通过定向内胚层的高效分化提高多能干细胞体外生成肝细胞样细胞的效率

doi:10.16352/j.issn.1001-6325.2023.07.1045

基础医学与临床 ›› 2023, Vol. 43 ›› Issue (7): 1045-1052.doi: 10.16352/j.issn.1001-6325.2023.07.1045

通过定向内胚层的高效分化提高多能干细胞体外生成肝细胞样细胞的效率

闫煜聪, 杨淑春, 贾玉艳, 黄粤^*

中国医学科学院基础医学研究所北京协和医学院基础学院医学遗传学系医学分子生物学国家重点实验室,北京 100005

收稿日期:2023-03-27 修回日期:2023-05-23 出版日期:2023-07-05 发布日期:2023-07-05
通讯作者: ^*huangyue@pumc.edu.cn
基金资助:
国家自然科学基金(32170840)

Improving the generation of hepatocyte-like cells from pluripotent stem cells via high-efficient differentiation to definitive endoderm

YAN Yucong, YANG Shuchun, JIA Yuyan, HUANG Yue^*

State Key Laboratory of Medical Molecular Biology,Department of Medical Genetics,Institute of Basic Medical Sciences CAMS, School of Basic Medicine PUMC,Beijing 100005,China

Received:2023-03-27 Revised:2023-05-23 Online:2023-07-05 Published:2023-07-05

摘要/Abstract

摘要： 目的优化多能干细胞(PSCs)体外定向分化为肝细胞样细胞(HLCs)的方法,提高得到HLCs的效率。方法根据体内肝细胞发育的规律,优化多能干细胞逐级诱导分化成HLCs过程中使用的细胞因子,制定出更高效率的肝细胞定向分化体系,并在人胚胎干细胞(hESCs)系H9上进行分化实验。通过对分化不同阶段的细胞进行形态学观察、标志基因表达水平的RT-qPCR检测以及流式细胞术测量分析,综合评估得到HLCs的效率。结果在hESCs向定向内胚层(DE)分化阶段的培养基中同时添加细胞因子激活素A、骨形态发生蛋白4(BMP4)和成纤维细胞因子2(FGF2),能显著提高该阶段标志基因的表达量,荧光示踪和流式分选也表明最终得到的HLCs的比例明显增加。结论 DE的高效分化能提高PSCs向HLCs的分化效率。

关键词: 分化, 多能干细胞, 定向内胚层, 肝细胞样细胞

Abstract: Objective To optimize the method of generating hepatocyte-like cells (HLCs) from pluripotent stem cells (PSCs)in vitro. Methods Considering the development of hepatocyte in vivo, a more efficient method was developed by optimizing biochemical factors to the stepwise differentiate PSCs into HLCs. Then the differentiation experiments were performed with human embryonic stem cell (hESC)line H9. The efficiency of the generation of HLCs was evaluated by morphological observation at different stages of differentiation. RT-qPCR and flow cytometry assay were used to examine the marker genes expression. Results A method of high-efficient hESCs differentiation to definitive endoderm (DE) was developed by adding cytokines activin A, bone morphogenetic protein 4(BMP4)and fibroblast growth factor 2(FGF2). It highly improved mRNA expression of marker genes at different stages. Venus fluorescence tracer and the results of flow-cytometry also showed that the population proportion of HLCs was significantly increased. Conclusions The high-efficient differentiation to DE improves differentiation efficiency of PSCs into HLCs.

Key words: differentiation, pluripotent stem cells, definitive endoderm, hepatocyte-like cell

中图分类号:

闫煜聪, 杨淑春, 贾玉艳, 黄粤. 通过定向内胚层的高效分化提高多能干细胞体外生成肝细胞样细胞的效率[J]. 基础医学与临床, 2023, 43(7): 1045-1052.

YAN Yucong, YANG Shuchun, JIA Yuyan, HUANG Yue. Improving the generation of hepatocyte-like cells from pluripotent stem cells via high-efficient differentiation to definitive endoderm[J]. Basic & Clinical Medicine, 2023, 43(7): 1045-1052.

参考文献 16

[1]	Satriano L, Lewinska M, Rodrigues PM, et al. Metabolic rearrangements in primary liver cancers: cause and consequences[J]. Nat Rev Gastroenterol Hepatol, 2019, 16: 748-766.
[2]	Sia D, Villanueva A, Friedman SL, et al. Liver cancer cell of origin, molecular class, and effects on patient prognosis[J]. Gastroenterology, 2017, 152: 745-761.
[3]	Forner A, Reig M, Bruix J.Hepatocellular carcinoma[J]. Lancet, 2018, 391: 1301-1314.
[4]	Luce E, Messina A, Duclos Vallee JC, et al. Advanced techniques and awaited clinical applications for human pluripotent stem cell differentiation into hepatocytes[J]. Hepatology, 2021, 74: 1101-1116.
[5]	Mun SJ.Generation of expandable human pluripotent stem cell-derived hepatocyte-like liver organoids[J]. J Hepatol, 2019, 71: 970-985.
[6]	Si Tayeb K.Highly efficient generation of human hepatocyte-like cells from induced pluripotent stem cells[J]. Hepatology, 2010, 51: 297-305.
[7]	Thompson WL, Takebe T.Human liver model systems in a dish[J]. Dev Growth Differ, 2021, 63: 47-58.
[8]	Touboul T.Generation of functional hepatocytes from human embryonic stem cells under chemically defined conditions that recapitulate liver development[J]. Hepatology, 2010, 51: 1754-1765.
[9]	Xinaris C, Brizi V, Remuzzi G.Organoid models and applications in biomedical research[J]. Nephron, 2015, 130: 191-199.
[10]	Ninomiya H, Mizwko K, Terada R, et al. Improved efficiency of definitive endoderm induction from human induced pluripotent stem cells in feeder and serum-free culture system in vitro[J].Cell Dev Biol Anim, 2015,51:1-8.
[11]	Loh KM,Ang LT, Zhang J, et al. Efficient endoderm induction from human pluripotent stem cells by logically directing signals controlling lineage bifurcations[J]. Cell Stem Cell, 2014,14:237-252.
[12]	Li S.Genetic and chemical screenings identify HDAC3 as a key regulator in hepatic differentiation of human pluripotent stem cells[J]. Stem Cell Reports, 2018, 11: 22-31.
[13]	Akbari S, Arslan N, Senturk S, et al. Next-generation liver medicine using organoid models[J]. Front Cell Dev Biol, 2019, 7: 345, doi: 10.3389/fcell.2019.00345.
[14]	Kretzschmar K, Clevers H.Organoids: modeling development and the stem cell niche in a dish[J]. Dev Cell, 2016, 38:590-600.
[15]	Fatehullah A, Tan SH, Barker N.Organoids as an in vitro model of human development and disease[J]. Nat Cell Biol, 2016, 18:246-254.
[16]	Li M, Izpisua Belmonte JC.Organoids-preclinical models of human disease[J]. N Engl J Med, 2019, 380: 569-579.

通过定向内胚层的高效分化提高多能干细胞体外生成肝细胞样细胞的效率

Improving the generation of hepatocyte-like cells from pluripotent stem cells via high-efficient differentiation to definitive endoderm

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