SMAD2和SMAD3在人胚胎干细胞分化过程中的不同调控作用

doi:10.3969/j.issn.1674-8115.2022.04.006

上海交通大学学报（医学版） ›› 2022, Vol. 42 ›› Issue (4): 443-454.doi: 10.3969/j.issn.1674-8115.2022.04.006

SMAD2和SMAD3在人胚胎干细胞分化过程中的不同调控作用

赵冰楠¹(), 马双羽¹(), 胥春龙²(), 王琼¹()

^1.上海交通大学医学院组织胚胎学与遗传发育学系，上海市生殖医学重点实验室，上海市“细胞稳态调控与疾病”前沿科学研究基地，上海 200025
^2.上海脑科学与类脑研究中心，上海 200031

收稿日期:2022-01-19 接受日期:2022-04-10 出版日期:2022-04-28 发布日期:2022-04-28
通讯作者: 胥春龙,王琼 E-mail:bingnanzhao@126.com;sherrysyma@163.com;larrylord@163.com;wangqiong@shsmu.edu.cn
作者简介:赵冰楠(1997—)，女，硕士生；电子信箱：bingnanzhao@126.com
马双羽(1990—)，女，助理研究员，电子信箱：sherrysyma@163.com。第一联系人：为共同第一作者。
基金资助:
国家自然科学基金面上项目(31771512)

Different roles of SMAD2 and SMAD3 in human embryonic stem cell differentiation

ZHAO Bingnan¹(), MA Shuangyu¹(), XU Chunlong²(), WANG Qiong¹()

^1.Department of Histoembryology, Genetics and Developmental Biology, Shanghai Key Laboratory of Reproductive Medicine, Shanghai Frontiers Science Center of Cellular Homeostasis and Human Disease, Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China
^2.Shanghai Center for Brain Science and Brain-Inspired Technology, Shanghai 200031, China

Received:2022-01-19 Accepted:2022-04-10 Online:2022-04-28 Published:2022-04-28
Contact: XU Chunlong,WANG Qiong E-mail:bingnanzhao@126.com;sherrysyma@163.com;larrylord@163.com;wangqiong@shsmu.edu.cn
Supported by:
National Natural Science Foundation of China(31771512)

摘要/Abstract

摘要：

目的·探究转化生长因子β（transforming growth factor β，TGFβ）家族下游的关键转录因子SMAD蛋白（drosophila mothers against decapentaplegic protein）——SMAD2和SMAD3在人胚胎干细胞（human embryonic stem cell，hESC）体外分化过程中的不同作用。方法·在hESC中，通过蛋白质印迹法（Western blotting）检测SMAD2和SMAD3在分化过程中的蛋白表达水平。通过CRISPR/Cas9（clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9）技术构建SMAD2敲除（SMAD2 knockout，SMAD2KO）和SMAD3敲除（SMAD3 knockout，SMAD3KO）的细胞系。利用免疫荧光染色检测SMAD蛋白丢失对多能性因子八聚体结合转录因子4（octamer-binding transcription factor 4，OCT4）和性别决定区Y框蛋白2（sex determining region Y-box 2，SOX2），神经外胚层（neuroectoderm，NE）标志因子配对盒因子6（paired box 6，PAX6）和性别决定区Y框蛋白1（sex determining region Y-box 1，SOX1）以及中内胚层（mesendoderm，ME）标志因子SOX17的表达影响；同时使用流式细胞术鉴定SMAD2敲除或SMAD3敲除对NE标志因子PAX6以及ME标志因子SOX17、脱中胚蛋白（eomesodermin，EOMES）和C-X-C模体趋化因子受体4（C-X-C motif chemokine receptor 4，CXCR4）表达的影响。在SMAD2KO和SMAD3KO细胞中分别过表达SMAD2和SMAD3质粒，通过Western blotting检测SMAD2和SMAD3蛋白回补的效果，并利用实时定量PCR（real-time quantitative reverse transcription PCR，qRT-PCR）检测SMAD蛋白回补后对分化过程中的ME关键因子EOMES、叉头框转录因子A2（forkhead box A2，FOXA2）和GSC同源盒（goosecoid homeobox，GSC）表达的影响。结果·虽然SMAD2和SMAD3的蛋白序列较为相似，但是SMAD2才是hESC中主要表达的蛋白因子。免疫荧光染色结果表明，SMAD2敲除和SMAD3敲除并不会影响hESC多能性因子OCT4和SOX2的表达；同时流式细胞术发现，SMAD蛋白的缺失也不会影响NE标志因子PAX6和SOX1的表达。SMAD3敲除对hESC向ME分化没有显著的影响，而SMAD2敲除严重阻碍了hESC表达ME标志因子SOX17、EOMES和CXCR4。通过qRT-PCR检测显示在SMAD2KO细胞中过表达外源性SMAD2可以有效地恢复ME基因EOMES、FOXA2和GSC的表达，而在SMAD3KO细胞中回补SMAD3对这些ME基因的表达无影响。结论·SMAD2和SMAD3在hESC向ME分化过程中的调控作用不同。SMAD2而非SMAD3是决定hESC表达ME基因的关键。

关键词: 人胚胎干细胞, 中内胚层分化, SMAD2, SMAD3

Abstract:

Objective·To investigate the different roles of SMAD proteins (drosophila mothers against decapentaplegic proteins)—SMAD2 and SMAD3, the major downstream effectors of the transforming growth factor β (TGFβ) family, in the differentiation process of human embryonic stem cells (hESCs) in vitro.

Methods·The protein levels of SMAD2 and SMAD3 were measured by Western blotting in hESCs. The SMAD2 knockout (SMAD2KO) and SMAD3 knockout (SMAD3KO) cell lines were constructed in hESCs by CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9) technology. Immunofluorescence staining was used to detect the effect of SMAD protein loss on the expression of pluripotent factors octamer-binding transcription factor 4 (OCT4) and sex determining region Y-box 2 (SOX2), neuroectoderm (NE) factors paired box 6 (PAX6) and sex determining region Y-box 1 (SOX1), and mesendoderm (ME) factor SOX17. Flow cytometry was used to identify the expression changes of NE factor PAX6 and ME factor SOX17, eomesodermin (EOMES) and C-X-C motif chemokine receptor 4 (CXCR4) upon SMAD2 or SMAD3 loss. SMAD2 and SMAD3 plasmids were overexpressed in SMAD2KO and SMAD3KO cell lines, respectively, which were verified by Western blotting. Real-time quantitative reverse transcription PCR (qRT-PCR) was used to check whether the recoveries of SMAD proteins in SMADKO cell lines could restore the expression of ME key factors, like EOMES, forkhead box A2 (FOXA2) and goosecoid homeobox (GSC) during differentiation.

Results·Although the protein sequences of SMAD2 and SMAD3 were similar, SMAD2 was the main factor expressed in hESCs. SMAD2 knockout and SMAD3 knockout did not affect the expression of pluripotent factors OCT4 and SOX2 from immunofluorescence staining results, nor the expression of NE factors PAX6 and SOX1 combined with flow cytometry results. SMAD3 knockout had no significant effect on the differentiation of hESCs into ME, while SMAD2 knockout severely hindered the expression of ME specific factors such as SOX17, EOMES and CXCR4. By qRT-PCR analysis, overexpression of exogenous SMAD2 in SMAD2KO cells can effectively restore the expression of ME genes including EOMES, FOXA2 and GSC, while replenishment of SMAD3 in SMAD3KO cells almost had no effect.

Conclusion·SMAD2 and SMAD3 play different roles in the ME differentiation of hESCs. SMAD2, but not SMAD3, is critical for the expression of ME genes in hESCs.

Key words: human embryonic stem cell (hESC), mesendoderm differentiation, SMAD2, SMAD3

中图分类号:

R329.2

赵冰楠, 马双羽, 胥春龙, 王琼. SMAD2和SMAD3在人胚胎干细胞分化过程中的不同调控作用[J]. 上海交通大学学报（医学版）, 2022, 42(4): 443-454.

ZHAO Bingnan, MA Shuangyu, XU Chunlong, WANG Qiong. Different roles of SMAD2 and SMAD3 in human embryonic stem cell differentiation[J]. Journal of Shanghai Jiao Tong University (Medical Science), 2022, 42(4): 443-454.

图/表 6

表1 qRT-PCR引物序列

Tab 1 Primer sequences for qRT-PCR

Gene	Forward primer （5'→3'）	Reverse primer （5'→3'）
GAPDH	GGAGCCAAACGGGTCATCATCTC	GAGGGGCCATCCACAGTCTTCT
EOMES	CGGCCTCTGTGGCTCAAA	AAGGAAACATGCGCCTGC
FOXA2	GGGAGCGGTGAAGATGGA	TCATGTTGCTCACGGAGGAGTA
GSC	AACGCGGAGAAGTGGAACAAG	CTGTCCGAGTCCAAATCGC

图1 SMAD2和SMAD3的表达模式Note：A. Scheme of human SMAD2 and SMAD3 protein structure and their amino acid (aa) sequences alignment. SMAD2 and SMAD3 were highly conserved except an N-terminal 10 aa insert (green) and 30 aa encoded by E3 (yellow) in SMAD2 MH1. B. RNA expression of Smad2 and Smad3 in mouse embryo E6.5?E7.5 from iTranscriptome. A—anterior; L—left lateral; R—right lateral; P—posterior. C. Western blotting of SMAD2 and SMAD3 expression in hESCs at indicated time point after CHIR and AC treatment for ME differentiation and blotted with anti-SMAD2/3, anti-SMAD2 or anti-SMAD3 antibodies. Arrows indicate SMAD2 and SMAD3 bands, respectively. Tubulin was used as an internal control. D. Fluorescence intensities of SMAD2 and SMAD3 from Western blotting in Fig 1C were measured by Odyssey imaging system.

Fig 1 Expression pattern of SMAD2 and SMAD3

图2 单一敲除 SMAD2 或者 SMAD3 对hESC多能性因子表达的影响Note：A. The scheme showed the genomic sequences of SMAD2KO and SMAD3KO cell lines compared to wildtype hESC (WT). B. Western blotting analysis of SMAD2 and SMAD3 expression in WT, SMAD2KO and SMAD3KO cell lines. Tubulin was used as a loading control. C. Immunofluorescence analysis of OCT4 and SOX2 in WT, SMAD2KO and SMAD3KO cell lines.

Fig 2 Influence of SMAD2 knockout or SMAD3 knockout on the expression of pluripotent factors in hESCs

图3 SMAD2 敲除和 SMAD3 敲除对hESC向NE分化的影响Note：A. Diagram of hESC to NE differentiation from D0 to D8. Dash line indicates differentiation from D3 to D8. Flow cytometry analysis of WT, SMAD2KO and SMAD3KO cells upon NE differentiation induction on D8. B. Immunofluorescence analysis of OCT4, PAX6 and SOX1 in WT, SMAD2KO and SMAD3KO cell lines on D8.

Fig 3 Effect of SMAD2 knockout or SMAD3 knockout on the NE differentiation of hESC

图4 SMAD2 敲除和 SMAD3 敲除对hESC向DE分化的影响Note：A. Diagram shows the procedure of hESC to DE differentiation in 3 d with addition of CHIR and/or AC. Flow cytometry analysis of WT, SMAD2KO and SMAD3KO cells upon DE differentiation induction on D3. B. Immunofluorescence analysis of SMAD2 and SOX17 in WT, SMAD2KO and SMAD3KO cell lines on D3.

Fig 4 Effect of SMAD2 knockout or SMAD3 knockout on the DE differentiation of hESC

图5 过表达外源性SMAD2对SMAD2KO细胞向ME分化的影响Note：A. After ME differentiation for 1 d, mRNA levels of EOMES, GSC and FOXA2 in WT, SMAD2KO and SMAD3KO cells were measured by qRT-PCR. ①P=0.000, compared with the WT group. B. Western blotting analysis of SMAD2/3 protein levels in WT, SMAD2KO cells with dox-inducible expression of SMAD2 or empty vector control, and SMAD3KO cells with dox-inducible expression of SMAD3 or empty vector control. V—empty vector control. Tubulin was used as a loading control. C. qRT-PCR analysis of EOMES, GSC and FOXA2 mRNA levels in the indicated cell lines. Assays were performed with dox treatment before (D0) or after CHIR+AC induced differentiation for 1 d (D1). ①P=0.005, ②P=0.000, compared with the WT group; ③P=0.002, ④P=0.000, compared with the SMAD2KO+V group (SMAD2KO cells expressing empty vector) .

Fig 5 Effect of overexpressing exogenous SMAD2 in SMAD2KO cells on the ME differentiation

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SMAD2和SMAD3在人胚胎干细胞分化过程中的不同调控作用

Different roles of SMAD2 and SMAD3 in human embryonic stem cell differentiation

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