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

Abstract

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 (IFNα) 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 IFNα 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.

Symmetric and asymmetric activation of hematopoietic stem cells

“…Because molecular differences between daughter cells can be explained by stochastic processes, cell-cell interaction, signals from the microenvironment, or ACD, an important next step will be to integrate single-cell genomics and live-cell imaging to obtain further mechanistic insights. [63], CD133 [64], CDC42 [57] [67 ]: The low number of purified HSCs, their high motility and low adhesion pose major technical challenges to capture HSCs during divisions. Most candidates were therefore not analyzed in HSCs but other cell types.…”

“…To date, NUMB [28 ▪ ,33,59], CD53 [60], CD62L [60], CD63 [28 ▪ ,60], CD71 [60], AP2A2 [28 ▪ ,61], TIE-2 [62], Myosin-II [63], CD133 [64], CDC42 [57], LAMP-1 [28 ▪ ], LAMP-2 [28 ▪ ], LC3β [28 ▪ ], TMRM [65 ▪▪ ], COX8a [66 ▪▪ ], GLUT1 [67 ▪ ], JAM3 [67 ▪ ] and HK2 [67 ▪ ] were postulated to be asymmetrically inherited during HSPC divisions. However, due to technical shortcomings used for their identification, most require additional experimental confirmation [51 ▪ ]: The low number of purified HSCs, their high motility and low adhesion pose major technical challenges to capture HSCs during divisions.…”

“…It was recently shown that proliferative stress induces asymmetries in paired daughter cell proliferation and marker expression in vitro after in vivo administration of Interferon-α [67 ▪ ]. Although the authors suggest that Glut1, JAM3, and HK2 are asymmetrically inherited during HSC in vitro divisions, their levels were quantified 1–2 h after division.…”

JAM-C/Jam-C Expression Is Primarily Expressed in Mouse Hematopoietic Stem Cells

“…Very recent data have shown that JAM-C was asymmetrically distributed upon HSC division after in vivo IFNα activation, the paired daughter cells expressing JAM-C being the most immature one. 20 This demonstrates that JAM-C is a robust marker/tool to decipher mechanisms controlling HSC maintenance and activation within the BM microenvironment.…”

New Insights into Hematopoietic Stem Cell Expansion to Stimulate Repopulation of the Adult Blood System for Transplantation

“…After BM transplantation, only the successful homing of HSCs can ensure that the long-term repopulation of the blood system is initiated. Thus, improving low efficacy in the engraftment of clinical hematopoietic transplantation has become an important challenge in the treatment of previously incurable hematopoietic diseases over the past few decades [ 4 ]. For years, available sources of HSCs are the BM, circulating peripheral blood and umbilical cord blood (UCB).…”