ng/ml FGF8 and 10-15ng/ml bFGF to continue to induce the differentiation of the cells after the induced differentiation of the step 3 into endothelial cells.
2. The method for inducing committed endothelial differentiation of human pluripotent stem cells according to claim 1, wherein the concentration of WNT3a and the concentration of BMP2 in the differentiation medium in step 2 and step 3 are 10ng/ml and 10ng/ml respectively.
3. The method for inducing committed endothelial differentiation of human pluripotent stem cells according to claim 1, wherein the concentration of FGF8 in the medium of step 4 is 10 ng/ml.
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