Microwell and impedance-based approaches to isolate single cells for life sciences

Cellular heterogeneity is ubiquitous in any cell population. Understanding these variations is key to answering fundamental questions in immunology, developmental biology, cancer or stem cell biology. For instance, single-cell in vitro and in vivo assays have been used to formally demonstrate the properties of stem and cancer cells.. Current strategies for single-cell isolation involve a trade-off between simplicity of use and single-cell yield. Moreover, few of these methods allows the traceability of the single-cell isolation for further quality control. In this thesis, we explored novel devices to isolate single cells for uses in the life sciences. We firstly investigated a new approach for gentle and size-based isolation of single cells. In this approach, cells move by gravity and size-selective trapping is achieved when a cell encounters a microwell. Models were established to predict the cell velocity and trapping on the tilted microwell array. The cell and cell trapping velocity were then experimentally characterized with respect to tilting angle and microwell size. This study showed that size-based trapping of individual cells is possible using tilted microwell arrays. Trapping of single cells in microwells was achieved and was shown to be influenced by the size of the wells and the tilting angle. Strikingly, this study showed that this approach is highly reliable, as the trapping of multiple cells within a unique well was rare (0.97% doublets). We secondly developed and validated an instrumented pipette for single-cell dispensing featuring a disposable sensing tip and a traceable process of the single-cell isolation, coupled to an impedance-based Quality Control (QC). A scalable manufacturing process was developed to produce batches of hundreds of disposable sensing tips at minimal costs. We then demonstrated the instrumented pipetteâs capacity to dispense single cells with a simple push of a button. Finally, we showed that impedance-based QC is a reliable method to improve confidence of the single-cell isolation process (single-cell efficiency after QC = 52 %). More importantly, none of the wells that passed QC contained more than one cell. Lastly, we demonstrated the instrumented pipetteâs ability to clone human epidermal stem cells (normal and diseased), human squamous carcinoma stem cells and rat hair follicle multipotent stem cells. In further investigations, we assessed through state-of-the-art in vitro and in vivo assays that the cells in the initiated clones maintained stem cell properties. The original devices developed in this thesis have shown to be suitable for use in the life science. Microwell array tilting is a gentle and simple approach to isolate single cells with a high level of confidence. Ultimately, this approach could be integrated into simple and cost-effective technologies able to isolate single targeted cells based on their morphological characteristics and without previous labelling. The instrumented pipette is an intuitive device coupled with inexpensive disposable sensing tips which are non-detrimental for stem cells. Furthermore, the instrumented pipette provides a reliable traceability and quality control of the single-cell isolation. To conclude, both devices will help to contribute to the dissemination of the single-cell approach in the life sciences.

Early Fate Choice Determines in vivo Engraftment and in vitro Behavior of Epidermal Stem Cells

François Gorostidi

Regenerative medicine aims at using stem cells to restore or establish lost, damaged, diseased, or aging tissues and organs function. The transplantation of cultured epidermal autograft (CEA) has saved the lives of many burned patients over the last 30 years, but the resulting epidermis is far from perfect, and clinical outcomes remain unpredictable. The engraftment of cultured epidermal stem cells is poorly understood, and, as a result, suboptimal. Using a large animal model, the pig, we recapitulated the clinical settings and outcomes of human CEA transplantation. With this model, we study the engraftment process, and we demonstrate that cultured epidermal stem cells often, but not always, favor the choice of differentiation over self-renewal when transplanted on full thickness wounds. Differences in this early fate choice are likely to explain the variability of clinical outcomes. We developed a system of in vitro live cell imaging (LCI) to study the fate choices of multiple individual cells in a microenvironment that is much more controlled than the grafting bed of a burned patient. We demonstrate that keratinocytes have a very heterogeneous behavior in vitro. With a fluorescent reporter of cell cycle progression (FUCCI), we show the influence of the cell cycle on early fate choices of cultured keratinocytes. By introducing a ROCK inhibitor in the culture medium, we show that keratinocyte’s early fate choices can be impacted so that they show greater clonogenicity and growth potential. The unique combination of large animal CEA transplantation with individual cell LCI demon- strate the critical role of early fate choice, both for epidermal stem cell engraftment and in vitro behavior. The possibility to influence the early fate choices of stem cells by modifying the microenvironment has a great potential to improve the engraftment and the ex vivo ampli- fication of stem cells, the two limiting steps of cell replacement therapy.

EPFL2012

Secreted semaphorin 5A suppressed pancreatic tumour burden but increased metastasis and endothelial cell proliferation

Anguraj Sadanandam, Stephan Wullschleger

BACKGROUND: Our earlier reports demonstrated that membrane-bound semaphorin 5A (SEMA5A) is expressed in aggressive pancreatic cancer cells and tumours, and promotes tumour growth and metastasis. In this study, we examine whether (1) pancreatic cancer cells secrete SEMA5A and (2) that secreted SEMA5A modulates certain phenotypes associated with tumour progression, angiogenesis and metastasis through various other molecular factors and signalling proteins. METHODS AND RESULTS: In this study, we show that human pancreatic cancer cell lines secrete the extracellular domain (ECD) of SEMA5A (SEMA5A-ECD) and overexpression of mouse Sema5A-ECD in Panc1 cells (not expressing SEMA5A; Panc1-Sema5-AECD; control cells - Panc1-control) significantly increases their invasion in vitro via enhanced ERK phosphorylation. Interestingly, orthotopic injection of Panc1-Sema5A-ECD cells into athymic nude mice results in a lower primary tumour burden, but enhances the micrometastases to the liver as compared with Panc1-control cells. Furthermore, there is a significant increase in proliferation of endothelial cells treated with conditioned media (CM) from Panc1-Sema5A-ECD cells and a significant increase in microvessel density in Panc1-Sema5A-ECD orthotopic tumours compared with those from Panc1-control cells, suggesting that the increase in liver micrometastases is probably due to increased tumour angiogenesis. In addition, our data demonstrate that this increase in endothelial cell proliferation by Sema5A-ECD is mediated through the angiogenic molecules - interleukin-8 and vascular endothelial growth factor. CONCLUSION: Taken together, these results suggest that a bioactive, secreted form of Sema5A-ECD has an intriguing and potentially important role in its ability to enhance pancreatic tumour invasiveness, angiogenesis and micrometastases. British Journal of Cancer (2012) 107, 501-507. doi: 10.1038/bjc.2012.298 www.bjcancer.com Published online 10 July 2012 (C) 2012 Cancer Research UK

Nature Publishing Group2012

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