Arterial cells support the development of human hematopoietic progenitors in vitro via secretion of IGFBP2

Abstract

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.

Modelling the erythroblastic island niche of dyserythropoietic anaemia type IV patients using induced pluripotent stem cells

“…We identified 4 genes that encoded secreted factors in this category, ANGPTL7 , ABI3BP , FDCSP , and IGFBP6 . IGFBP6 was particularly interesting due to the emerging literature about the role of the IGFBP family member IGFBP2 in haematopoiesis and erythropoiesis ( Petazzi et al, 2022 ), and therefore IGFBP6 expression was confirmed via qRT-PCR and was significantly upregulated in 4OH-tamoxifen treated iKLF1-WT but not iKLF1-E325K (iCDA4.1 and iCDA4.20) iPSC-DMs ( Figure 5A ). IGFBP6 was added in combination with NRG1, NOV, CCL13 and TNFSF10 to UCB-derived CD34 + cells under the erythroid cell differentiation conditions.…”

<em>In Vitro</em> Model of Fetal Human Vessel On-chip to Study Developmental Mechanobiology

“…A limitation of the protocol is that the endothelial cells derived We have previously shown that iPSC-derived endothelial cells mimic the heterogeneous arteriovenous cellular identities 27 similar to that observed in the fetal dorsal aorta 28 , 29 , 30 . This is of particular importance in the context of vessel development and cellular specification, known to be controlled by blood circulation.…”