Molecular Diversity of Midbrain Development in Mouse, Human, and Stem Cells

Understanding human embryonic ventral midbrain is of major interest for Parkinson's disease. However, the cell types, their gene expression dynamics, and their relationship to commonly used rodent models remain to be defined. We performed single-cell RNA sequencing to examine ventral midbrain development in human and mouse. We found 25 molecularly defined human cell types, including five subtypes of radial glia-like cells and four progenitors. In the mouse, two mature fetal dopaminergic neuron subtypes diversified into five adult classes during postnatal development. Cell types and gene expression were generally conserved across species, but with clear differences in cell proliferation, developmental timing, and dopaminergic neuron development. Additionally, we developed a method to quantitatively assess the fidelity of dopaminergic neurons derived from human pluripotent stem cells, at a single-cell level. Thus, our study provides insight into the molecular programs controlling human midbrain development and provides a foundation for the development of cell replacement therapies.

A Novel 3D hydrogel based microfluidic cell culture platform

Keshav Krishnamani

Breakthroughs in health care over the coming decades may well consist of cell based therapeutics, potentially allowing the repair and replacement of damaged tissues and organs. Recently developed induced pluripotency methods as well as advances in stem cell biology show hope of such a reality. But such prospects come with the need to have a robust understanding and control over cell development and differentiation processes. Cells in the body develop in tightly controlled and specialized micro-environments that provide many complex cues and interactions that guide cell fate by directing gene expression. But current cell culture techniques that are most widely used to study the cell outside the body have not significantly changed from when first started over a century ago, using simple containers and flasks with plastic surfaces. Many studies have shown that three-dimensions (3D) over conventional 2D cell culture alone, showed to have increased levels of cell organization, morphology and function. By combining novel biomaterials and microfabrication methods, this project aims to create a tool that allows the study of cells within a simplified 3D environment. With the potential to build up the physical and soluble complexity of this environment in a tailor made fashion, a "dissection" of the functions and influences of external factors may be understood. Such a tool can also be used for the development of cell based models, highly relevant for drug discovery application as well as potentially for the development of cell based therapeutics. We have developed a novel microfluidic 3D cell culture platform that allows controlled soluble factor perfusion in an enzymatically formed and cell responsive PEG-based hydrogel. Soft lithographic processes were used to mold microchannels on a layer of hydrogel. In a second step, a flat hydrogel layer is placed on top to enclose and form the microchannels . The system allows the control over soluble factor delivery and it is able to perform long-term cell culture and generate user controlled gradients of soluble factors. With continuous perfusion, metabolic waste is constantly removed and cells are supplied with fresh medium. The platform also can allow creation of tailor made environments building up complexity in a controlled fashion. Higher throughput is also achieved by arraying individual culture chambers. The device was applied towards the culture of mouse embryonic stem cells (mESC) in order to demonstrate their self-renewal capacity on the microfluidic platform. The effects of some cytokine factors were also studied. Meaningful readouts such as fluorescence are easily used for analysis and cells can possibly be extracted from the device for further downstream analysis and processing. Through this device we show that microfluidics technologies combined with biomaterials enables the creation of platforms that are biologically relevant for the study and understanding of cell processes from cancer to stem cell development, processes which are fundamentally regulated by interactions with the microenvironment. The platform developed may also be used to develop human tissue models perhaps more relevant than current animal based testing, providing new tools for drug development and also potentially new approaches for developing cell based therapeutics

2009

KRAB/KAP1 Regulation of Embryonic Stem Cell Pluripotent Self-renewal

Andrea Corsinotti

KRAB-ZFPs constitute the largest family of transcription factors (TFs) encoded by the mouse genome and are known to interact with the co-repressor KAP1. KRAB/KAP1-mediated regulation is essential for several development- and ESC- specific functions, in particular regulation of pluripotent self-renewal. We performed an expression analysis in mouse pluripotent ESCs and differentiated cells. A large number of KRAB-ZFPs was expressed in pluripotent cells and many of them were differentially regulated in other cell types, suggesting that a set of KRAB-ZFPs can be involved in ESC-specific functions. We identified a set of candidate genes, including Zfp459, which show a pluripotency-specific expression pattern. Zfp459 depletion in ESCs induced transcriptional perturbation of pluripotency- and differentiation-associated genes, even if their ability to self-renew or to differentiate was not impaired, and its expression was not required for the formation of blastocysts ex vivo. To understand the precise role of Zfp459 in ESCs further experiments for the identification of its targets are still required. We used a lentiviral vector (LV) to over-express in ESCs ZFP57, which had been previously demonstrated to play a role in regulating genomic imprinting, in fusion with HA-tags. ChIP-seq experiments using a HA antibody demonstrated that ZFP57-HA binds all the known and predicted imprinted loci and identified a hexanucleotide consensus in most of the ZFP57 binding sites co-occupied also by KAP1 and its associated factor SetDB1. We also demonstrated that our LV based expression system could be exploited to perform ChIP-seq analyses in ESCs of TFs, such as Zfp459, for which specific antibodies are not available. Finally, when we cultured ESCs in different conditions, we observed that KAP1 recruitment upstream of Nanog, which had been previously observed, was dependent on the MAPK and GSK3 signaling pathways. We hypothesized that this could be due to the presence in these conditions of ESCs expressing heterogeneous Nanog levels. However, when Nanoghigh and Nanoglow cells were sorted, both the populations shown a comparable enrichment for KAP1 binding upstream of Nanog. The precise role for the KAP1-mediated regulation of Nanog in ESCs and its dependence on extrinsic signaling pathways remains unclear, even if we hypothesize that it can depend on the mono-/bi-allelic expression of Nanog in ESCs and during early development.

EPFL2012

Culture and Characterization of Human and Porcine Urothelial Progenitor Cells

Hans Mattias Larsson

An estimated 400 million people worldwide suffer from bladder diseases. However, little is known at the cellular level as to what causes or initiates many of these diseases. Therefore, this thesis has focused on understanding one cell type within the bladder wall, the urothelial cell. Urothelial cells are capable of keeping urine from entering the blood stream, which can be only a few microns away. Until now, an urothelial stem cell, or holoclone, with the capacity to self-renew and differentiate into fully mature urothelial cells has not been isolated. Therefore, we have tried to find a new adult epithelial stem cell population in vitro that could be useful for regenerative medicine applications. Cells were isolated from both human and porcine tissues from various locations within the urinary tract. We found that a porcine bladder urothelial holoclone had the capacity to self-renew extensively and to differentiate into fully differentiated urothelial cells. In addition, from single cell analysis, we also found a human urothelial holoclone but could not yet show that this human urothelial holoclone could differentiate. Thus, we believe the potential exists to isolate urothelial holoclones and use them in engineered tissue constructs. Furthermore, work was also focused on evaluating a molecular biotechnology tool to sort live cells based on mRNA expression as a novel means to isolate holoclones. Molecular beacons are hairpin structures that can sense mRNA present within a cell, and this approach to sort live cells has not been investigated in great detail. We designed and found a molecular beacon specific to Sox2 mRNA that could sort live Sox2-positive cells from a mixed cell population. This sorted Sox2-positive population showed specific phenotypic behavior that has only previously been shown with sorted cells based on protein surface markers. We hope that this work can aid in the design of tissue-engineered constructs that incorporate urothelial holoclones for patients that need bladder replacements. Thus, a molecular beacon sorting strategy could be used to isolate urothelial holoclones that could be embedded in engineered urotheliums. In addition, a molecular beacon strategy to analyze the number of urothelial holoclones that survive in an implanted urothelium could potentially enhance the success rate of implanted tissues for regenerative medicine applications in the bladder.

EPFL2012

A Novel 3D hydrogel based microfluidic cell culture platform

Keshav Krishnamani

2009

KRAB/KAP1 Regulation of Embryonic Stem Cell Pluripotent Self-renewal

Andrea Corsinotti

EPFL2012

Culture and Characterization of Human and Porcine Urothelial Progenitor Cells

Hans Mattias Larsson

EPFL2012