In vitro Fate Mapping of Single Hematopoietic Stem Cells

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.

Modeling hematopoietic stem cell dynamics in bioengineered niches

Sonja Giger

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.

EPFL2019

Microtechnologies to Map the Fate of Single Stem Cells and their Progeny

Stefan Kobel

Since stem cells have the unique ability to produce more of themselves (i.e. to "self-renew") and to generate specialized tissue cells, they are an ideal source of cells for regenerative medicine and in vitro tissue models. In order to fully exploit this potential, we have to better understand the mechanisms that regulate stem cell behavior at a single cell level. However, current in vitro methodologies to study single cells are limited by their throughput, do not afford to distinguish the fate of daughter cells or track the progeny of a single cell over long time periods. This thesis addresses these shortcomings by developing miniaturized devices to handle and analyze single live stem cells. To this end, a micro-structured platform was first established that consisted of miniaturized wells with diameters of 100 µm that were arranged in a regular grid. Single hematopoietic stem cells (HSCs) were then seeded into these "microwell arrays" and, since the HSCs could not leave the microwells, they could be tracked over many days, and their dynamics and proliferation rates could be studied. However, in order to analyze the daughter cells of these single HSCs individually, the daughter cells have to be physically separated by micromanipulation. For this purpose, a microdevice was developed to reliably manipulate single cells using hydrodynamic single cell trapping. This platform was thoroughly characterized using computational simulations combined with particle flow velocimetry and live-cell time-lapse microscopy. The optimization of design parameters towards increasing trapping efficiency ensured a nearly perfect efficiency of single cell trapping (97%) and a successful re-capture of daughter cells generated by dividing mother cells. Furthermore, it was demonstrated that cell trapping under very low flow is sufficiently gentle to enable long-term cell trapping in the microfluidic environment. To identify and track captured cells in these microfluidic single cell traps, automated image analysis tools were developed. A reliable and fast algorithm was generated to identify the borders of microchannels that were indicative for the position of single cell traps. These detected edges were used to individualize the single cell traps with a sub-pixel resolution such that it was possible to segment single HSCs on the chip by thresholding. Accordingly, single HSCs were discovered with efficiencies as high as >95%, allowing the automated tracking of microfluidic single cell trapping, as well as the detection of cell cycle phases of trapped single HSCs engineered with dual fluorescence reporter system marking G1 and S/G2-M. To actively micromanipulate single cells for downstream cell-fate analyses, on-chip valves were integrated on the microfluidic platform to precisely control the direction of medium flow in the microfluidic channels. This approach enabled a proof-of-concept study to demonstrate a more complex single cell manipulation: Cells were first captured in single cell traps and transferred into a culture chamber for clonal expansion. Subsequently, the progeny of these single cells was removed from the culture chamber and trapped in a series of single cells traps. Importantly, since these traps could be addressed individually, a reliable physical separation of the daughter cells was achieved. To enable individual analyses of these separated daughter cells, the last aim of this thesis was to interface a microfluidic chip with standard multititer plates in order to efficiently recover the cells or the cDNA of the single cell generated on-chip from extracted mRNA. For this purpose, the bottom of the chip was equipped with an array of access holes that matched the layout of a 1536-well plate. These access holes were linked to a single cell trap via an analysis chamber, where the HSCs were lysed and their mRNA was reverse transcribed using bead-coupled primers. In this vain, the obtained cDNA was coupled to these beads that could then removed from the chip into a 1536-well by centrifugation. These beads could then be analyzed using standard bench-top PCR machines. In conclusion, this thesis presented several microfluidic modules to handle and analyze single stem cells in high-throughput. In a proof-of-concept study, these modules were applied to automatically capture single cells, culture them, and subsequently separate the daughter cells for downstream cell-fate analyses, thereby greatly enhancing the throughput compared to manual micromanipulation. During the development of this system, it became apparent how crucial reliable and gentle cell handling is to perform complex experimental tasks with live cells on microfluidic chips. The further integration of such microfluidic modules into multi-functional lab-on-a-chip devices will open the door to experimental paradigms previously not imaginable and promises novel insights in stem cell biology.

EPFL2012

The role of recruited bone-marrow derived mesenchymal stem cells in the establishment of the tumor stroma and a growth-supportive environment

Jean-Paul Abbühl

The Mesenchymal Stem Cell (MSC) is a self-renewing multipotent progenitor originally found in the bone marrow. It has drawn strong interest from translational research because of the multipotency of MSCs, their immunosuppression, their intrinsic homing and their promotion of wound healing in human patients. Currently available methods rely on in vitro culture for the isolation and expansion of MSCs and most studies used these cells for their investigation. Fibroblasts constitute the connective tissue and support epithelial or hematopoietic cells. Although they have already been extensively investigated, it is not clear whether stem cells exist in vivo which specifically regenerate fibroblasts. MSCs are a potential candidate, because of their fibroblastic shape in vitro, their differentiation towards several mesodermal lineages and their supportive function. We aim to characterize the in vivo differentiation potential of MSCs toward fibroblasts in normal and tumor tissues. A new transplantation approach was developed in order to transfer labeled MSCs in the bone marrow and assess their recruitment and lineage contribution in vivo. Long-term engraftment was accomplished by isolating and transplanting these cells within one day. To this end, strategies for their purification, preconditioning of the host and transplantation methodologies were established during this thesis. We were able to enrich MSCs over 1000 fold by combining a new extraction protocol, a negative selection by MACS and a positive selection by FACS. Cloning of sorted MSCs was performed to confirm their multipotency at single cell level. GFP-expressing MSCs were able to engraft in host mice tolerant for GFP and their expansion in vivo was stimulated by block of GSK3 activity. Altogether, the chimeric MSC model was used to track the recruitment of bone-marrow derived MSCs into spontaneous breast tumors. The tumor microenvironment encompasses several non-tumorigenic cells interacting with cancer cells. Among them, cancer-associated fibroblasts are known to participate in carcinogenesis. Whilst this heterogeneous stromal population has been extensively investigated, their source(s) remain elusive. I now propose tumoral MSCs as one source of cancer-associated fibroblast, based on their ability to self-renew and reconstitute stromal heterogeneity upon serial transplantation. Finally, I introduced the new concept that the multipotency of MSCs is subjugated by cancer cells to meet their requirements for enhanced tumor progression and prepare for metastasis. I found MSCs present in the tumor microenvironment to differentiate into cancer-associated osteoblasts and produce hydroxyapatite-mineralization in vivo. Microcalcification is an accepted poor prognostic feature in breast cancer patients but its function and origin is not documented. I showed that hydroxyapatite was able to promote proliferation of tumor cells and increased intracellular calcium concentration. Lastly, cancer-associated osteoblast and hydroxyapatite were able to promote tumor growth and metastasis in vivo. To conclude, bone marrow derived MSCs are circulating stem cells that participate in the establishment of the pathogenic stroma in breast cancer. Owing to their multipotency, MSCs give rise to cancer-associated osteoblasts that induce pro-malignant mineralization in situ. Subsequently, cancer cells harbor increased intracellular calcium concentration, enhanced proliferation and metastasis. We foresee the development of new stroma-targeted therapies that prevent the mineralization of tumor tissue and reduce metastasis in breast cancer patients.

EPFL2014