Ability to form Sox17-induced hematopoietic cell clusters varies among distinct hematopoietic sites during...

Definitive hematopoietic stem cells (HSCs) emerge within intra-aortic hematopoietic cell clusters (IAHCs) located in the dorsal aorta of the aorta-gonad-mesonephros (AGM) region during midgestation in the mouse embryo. Thereafter, HSCs migrate to the fetal liver (FL) and finally settle in the bone marrow (BM). We previously showed that the transcription factor Sox17 is expressed in IAHCs. Overexpression of the Sox17 gene in IAHC cells induces the formation of cell clusters in vitro that resemble IAHCs and retain hematopoietic potential. In addition, a previous report showed that Sox17-transduced hematopoietic stem/progenitor cells (HSPCs) in the BM maintained multipotency. However, whether the ability to form such cell clusters differs among hematopoietic sites has not been fully examined.

We examined whether viral overexpression of the Sox17 gene in HSPCs derived from the AGM region, the FL, and the BM leads to the formation of cell clusters. To identify the candidate genes involved in cluster formation, we performed RNA sequencing (RNA-seq) analysis on Sox17-ERT-transduced cells from the AGM region and the BM cultured with or without tamoxifen. We further analyzed the ability of one candidate gene, the Procr gene, to support cluster formation and in vitro hematopoietic activity.

A large number of multilineage colonies were observed in Sox17-ERT-transduced tamoxifen-treated cells in all tissues examined. However, BM cells generated significantly fewer clusters than embryonic tissues under identical experimental conditions. RNA-seq analysis revealed several genes that were highly expressed in Sox17-ERT-transduced tamoxifen-treated cells from the AGM region. The Procr gene, one of these genes, was expressed in IAHCs and was found to contribute to cluster formation and to be associated with the maintenance of in vitro hematopoietic activity in Sox17-transduced cells of the AGM region.

Our results revealed that the efficiency of cluster formation varies depending on the developmental origin of hematopoietic cells, suggesting that cluster formation and colony-forming activity can be partially dissociated during the transition from fetal to adult hematopoiesis. Moreover, the Procr gene appears to contribute to cluster formation and to be associated with enhanced in vitro hematopoietic activity in midgestation mouse embryos.