Proliferation of hematopoietic stem cells differentiated from embryonic stem cells via sustained Wnt pathway activation

doi:10.3969/j.issn.2095-4344.2014.06.010

Abstract

Abstract:

BACKGROUND:A variety of embryonic stem cells induction and differentiation systems have been established so far, while the research that promotes embryonic stem cells to differentiate into hematopoietic stem cells is still at an initial stage, and the induction efficiency needs to be improved.

OBJECTIVE: To active the Wnt/β-catenin signal pathway in mouse embryonic stem cells with exogenous win3a as an inducer, and then to observe whether the activation of this pathway will promote the directional differentiation of embryonic stem cells into hematopoietic progenitor cells.

METHODS:The ES-E14TG2a mouse stem cells were cultured with the exogenous wnt3a (100 µg/L) for 21 days, the content of β-catenin was tested by cell immunofluorescence and western blot, and expression of Wnt downstream target gene was detected by quantitative reverse transcription PCR to determine the activation of Wnt/β-catenin signal pathway. Single-layer adherent culture method was used to induce the directional differentiate of above-mentioned cells into hematopoietic stem cells, and detection of hematopoietic development associated surface marker CD34⁺/Sca-1⁺ was achieved by flow cytometry;

meanwhile, the expression of hematopoietic associated gene was measured by quantitative reverse transcription PCR.

RESULTS AND CONCLUSION: We found that β-catenin accumulated in ES-E14TG2a mouse stem cells after cultured with wnt3a (100 µg/L) for 21 days; the expressions of Wnt downstream target genes such as Pitx2, Frizzled, Sox17 and Oct4 showed the different degrees in increase, meaning the activation of Wnt/β-catenin signal pathway. Furthermore, during the time that we used single-layer adherent culture method to induce hematopoietic stem cells, the CD34⁺/Sca-1⁺ cells accounted for 20.2% of total cells at day 14, and control cells only accounted for 11.9%. Again, expression quantity of hematopoietic associated gene BMP4, FLK2 and CD34 increased while Smad5 was suppressed significantly. Our data suggest that sustaining action by wnt3a will active Wnt/β-catenin signal pathway, and also promote the directional differentiation of ES-E14TG2a mouse stem cells into hematopoietic progenitor cells.

中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程

全文链接：

Key words: stem cells, embryonic stem cells, hematopoietic stem cells, cell differentiation, cell proliferation

CLC Number:

R394.2

Lin Fang, Jin Jing-jun, Zhang Tao, Ji Bin-feng, Chen Yong. Proliferation of hematopoietic stem cells differentiated from embryonic stem cells via sustained Wnt pathway activation[J]. Chinese Journal of Tissue Engineering Research, 2014, 18(6): 880-887.

Figures/Tables 5

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