Automated analysis of single stem cells in microfluidic traps

We report a reliable strategy to perform automated image cytometry of single (non-adherent) stem cells captured in microfluidic traps. The method rapidly segments images of an entire microfluidic chip based on the detection of horizontal edges of microfluidic channels, from where the position of the trapped cells can be derived and the trapped cells identified with very high precision (>97%). We used this method to successfully quantify the efficiency and spatial distribution of single-cell loading of a microfluidic chip comprised of 2048 single-cell traps. Furthermore, cytometric analysis of trapped primary hematopoietic stem cells (HSC) faithfully recapitulated the distribution of cells in the G1 and S/G2-M phase of the cell cycle that was measured by flow cytometry. This approach should be applicable to automatically track single live cells in a wealth of microfluidic systems.

Deciphering Metabolic Regulation of Hematopoietic Stem Cell Fate

Mukul Girotra

Hematopoietic stem cells (HSCs) are responsible for life-long production of all mature blood cells. This unique characteristic makes them an ideal candidate for cell-based therapies to treat various hematological malignancies. Their extensive use in the clinic is often hampered due to insufficient number of cells obtained from donors. Countless attempts to expand HSCs in vitro have failed, primarily due to our inability to recapitulate key features of the native bone marrow microenvironment, termed niche, in a dish. The absence of important niche signals in vitro results in rapid proliferation of HSCs with a concomitant loss of their long-term multi lineage blood reconstitution potential. The niche in the bone marrow involves a highly complex network of physical and biochemical signals that, in concert with cell-intrinsic mechanisms, is believed to control HSC fate choices. Moreover, the hypoxic conditions in the niche presents an extreme metabolic environment, imposing HSCs to attain a distinct metabolic identity as compared to their differentiated progeny. However, despite decades of research it is currently very poorly understood how HSCs take the decision to either undergo self-renewal or differentiation. Insights into the mechanisms regulating HSC fate choices are key to design better strategies for HSC maintenance and expansion in vitro for use in clinical transplantations. The overall goal of this thesis is to employ innovative experimental tools to explore the role of metabolism in regulating HSC fate choices. In the first part of this thesis a versatile cell-tracking assay was developed to follow HSC divisions in vitro. A combination of cell tracking and immunostaining was used to systematically map phenotypic changes in HSCs up to four divisions, under defined culture conditions imposing specific fates. We found that the proportion of cells maintaining an HSC phenotype decreased with increasing number of cell divisions, supporting the notion that faster cycling results in HSC exhaustion. In the second part of this thesis, we for the first time identify a link between mitochondrial metabolism and HSC fate decision. Using flow cytometry and long-term blood reconstitution assays low mitochondrial activity was established as a reliable marker of functional HSCs, independent of their cell cycle state. Consequently, we could use this marker to reliable identify self-renewing HSCs from heterogeneous in vitro cultures. Strikingly, we found that HSC fate could be altered by artificially modulating their mitochondrial activity in vitro. These results suggest that mitochondrial activity is a determinant of HSC fate. The last part of this thesis describes an experimental paradigm to analyze in vivo niche-instructed fate choices in paired HSC daughter cells. Live single cell imaging revealed a significant increase in asynchronous divisions in niche activated HSCs compared to control cells, suggesting a possible involvement of niche-instructed asymmetric cell division program. Indeed, a significantly higher level of asymmetric gene expression was found in paired daughter cells arising from niche-instructed HSCs. This analysis led to the identification of 12 asymmetrically expressed genes, among them were key enzymes belonging to glycolytic and mitochondrial TCA cycle metabolic pathways. Altogether, this thesis successfully employed unique experimental strategies to provide an intriguing link between metabolism and HSC fate choices.

EPFL2015

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

Measurement of Mitochondrial Mass and Membrane Potential in Hematopoietic Stem Cells and T-cells by Flow Cytometry

George Coukos, Mukul Girotra, Alexandre Harari, Olaia Maria Naveiras Torres-Quiroga, Nicola Vannini

A fine balance of quiescence, self-renewal, and differentiation is key to preserve the hematopoietic stem cell (HSC) pool and maintain lifelong production of all mature blood cells. In recent years cellular metabolism has emerged as a crucial regulator of HSC function and fate. We have previously demonstrated that modulation of mitochondrial metabolism influences HSC fate. Specifically, by chemically uncoupling the electron transport chain we were able to maintain HSC function in culture conditions that normally induce rapid differentiation. However, limiting HSC numbers often precludes the use of standard assays to measure HSC metabolism and therefore predict their function. Here, we report a simple flow cytometry assay that allows reliable measurement of mitochondrial membrane potential and mitochondrial mass in scarce cells such as HSCs. We discuss the isolation of HSCs from mouse bone marrow and measurement of mitochondrial mass and membrane potential post ex vivo culture. As an example, we show the modulation of these parameters in HSCs via treatment with a metabolic modulator. Moreover, we extend the application of this methodology on human peripheral blood-derived T cells and human tumor infiltrating lymphocytes (TILs), showing dramatic differences in their mitochondrial profiles, possibly reflecting different T cell functionality. We believe this assay can be employed in screenings to identify modulators of mitochondrial metabolism in various cell types in different contexts.