In Vivo Pre-Instructed HSCs Robustly Execute Asymmetric Cell Divisions In Vitro

Hematopoietic stem cells (HSCs) are responsible for life-long production of all mature blood cells. Under homeostasis, HSCs in their native bone marrow niches are believed to undergo asymmetric cell divisions (ACDs), with one daughter cell maintaining HSC identity and the other committing to differentiate into various mature blood cell types. Due to the lack of key niche signals, in vitro HSCs differentiate rapidly, making it challenging to capture and study ACD. To overcome this bottleneck, in this study, we used interferon alpha (IFN alpha) treatment to "pre-instruct" HSC fate directly in their native niche, and then systematically studied the fate of dividing HSCs in vitro at the single cell level via time-lapse analysis, as well as multigene and protein expression analysis. Triggering HSCs' exit from dormancy via IFN alpha was found to significantly increase the frequency of asynchronous divisions in paired daughter cells (PDCs). Using single-cell gene expression analyses, we identified 12 asymmetrically expressed genes in PDCs. Subsequent immunocytochemistry analysis showed that at least three of the candidates, i.e., Glut1, JAM3 and HK2, were asymmetrically distributed in PDCs. Functional validation of these observations by colony formation assays highlighted the implication of asymmetric distribution of these markers as hallmarks of HSCs, for example, to reliably discriminate committed and self-renewing daughter cells in dividing HSCs. Our data provided evidence for the importance of in vivo instructions in guiding HSC fate, especially ACD, and shed light on putative molecular players involved in this process. Understanding the mechanisms of cell fate decision making should enable the development of improved HSC expansion protocols for therapeutic applications.