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

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

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

Hydrogel Microfluidics to Control Stem Cell Fate

Steffen Cosson

Biomolecular signaling is of utmost importance in governing many biological processes such as morphogenesis during tissue development where biomolecules regulate key cell-fate decisions. In vivo, these factors are presented in a spatiotemporally tightly controlled fashion and in the context of a soft and hydrated microenvironment. Although state-of-the-art microfluidic technologies allow precise biomolecule delivery in time and space, long-term (stem) cell culture at the micro- scale is far from ideal due to issues related to medium evaporation, limited space for cell growth, shear stress and a lack of cell-instructive microenvironments that might adversely impact cell-fate. As a result, microfluidic cell culture systems are not yet suitable to unravel complex multicellular phenomena. This may explain why they are not yet widely used in biology laboratories. Consequently, the overall goal of this thesis was to overcome this technology gap by developing next generation microfluidics through a combination of microfabrication and smart biomaterials. A special emphasis was placed on decoupling biomolecule presentation at micro-scale from macro-scale cell culture in order to enable long-term stem cell culture within user-friendly multiwell plate formats. First, a novel microfluidic concept was invented to rapidly immobilize linear protein gradients on the surface of poly(ethylene glycol) (PEG)-based hydrogels whose biophysical properties are reminiscent of natural extracellular matrices. This method allows efficient capture of steady-state gradients of tagged proteins (e.g. using Fc or biotin tags) in just a few minutes on engineered hydrogels that display the corresponding auxiliary proteins (e.g. ProteinA or NeutrAvidin). The selectivity and orthogonality of the chosen binding schemes enables the formation of parallel and orthogonal overlapping gradients of multiple proteins, which is impossible using existing platforms. After patterning, the microfluidic chip can be readily removed from the gel for cell culture applications. Using quantitative single-cell time-lapse microscopy, this platform was validated here by probing the effect of fibronectin concentration on the directionality and speed of cell migration. Next, the resolution, flexibility and throughput of the protein patterning on hydrogels was substantially enhanced by the introduction of hydrodynamic flow focusing, that is, the generation of user-defined patterns by spatially controlled step- wise deposition of biomolecules. To this end, a microfluidic device was conceived that enabled the generation of arrays of parallel and crossed overlapping gradients. Application of the platform to generate gradients of immobilized leukemia inhibitory factor (LIF) showed an influence of the LIF concentration on ESC self-renewal. This platform should be useful to systematically probe the effect of biomolecule dose, singly or in combinations, on stem cell behavior in vitro; however, it lacks the ability to dynamically vary the presentation of biomolecules that might be crucial to control stem cell fate towards establishing functional in vitro tissue models. To address this limitation, in the last part of the thesis work a hydrogel-based microfluidic chip was developed that decouples macro-scale cell culture on the gel surface from the precise spatiotemporal biomolecule delivery at the micro-scale, i.e. through the gel layer. The hydrogel chip, used here as a simple insert that is compatible with conventional multiwell plate formats, was optimized to support long- term ESC maintenance in both adherent format and as uniformly sized ESC aggregates termed embryoid bodies. The platform was successfully used for the spatially controlled neuronal commitment of ESC via delivery of gradients of the morphogen retinoic acid. Taken together, in this thesis several innovative microengineered cell culture platforms are presented which enable, perhaps for the first time, the long-term culture and manipulation of stem cell fate at the micro-scale. These systems should be useful to systematically probe in vitro the effect of biomolecule dose and delivery dynamics on stem cell behavior, ultimately facilitating the development of complex in vitro tissue models.

EPFL2012

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