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.

In vitro Fate Mapping of Single Hematopoietic Stem Cells

Aline Roch

The in vitro expansion of hematopoietic stem cells (HSC) for clinical applications is hampered by a rapid loss of HSC blood reconstitution capability in culture. While these rare cells can be stimulated to massively proliferate, cell divisions mostly result in differentiation caused by the lack of interactions with the native microenvironment, termed niche, in the bone marrow. Indeed, an increasing body of literature highlights the crucial role of instructive niche signals directing HSC fate in vivo. But how HSC fate, and in particular the delicate choice between self-renewal and differentiation divisions, is controlled by niche signaling cues remains unknown. Therefore, the goal of this thesis was to systematically characterize HSC fate decisions in vitro, and to utilize this knowledge to design artificial niches that maintain stemness. Differentiating HSCs first give rise to several transient populations of highly proliferative multipotent progenitors. Because reliable markers that distinguish HSCs from the earliest multipotent progenitors in vitro are lacking, it is currently not possible to discriminate between HSC self-renewal and commitment fate choices. The development and application of novel tools to probe HSC fate at the single cell level is therefore crucial, even more so because HSC populations are highly heterogeneous. In the first part of this thesis, a micro-engineered single cell analysis platform was employed to track in high-throughput the fate of individual HSCs by time-lapse microscopy. Single cell imaging revealed a surprising direct generation of megakaryocytes, cells that produce blood platelets, from phenotypic HSCs in the complete absence of a cell division. Megakaryocytic differentiation has previously been shown to occur very early in hematopoiesis and recent studies revealed the existence of a subpopulation of HSCs that is predetermined to undergo megakaryocytic differentiation. Our results suggest that some megakaryocyte progenitors may not be directly derived from HSCs but rather share the same cell surface marker repertoire such that they are indistinguishable from HSCs by state-of-the-art purification strategies. In order to discriminate between HSC self-renewal and commitment at single cell level, in the second part of this thesis gene expression signatures associated with the stem cell and multipotent progenitor cell states were established. Twelve differentially expressed genes marking the quiescent HSC state were identified, including four genes encoding cellcell interaction signals in the niche. Single cell multigene expression analysis performed on daughter cells, derived from single HSCs in serum-free culture, showed a rapid loss of the HSC identity with increasing number of cell divisions. In order to prevent such a dramatic loss of stemness in vitro, biomimetic microenvironments were engineered to display ligands of the newly identified niche components. Exposure of single HSCs to these artificial niches revealed a reduction in the mitotic activity of HSCs. Strikingly, in vivo transplantation of artificial niche-cultured HSCs resulted in long-term blood reconstitution of irradiated mice, demonstrating maintenance of functional HSC in vitro when exposed to critical cell-cell interaction signals found in native niches. Studies with invertebrates have shown that the pool of stem cells in vivo is controlled through asymmetric self-renewal divisions, resulting in two daughter cells with distinct fates, only one of which maintains stemness. Very little is known about asymmetric divisions of HSCs. Therefore, in the third part of this thesis, the symmetry of HSC divisions was systematically investigated in vitro. Using time-lapse imaging and single cell multigene expression analysis of paired daughter cells isolated by micromanipulation, approximately one third of all HSC was found to divide in an asymmetric fashion under serum-free culture conditions. Strikingly, paired daughter cells of cultured HSCs that were activated in vivo to exit their dormant state were found to divide mostly in an asymmetric fashion. These results shed light on the critical role of the in vivo microenvironment in specifying HSC fate and in particular asymmetric cell divisions. Altogether, this thesis demonstrates the power of single cell analyses to unravel stem cell fate decisions, and presents a novel approach to identify functional artificial niches to maintain HSCs in culture. This experimental paradigm should contribute to the development of better strategies to study and manipulate other stem cell types in culture. One exciting avenue is the expansion of human cord blood-derived HSCs, a cellular source that is particularly plagued by a lack of sufficient numbers for transplantation, with the aim of improving HSC therapies.

EPFL2014

Deciphering Metabolic Regulation of Hematopoietic Stem Cell Fate

Mukul Girotra

Hematopoietic stem cells (HSCs) are responsible for life-long production of all mature blood cells. This unique characteristic makes them an ideal candidate for cell-based therapies to treat various hematological malignancies. Their extensive use in the clinic is often hampered due to insufficient number of cells obtained from donors. Countless attempts to expand HSCs in vitro have failed, primarily due to our inability to recapitulate key features of the native bone marrow microenvironment, termed niche, in a dish. The absence of important niche signals in vitro results in rapid proliferation of HSCs with a concomitant loss of their long-term multi lineage blood reconstitution potential. The niche in the bone marrow involves a highly complex network of physical and biochemical signals that, in concert with cell-intrinsic mechanisms, is believed to control HSC fate choices. Moreover, the hypoxic conditions in the niche presents an extreme metabolic environment, imposing HSCs to attain a distinct metabolic identity as compared to their differentiated progeny. However, despite decades of research it is currently very poorly understood how HSCs take the decision to either undergo self-renewal or differentiation. Insights into the mechanisms regulating HSC fate choices are key to design better strategies for HSC maintenance and expansion in vitro for use in clinical transplantations. The overall goal of this thesis is to employ innovative experimental tools to explore the role of metabolism in regulating HSC fate choices. In the first part of this thesis a versatile cell-tracking assay was developed to follow HSC divisions in vitro. A combination of cell tracking and immunostaining was used to systematically map phenotypic changes in HSCs up to four divisions, under defined culture conditions imposing specific fates. We found that the proportion of cells maintaining an HSC phenotype decreased with increasing number of cell divisions, supporting the notion that faster cycling results in HSC exhaustion. In the second part of this thesis, we for the first time identify a link between mitochondrial metabolism and HSC fate decision. Using flow cytometry and long-term blood reconstitution assays low mitochondrial activity was established as a reliable marker of functional HSCs, independent of their cell cycle state. Consequently, we could use this marker to reliable identify self-renewing HSCs from heterogeneous in vitro cultures. Strikingly, we found that HSC fate could be altered by artificially modulating their mitochondrial activity in vitro. These results suggest that mitochondrial activity is a determinant of HSC fate. The last part of this thesis describes an experimental paradigm to analyze in vivo niche-instructed fate choices in paired HSC daughter cells. Live single cell imaging revealed a significant increase in asynchronous divisions in niche activated HSCs compared to control cells, suggesting a possible involvement of niche-instructed asymmetric cell division program. Indeed, a significantly higher level of asymmetric gene expression was found in paired daughter cells arising from niche-instructed HSCs. This analysis led to the identification of 12 asymmetrically expressed genes, among them were key enzymes belonging to glycolytic and mitochondrial TCA cycle metabolic pathways. Altogether, this thesis successfully employed unique experimental strategies to provide an intriguing link between metabolism and HSC fate choices.

EPFL2015

Metabolic and microenvironmental strategies to accelerate hematopoietic recovery post-aplasia

Vasco Ledebur de Antas de Campos

The procedure-associated mortality rate of bone marrow transplantations (BMT) remains close to 25%. The BMT is therefore only indicated for life-threatening diseases, with no replacement of this technology to be expected in the foreseeable future. Modest improvements such as the use of G-CSF to accelerate hematopoietic recovery significantly reduce mortality. Hematopoietic stem cells (HSC) reside in the bone marrow (BM) and are mainly quiescent, dividing only to self-renew. Via extracellular signals and interactions with other cells of the BM (microenvironment), a stable HSC pool is maintained, while hematopoietic progenitor cells (HPC) proliferate and differentiate to mature blood cell types. Marrow adipocytes (BMA) have recently been recognized as a hematopoietic regulatory entity, although the mechanisms by which it interacts with HSCs and HPCs (HSPC) in vivo, remain unknown. During the hematopoietic recovery period post-BMT, HSPCs increase their metabolism as a response to hematopoietic stress, partly modulated by the microenvironment. In this work we identified novel pharmacological approaches to accelerate hematopoietic recovery in the post-BMT period, addressed via two strategies: (i) by directly modulating HSC metabolism, and (ii) by regulating the differentiation of BMA. Increasing oxidative phosphorylation, combined with an impaired mitochondrial stress response, can severely compromise the regenerative capacity of HSCs. Here we demonstrate that the NAD+-boosting agent Nicotinamide Riboside (NR) reduces mitochondrial activity within HSCs through increased mitophagy, the recycling mechanism of damaged mitochondria. We observed in vitro NR treatment of HSCs to lead to increased asymmetric HSC divisions, which resulted in a significantly enlarged pool of progenitor cells, without HSC exhaustion. Dietary supplementation of NR to mice undergoing BMT accelerated blood recovery and improved survival. We therefore established a novel link between HSC mitochondrial stress, mitophagy and stem cell fate decision. Recent studies have suggested BMA maintain HSCs quiescent, which, may be detrimental to the increased expansion of HPCs in the recovery phase post-BMT. BM stromal cells (BMSC) are the cellular precursors of both adipocytes and osteoblasts, and they have been strongly implicated in supporting hematopoiesis by secreting cytokines such as SCF and Cxcl12. We observed that adipocytes quickly accumulate the marrow space of mice undergoing BMT, followed by an expansion hematopoiesis-supportive multipotent Sca1+/CD24+ stromal cells, coinciding with hematopoietic recovery. We developed a novel screening platform using Digital Holographic Microscopy (DHM), quantifying in vitro adipocytic differentiation non-invasively, allowing for follow-up co-cultures with HSPCs. Using the OP9 BMSC-line, we modulated adipocyte differentiation prior to co-culture using a pharmacological approach and observed that high adipocytic content was associated to a decrease in HPC expansion. We perfomed a screening of over 4000 FDA-approved drugs and natural compounds and identified one to efficiently prevent adipocytic differentiation and maintain the hematopoietic supportive capacity of OP9 stroma. Altogether, this work opens the door to alternative strategies accelerating hematopoietic reconstitution and unveils the potential to improve recovery of patients undergoing BMT.

EPFL2019