Arterial cells support the development of human hematopoietic progenitors in vitro via secretion of IGFBP2
Arterial cells support the development of human hematopoietic progenitors in vitro via secretion of IGFBP2
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DOI：
10.1101/2022.10.04.510611
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发表时间：
2022-10
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影响因子：
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通讯作者：
P. Petazzi;T. Ventura;F. P. Luongo;Alisha May;H. Taylor;N. Romanò;L. Forrester;P. Menéndez;A. Fidanza
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文献类型：
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作者：
P. Petazzi;T. Ventura;F. P. Luongo;Alisha May;H. Taylor;N. Romanò;L. Forrester;P. Menéndez;A. Fidanza
造血干细胞和祖细胞在发育过程中从位于各个部位的造血内皮发育，包括造血干细胞（HSC）出现的背主动脉。这一过程已被证明是特别具有挑战性的重演体外从多能干细胞和进一步的研究，需要在体外查明失踪的刺激。在这里，我们比较了iPSC衍生的内皮细胞和体内HSC引发的生血内皮细胞，并确定了9个转录因子在体外产生的细胞中以显著较低的水平表达。使用一种新的DOX诱导的CRISPR激活系统，我们在体外分化过程中诱导了这些基因的表达。为了研究由靶基因的激活诱导的表型变化，我们采用单细胞RNA测序与在测序管道内可检测的工程化gRNA组合。我们的数据显示动脉内皮细胞的显著扩增与较高的体外祖细胞活性相关。扩张的动脉簇以IGFBP2的高表达为标志，并且它与显示细胞周期进展增加的生血簇不同。我们证明，向分化的PSC中添加IGFBP 2会产生更多数量的功能性祖细胞，从而确定动脉细胞通过IGFBP 2旁分泌信号传导对血液祖细胞的出现发挥支持作用。
Hematopoietic stem and progenitor cells develop from the hemogenic endothelium located in various sites during development, including the dorsal aorta from where Hematopoietic Stem Cells (HSCs) emerge. This process has proven especially challenging to recapitulate in vitro from pluripotent stem cells and further studies are needed to pinpoint the missing stimuli in vitro. Here, we compared iPSC-derived endothelial cells and in vivo HSC-primed hemogenic endothelium and identified 9 transcription factors expressed at significantly lower levels in cells generated in vitro. Using a novel DOX-inducible CRISPR activation system we induced the expression of those genes during in vitro differentiation. To study the phenotypical changes induced by the activation of target genes, we employed single cell RNA sequencing in combination with engineered gRNA that are detectable within the sequencing pipeline. Our data showed a significant expansion of arterial-fated endothelial cells associated with a higher in vitro progenitor activity. The expanded arterial cluster was marked by high expression of IGFBP2 and it was distinct from the hemogenic cluster that showed increased cell cycle progression. We demonstrated that the addition of IGFBP2 to differentiating PSCs resulted in a higher number of functional progenitors, identifying the supporting role of arterial cells play to the emergence of blood progenitors via IGFBP2 paracrine signalling.