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

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.

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 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

EPFL2018

Development of a microfluidic on-chip reverse transcription module for the analysis of hematopoietic single stem cells

Viktoria Stepanova

Hematopoietic stem cells (HSCs) have a unique ability to self-renew and produce differentiated progeny throughout the entire life of the human body. However, present clinical use of HSCs in regenerative medicine is largely hindered by our limited understanding of the mechanisms that direct stem cell fate both in vivo and in vitro. Genetic profiling of single stem cells is an appealing method to gain a deeper understanding of these processes by dissecting the relationship between the cellular phenotype and its gene expression signature. To do so, classical methods rely on the use of bench-top assays which either lack the required efficiency for the analysis of low-copy transcripts such as transcription factors, or the ability to capture the dynamics of a stem cell population, for instance during cell division. Here, we report a novel microfluidic device for the on-chip reverse transcription (RT) of single stem cells coupled with a chip-to-world interface that enables reliable extraction of samples for virtually any bench-top assay. The designed microfluidic module allows the trapping of single cells, their lysis and mRNA capture, followed by solid-phase cDNA synthesis using the Dynabead® system. We have successfully validated and characterized all major stages of the on-chip RT protocol and obtained single-cell nested RT-PCR readouts with an efficiency superior to bench-top assays. Live single stem cells have also been extracted and analyzed via this method, thus demonstrating that the developed device can act as much as a Lab-on-a-Chip system as a micro-manipulator for single cell microfluidic handling. Furthermore, we have laid the foundations for the integration of our RT module with on-chip cell culture for automated stem cell progeny capture during division. Such a device will help to study heterogeneities not only within cell populations, but also within the progeny of a single cell.

2011