1013 – CAPTURING THE TRANSCRIPTOME OF THE FIRST FUNCTIONAL HEMATOPOIETIC STEM CELLS

Abstract

Cell fate and behavior are regulated by dynamic intrinsic and extrinsic processes. These processes affect single cells to produce a fully integrated organism with distinct functional cell types and tissues. Experimental hematology provides insight into the independent iterations of blood cell development (primitive, definitive progenitor and definitive stem) in the different embryonic sites/times prior to adult hematopoietic system formation. Hematopoietic stem cell (HSC) generation occurs in a short developmental time window from a natural transdifferentiation of aortic endothelial-to-hematopoietic cells (EHT). The Gata2 transcription factor is a dosage-dependent intrinsic regulator of EHT controlled by signals from the extrinsic developmental niche. Hematopoietic progenitor cells (HPC) are also generated by EHT, although at an earlier stage. So, what mechanistically determines HPC or HSC fate during EHT? Vital time-lapse imaging reveals highly dynamic Gata2 expression changes in cells emerging during EHT. We used Gata2 levels to examine the "inner" HSC transcriptional regulatory network. Single cell iterative RNAseq, index-sorting and in vivo/vitro hematopoietic analyses show that the transcriptome of the first aortic HSCs is unique compared to HPCs. The observed heterogeneity and quantitative expression differences of pivotal EHT/HSC genes suggest that establishment of functional hematopoietic fate is a stochastic process. Additionally, dynamic changes occur in the aortic niche. Transient cell-cell interactions occur between motile yolk sac HPC-derived macrophages and emerging aortic hematopoietic cells to affect hematopoietic output/function. The extrinsic signals from this new pro-inflammatory macrophage subset, together with intrinsic dynamic Gata2 expression changes in endothelial cells could assist strategies for ex vivo HSC generation.