应用流式细胞Index Sorting技术研究小鼠胚胎生血内皮细胞
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摘要
目的:应用流式Index Sorting技术鉴定小鼠胚胎期d10(E10)的主动脉中生血内皮细胞(HEC)的表面分子表达特征,并结合体外孵育实验及免疫组化染色验证HEC的生血潜能。方法:已有研究显示细胞表面分子Kit和CD47均能标记造血相关群体。在此基础上,应用流式细胞Index Sorting技术分选单个分子表型为CD41~-CD43~-CD45~-CD31~+Kit~+的主动脉内皮细胞,并与基质细胞OP9-DL1共孵育诱导培养7 d,进而结合单个内皮细胞的流式分选特征参数,及诱导后生成CD45~+造血细胞和CD31~+内皮细胞的效率,综合回溯分析Kit和CD47富集HEC的效果。结果:具有生血潜能的HEC均富集在CD47~+内皮细胞群体中,CD47分子将HEC群体的精度提高1.5倍;CD47~+内皮群体中,仍有60%以上的内皮不具备生血能力,提示进一步探索寻找富集HEC表面标志的必要性。结论:应用流式细胞Index Sorting技术发现CD47分子能显著富集小鼠胚胎期HEC,为进一步精准富集并研究HEC提供了重要的技术手段。
Objective:The aortic vascular endothelial cells of mouse embryonic day 10(E10) contains extremely rare hemogenic endothelial cells(HEC) population, and there is still no effective cell surface molecule for enrichment. We try to use flow cytometric Index Sorting technique to identify the surface marker molecules expression characteristics of HEC during this period, and to use the in vitro co-culture assay and immunohistochemical staining technique to verify the hematopoietic differentiation potential of HEC.Methods:Previous studies have shown that cell surface molecules Kit and CD47 can label hematopoietic-competent populations. On this basis, flow cytometric Index Sorting technique is used to separate single aortic endothelial cells with molecular phenotype of CD41~-CD43-CD45~-CD31~+ Kit~+ ,and the cells were incubated with stromal cell OP9-DL1 for 7 days. Finally, combined with the flow sorting characteristic parameters of individual endothelial cells and the efficiency of induced CD45~+ hematopoietic cells and CD31~+ endothelial cells, the effects of Kit and CD47 on HEC enrichment were comprehensively analyzed retrospectively.Results:Retrospective expression analysis of flow cell Index Sorting data and in vitro hematopoietic induction showed that HEC with hematopoietic potential were enriched in CD47~+ endothelial cell population, and CD47 molecules significantly enriched the accuracy of HEC population by more than 1.5 fold. At the same time, it is also found that more than 60% of the CD47~+ endothelium can not be induced to hematopoietic cells, suggesting that it is necessary to further explore more accurate surface markers for HEC.Conclusion:Using flow cytometry Index Sorting technology, for the first time we found that CD47 molecule can significantly enrich HEC in mouse embryo, which provides an important technical method for further accurate enrichment and research of HEC.
Objective:The aortic vascular endothelial cells of mouse embryonic day 10(E10) contains extremely rare hemogenic endothelial cells(HEC) population, and there is still no effective cell surface molecule for enrichment. We try to use flow cytometric Index Sorting technique to identify the surface marker molecules expression characteristics of HEC during this period, and to use the in vitro co-culture assay and immunohistochemical staining technique to verify the hematopoietic differentiation potential of HEC.Methods:Previous studies have shown that cell surface molecules Kit and CD47 can label hematopoietic-competent populations. On this basis, flow cytometric Index Sorting technique is used to separate single aortic endothelial cells with molecular phenotype of CD41~-CD43-CD45~-CD31~+ Kit~+ ,and the cells were incubated with stromal cell OP9-DL1 for 7 days. Finally, combined with the flow sorting characteristic parameters of individual endothelial cells and the efficiency of induced CD45~+ hematopoietic cells and CD31~+ endothelial cells, the effects of Kit and CD47 on HEC enrichment were comprehensively analyzed retrospectively.Results:Retrospective expression analysis of flow cell Index Sorting data and in vitro hematopoietic induction showed that HEC with hematopoietic potential were enriched in CD47~+ endothelial cell population, and CD47 molecules significantly enriched the accuracy of HEC population by more than 1.5 fold. At the same time, it is also found that more than 60% of the CD47~+ endothelium can not be induced to hematopoietic cells, suggesting that it is necessary to further explore more accurate surface markers for HEC.Conclusion:Using flow cytometry Index Sorting technology, for the first time we found that CD47 molecule can significantly enrich HEC in mouse embryo, which provides an important technical method for further accurate enrichment and research of HEC.
引文
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[11]Schulte R,Wilson N K,Prick J C,et al.Index sorting resolves heterogeneous murine hematopoietic stem cell populations[J].Exp Hematol,2015,43(9):803-811.
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[13]Mark P,Tolk A,Lux C A,et al.Difference in proliferation potential and gene expression pattern of c-kit+HSC subpopulations[J].J Stem Cells Regen Med,2010,6:99-100.
[2]Dzierzak E,Bigas A.Blood development:hematopoietic stem cell dependence and independence[J].Cell Stem Cell,2018,22(5):639-651.
[3]Dzierzak E,Speck N A.Of lineage and legacy:the development of mammalian hematopoietic stem cells[J].Nat Immunol,2008,9(2):129-136.
[4]Zovein A C,Hofmann J,Lynch M,et al.Fate tracing reveals the endothelial origin of hematopoietic stem cells[J].Cell Stem Cell,2008,3(6):625-636.
[5]Samokhvalov I M,Samokhvalova N I,Nishikawa S.Cell tracing shows the contribution of the yolk sac to adult haematopoiesis[J].Nature,2007,446(7139):1056-1061.
[6]Bertrand J Y,Chi N C,Santoso B,et al.Haematopoietic stem cells derive directly from aortic endothelium during development[J].Nature,2010,464(7285):108-111.
[7]Boisset J C,Cappellen W,Andrieu-Soler C,et al.In vivo imaging of haematopoietic cells emerging from the mouse aortic endothelium[J].Nature,2010,464(7285):116-120.
[8]Solaimani Kartalaei P,Yamada-Inagawa T,Vink C S,et al.Whole-transcriptome analysis of endothelial to hematopoietic stem cell transition reveals a requirement for Gpr56 in HSC generation[J].J Exp Med,2015,212(1):93-106.
[9]Swiers G,Baumann C,O′Rourke J,et al.Early dynamic fate changes in haemogenic endothelium characterized at the single-cell level[J].Nat Commun,2013,4:2924.
[10]Zhou F,Li X,Wang W,P.et al.Tracing haematopoietic stem cell formation at single-cell resolution[J].Nature,2016,533(7604):487-492.
[11]Schulte R,Wilson N K,Prick J C,et al.Index sorting resolves heterogeneous murine hematopoietic stem cell populations[J].Exp Hematol,2015,43(9):803-811.
[12]Wilson N K,Kent D G,Buettner F M,et al.Combined single-cell functional and gene expression analysis resolves heterogeneity within stem cell populations[J].Cell Stem Cell,2015,16(6):712-724.
[13]Mark P,Tolk A,Lux C A,et al.Difference in proliferation potential and gene expression pattern of c-kit+HSC subpopulations[J].J Stem Cells Regen Med,2010,6:99-100.