Deciphering Metabolic Regulation of Hematopoietic Stem Cell Fate

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.

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

Measurement of Mitochondrial Mass and Membrane Potential in Hematopoietic Stem Cells and T-cells by Flow Cytometry

George Coukos, Mukul Girotra, Alexandre Harari, Olaia Maria Naveiras Torres-Quiroga, Nicola Vannini

A fine balance of quiescence, self-renewal, and differentiation is key to preserve the hematopoietic stem cell (HSC) pool and maintain lifelong production of all mature blood cells. In recent years cellular metabolism has emerged as a crucial regulator of HSC function and fate. We have previously demonstrated that modulation of mitochondrial metabolism influences HSC fate. Specifically, by chemically uncoupling the electron transport chain we were able to maintain HSC function in culture conditions that normally induce rapid differentiation. However, limiting HSC numbers often precludes the use of standard assays to measure HSC metabolism and therefore predict their function. Here, we report a simple flow cytometry assay that allows reliable measurement of mitochondrial membrane potential and mitochondrial mass in scarce cells such as HSCs. We discuss the isolation of HSCs from mouse bone marrow and measurement of mitochondrial mass and membrane potential post ex vivo culture. As an example, we show the modulation of these parameters in HSCs via treatment with a metabolic modulator. Moreover, we extend the application of this methodology on human peripheral blood-derived T cells and human tumor infiltrating lymphocytes (TILs), showing dramatic differences in their mitochondrial profiles, possibly reflecting different T cell functionality. We believe this assay can be employed in screenings to identify modulators of mitochondrial metabolism in various cell types in different contexts.

2019

In vitro fate mapping of stem cell development at the single cell level

Vincent Trachsel

Hematopoietic stem cells (HSCs) are responsible for the continuous production of all blood cells. This unique ability has made it possible to successfully use HSCs in the clinical setting to remedy various blood disorders. However, despite six decades of research, the full potential of HSCs to treat patients with hematological malignancies is still restricted by the low number of functional stem cells that can be isolated from different sources. In vitro HSC expansion has been widely considered a potential solution but has proven to be extremely difficult due to the rapid loss of functional potential of cells in the culture outside of their natural microenviron-mental niche. In order to achieve robust HSC expansion in vitro, it is crucial to better under-stand the mechanisms that regulate HSC fate choices. Moreover, the absence of reliable mark-ers for identifying functional HSCs on a culture dish necessitates the use of expensive and time-consuming in vivo assays. The discovery of predictive phenotypic markers for HSC potential could address this problem. Many platforms for analyzing HSC fate at the single-cell level have been reported, but they generally suffer from poor cell viability, limited throughput, unreliable phenotyping by immunocytochemistry, and the difficulty of performing long-term cell cultures. Additionally, these platforms are usually fabricated from polydimethylsiloxane (PDMS), a poly-mer known to have several disadvantages for cell-based applications. This thesis addresses these shortcomings through the development of a robust microchip sys-tem for the high-resolution, live-cell analysis of hundreds to thousands of single stem cells and their daughter cells. The platform combines key advantages of microfluidic technology (for cell lineage analysis) and hydrogel microwell array technology (for gentle cell capture and long-term culture). The platform is fabricated from two different layers of hydrogel to create a closed ar-ray of grooves, where cellsâ movements are restricted and grow along one axis. This design en-ables an unambiguous tracking of a single cell and all of its progeny over several generations. These two layers are produced from different biocompatible hydrogels: poly (2-hydroxyethyl methylacrylate) / trimethylolpropane trimethacrylate (pHEMA-TMPTMA), a copolymer acting as a topographically structured cellular substrate that can be closed with a polyethylene glycol (PEG) hydrogel lid. These two parts are combined together and covalently bonded to each oth-er after the seeding of live cells into the grooves of the bottom part. To assess the potential of the platform for single stem-cell analyses, HSCs were cultured and their behavior (including cell fate, proliferation kinetics, and functionality) were thoroughly characterized. To analyze imaging data obtained from single-cell time-lapse experiments performed on the platform, an algorithm was developed to automatically identify and track single HSCs and their daughter cells, efficiently extracting various parameters, such as single-cell proliferation kinet-ics, cell fate choices, and lineage relationships. Mitochondria have recently been identified as key modulators of HSC function; however, their specific role in fate regulation has remained obscure. Therefore, the single-cell analysis plat-form was applied to measure the distribution of mitochondrial mass in dividing HSCs and their progeny to assess a potential correlation with HSC fate choices. This