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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 β-oxidation. Malonyl-CoA was used to reversibly block fatty acid β-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.

Stem cells are characterized by their dual ability to reproduce themselves (self-renew) and specialize (differentiate), yielding a plethora of daughter cells that maintain and regenerate tissues. In contrast to their embryonic counterparts, adult stem cells retain their unique functions only if they are in intimate contact with an instructive microenvironment, termed stem cell niche. In these niches, stem cells integrate a complex array of molecular signals that, in concert with induced cell-intrinsic regulatory networks, control their function and balance their numbers in response to physiologic demands. This progress report provides a perspective on how advanced materials technologies could be used (i) to engineer and systematically analyze specific aspects of functional stem cells niches in a controlled fashion in vitro and (ii) to target stem cell niches in vivo. Such “artificial niches” constitute potent tools for elucidating stem cell regulatory mechanisms with the capacity to directly impact the development of novel therapeutic strategies for tissue regeneration.

Stem cells are characterized by their dual ability to self- renew and differentiate, yielding essentially unlimited numbers of progeny that can replenish tissues with either high turnover such as blood and skin or contribute to the regeneration of organs with less frequent remodeling such as muscle. In contrast to their embryonic counterparts, adult stem cells can only preserve their unique functions if they are in intimate contact with an instructive microenvironment, termed niche. Stem cells integrate a complex array of niche signals that regulate their fate, keeping them in a relatively quiescent state during homeostasis, or controlling their numbers via symmetric or asymmetric divisions in response to the regenerative demands of a tissue. This chapter provides an overview of the current state of knowledge of structural and functional hallmarks of mammalian stem cell niches and offers a perspective on how bioengineering principles could be used to deconstruct the niche and providing novel insights into the role of its specific components in the regulation of stem cell fate. Such “artificial niches” constitute powerful tools for elucidating stem cell regulatory mechanisms that should fuel the development of novel therapeutic strategies for tissue regeneration.