Modeling hematopoietic stem cell dynamics in bioengineered niches

Bone marrow transplantation is a well-established medical procedure for the treatment of various hematologic diseases. However, the relatively low number of hematopoietic stem cells (HSCs) that can be harvested, especially from umbilical cord blood, limits even broader applicability of the procedure. A better understanding of the biology of HSCs, and in particular how these rare cells are regulated by microenvironmental niches in the bone marrow, could ultimately enable robust in vitro expansion of HSCs. In this thesis, several novel strategies were developed to study and manipulate HSC biology in vitro, through the regulation of key niche factors, cellular metabolism, and the complex multicellular crosstalk in a niche-mimicking context. First, we made use of gastruloids, an embryonic stem cell-based model of early mouse development, to mimic key aspects of embryonic hematopoiesis in vitro. Simultaneously with the establishment of a vascular network, we detected by immunophenotyping the emergence of primitive blood progenitor cells during the late developmental stages of gastruloids. These embryonic blood progenitors were spatially localized close to a vascular-like plexus in the anterior portion of the gastruloid. Colony-forming assays demonstrated an interesting differentiation potential of these cells. These data demonstrate the potential of gastruloids as an easily accessible in vitro model to study blood development in an embryo-like context. Second, a bioengineering approach was used to study HSC fate choices upon exposure to niche-specific ligands in a well-defined artificial environment. A combination of time-lapse microscopy and single-cell multigene expression analysis was used to define differentiation and cell-cycle states of mouse and human HSCs. Strikingly, selected artificial niches reduced proliferation and maintained the long-term multilineage potential of HSCs in vitro. These artificial niches hold significant potential for the study of mechanisms involved in HSC fate regulation. Third, the influence of the metabolism on fate choices of adult HSCs was studied, specifically focusing on fatty acid Î²-oxidation. Malonyl-CoA was used to reversibly block fatty acid Î²-oxidation in mouse HSCs. In vitro treatment of HSCs with malonyl-CoA promoted their expansion and increased lymphoid reconstitution upon in vivo transplantation. This study sheds new light on the role of the metabolic environment in HSC regulation. Surprisingly, exposure to only a single, readily available metabolite was found to be sufficient to strongly influence HSC behavior. Fourth, a miniaturized multicellular culture system was developed to mimic the hallmarks of the dynamics of HSCs in their native bone marrow. Primary human mesenchymal stem/progenitor cells and endothelial cells were aggregated in a high-throughput manner in biomimetic hydrogel microwells to form self-organizing bone marrow organoids. Immunostaining demonstrated the formation of branched vascular networks, rendering the organoids permissive for the homing of human hematopoietic stem and progenitor cells. These results suggest that bone marrow organoids may be a suitable platform for the modeling of physiological and clinically-relevant stem cell dynamics in vitro. Altogether, this thesis presents several innovative approaches for modeling key features of native stem cell microenvironments that will contribute to a better understanding of the biology of HSCs and their regulatory niche.

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

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

In vitro modeling of complex microenvironmental regulation of stem cells

Yoji Tabata

Although stem cells hold tremendous potential for clinical applications, their in vitro manipulation remains very challenging. In vivo, stem cells reside in intricate 3D microenvironments, termed niche, in which many local and systemic extrinsic factors are integrated by the cells to induce controlled fate choices. Niche factors must not only be presented to stem cells in the correct composition, but also in a dynamic and spatially heterogeneous manner to evoke the appropriate response. However, state-of-the-art in vitro culture platforms fail to capture such microenvironmental complexity. In this thesis, a novel hydrogel-based microchip technology was invented to recapitulate in vitro the complexity of native stem cell niches. In this system, stem cells can be exposed to gradients of soluble or extracellular matrix (ECM)-tethered biomolecules. As a first proof of principle, this platform was used to locally influence the behavior of mouse embryonic stem cells (mESC) by exposing them to soluble, matrix-tethered or cell-secreted leukemia inhibitory factor (LIF). mESCs grown in synthetic 3D gels were found to respond to LIF gradients in a spatially regulated manner. In two subsequent studies, the microchip technology was utilized to recapitulate embryonic processes that are characterized by highly dynamic and spatially controlled morphogenetic processes that can currently not be modelled in a 3D context in vitro. First, an in vitro model of the developing central nervous system was conceived. In the developing vertebrate neural tube, dorsoventral identity of neural progenitors is defined by counter gradients of sonic hedgehog (Shh) and bone morphogenetic protein 7 (BMP7). Using the developing neural plate of chick embryos as a model system, a dedicated microchip was developed for exposing the neural progenitors in these tissues to gradients of Shh and BMP7. This approach allowed achieving, for the first time, ex vivo dorsoventral patterning. Secondly, a similar approach was employed towards modeling early human development in vitro. To this end, colonies of human embryonic stem cells (hESC) were exposed to opposing gradients of BMP4 and Noggin which induced mesendoderm differentiation exclusively in specific regions of the multicellular constructs. This study revealed the power of the developed platform to spatially control hESCs self-patterning. In a last set of experiments, the microchip technology was applied to mimic the complex molecular and cellular make-up of the bone marrow niche that regulates the activity of hematopoietic stem cells (HSC). A microchip system was successfully designed to capture the main anatomic features of native bone marrow niches with spatially separated compartments that supported either HSC dormancy or activation. Taken together, this thesis presents a set of innovative microengineering concepts for recapitulating the spatiotemporal complexity of native stem cell microenvironments. The platforms introduced here represent important additions to the currently rather limited set of in vitro tools for the study of complex multicellular phenomena in developmental biology, tissue engineering and regenerative medicine.

EPFL2017