Conversion of mouse neural stem cells into glioma cancer stem cells

Gliomas are the most common and lethal primary adult human brain cancers. The most lethal form of glioma is glioblastoma multiforme (GBM). Multiple apparently differentiated cell types are present within these tumours, including varying proportions of astrocytes and oligodendrocytes. The identification of new GBM subtypes, the role of the cell-of-origin and the role of commonly disrupted genes are active areas of investigation. Of particular interest to this laboratory is the recent realisation that GBMs may be driven by and initiated from tumorigenic stem cells. Our goal was to develop a mouse model of the disease based on genetic manipulation of in vitro neural stem (NS) cell lines. Importantly, this would enable us to define the contribution of each oncogenic pathway in altering the self-renewal of these cells. The original hypothesis was that the simultaneous activation of three critical pathways (RTK/PI3K, Rb and p53) would be required to confer a glioma-like phenotype. However, surprisingly we found that forced expression of the commonly amplified cyclin dependent kinase-4 (CDK4) was sufficient to elicit reduced dependency on growth factor signalling and a decreased sensitivity to the differentiation factor BMP4. Moreover, preliminary results indicate that the mRNA expression of the proto-oncogene Akt2 increases in CDK4 transfectants, suggestive of a feedback mechanism. We tested whether constitutive expression of Akt2 together with CDK4 increases the severity of the phenotype and initial results suggest this is the case. These findings provide encouragement that minimal genetic events will be required in order to transform NS cells in vitro. These cells can now be tested for in vivo tumorigenicity following transplantation. Finally, we have also generated CDK4 overexpressing cell lines in a specific NS cell variant which I identified as expressing an oligodendrocyte differentiation bias (MG1-7). We are therefore now in a position to assess whether differentiation behaviour is affected by the disruption of this pathway. Together these findings highlight the utility of mouse NS cells as a tool to study glioma

Assessing the role of Hes1 and identification of target genes in human and murine T-ALL

Silvia Wirth

The Notch signalling pathway is an ancient cell signalling mechanism that enables short-range communications between cells and controls a broad spectrum of cell fates and developmental processes. In the haematopoietic system Notch signalling has been linked to physiological, but also to pathological phenotypes of T cell development. Identification of numerous activating mutations within the NOTCH1 receptor in human T cell acute lymphoblastic leukaemia (T-ALL) patient samples demonstrated that aberrant activation of this signalling pathway is a frequent event in T-ALL. Active Notch signalling leads to the expression of a plethora of genes that might be drivers or passengers in tumorigenesis including Hes1, which encodes a transcriptional repressor. We have therefore studied Hes1 in the context of T-ALL in mouse models that mimic human disease and in human T-ALL cell lines with the aim to evaluate whether Hes1 affects disease development and disease maintenance and to characterize its function on a molecular level. The first part of this study is dedicated to the functional characterisation of Hes1 in vivo in T-ALL mouse models and in vitro in human T-ALL cell lines. Using a retroviral bone marrow transplant model of T-ALL in which progenitor cells are transduced with a retrovirus expressing constitutive active Notch1 intracellular domain (NICD) we could show that Hes1 is not required in established, late stage T-ALL cells, but that it promotes tumour progression in the early stages of the disease. We have also investigated the function of HES1 in human T-ALL and can show that the overexpression of dominant negative versions of Hes1 in two patient derived cell lines (T-ALL1, CUTLL-1) does not affect proliferation or apoptosis, emphasising that HES1 does not affect cell viability once cells are fully transformed to the leukemic state. In the second part of the study we set out to systematically delineate the gene regulatory networks down-stream of Hes1 by combining RNA-seq and ChIP-seq analysis. Comparing the gene expression of murine premalignant, early stage NICD induced CD4+CD8+ DP cells in the presence and absence of Hes1 by RNA-seq, we unexpectedly found a predominant upregulation of genes in cells expressing Hes1 indicating that Hes1 might rather act as a positive regulator of transcription than as a transcriptional repressor of distinct target genes. Hes1 might influence transcription through secondary or indirect effects as combinatorial analysis of RNA-seq and ChIP-seq led to the identification of only a small number of potential direct target genes that are transcriptionally regulated by Hes1. In summary, the role of Hes1 in promoting the early stages of the disease appears to be highly complex and is likely to involve both direct and indirect control of gene regulatory networks.

EPFL2014

Tissue engineering of the bladder

Catharina Adelöw

Stem cell use in bladder tissue engineering is a recently addressed area of investigation that has generated excitement as a novel way to restore and regenerate lost or damaged urinary bladder tissue. The remodelling of smooth muscle plays a significant role in bladder repair and regeneration. De-differentiation of smooth muscle cells from a contractile phenotype to a synthetic proliferative phenotype is characterised by smooth muscle hypertrophy and fibrosis, and leads to a poorly compliant bladder. To elucidate the underlying mechanisms of such phenomena, recent efforts in bladder tissue engineering have focused on understanding smooth muscle biology and associated mechanisms of bladder diseases. In this thesis we have designed a synthetic poly (ethylene glycol) (PEG) hydrogel for stem cell and drug delivery aimed to enhance control smooth muscle cell phenotype and thus assist in bladder repair and regeneration. We began by developing a method for isolation of cells from the bladder wall. Traditionally urothelium and smooth muscle cells are separated by dissection, and primary cultures are initiated in distinctly different medium formulations to obtain homogenous cultures and limit contaminating cells. However, the bladder wall harbours, apart from urothelial and smooth muscle cells, fibroblasts that have similar nutritional requirements and are likely candidates to contaminate smooth muscle cell cultures. Isolation of these three cell types by conjugation to magnetic beads and repeated separations allowed us to obtain pure cell populations. Furthermore, this technique permitted us to freely choose any specific medium formulation we wished to utilise. We then turned our attentions to engineering a materials system, by chemically and physically tuning a PEG hydrogel in terms of ligand concentration and matrix stiffness, to provide a three-dimensional environment for optimal spreading of human smooth muscle cells and mesenchymal stem cells. Cell viability, proliferation and differentiation were assessed in optimised hydrogels. Compared to cultures on traditional two-dimensional plastic, mesenchymal stem cells cultured within our three-dimensional hydrogels acquired a smooth muscle cell-like phenotype and smooth muscle cells obtained a less de-differentiated phenotype. In addition, we demonstrated cell-demanded gel degradation and deposition of newly synthesised extracellular matrix proteins. In order to modulate cell phenotypes further, we hypothesised that cell differentiation could be directed by the adhesion ligands made available within the matrix. In this manner, cells that were exposed to a matrix presenting certain integrin-binding ligands would begin expressing the corresponding integrins in order to adhere to the matrix. Consequently, we explored the effect of various specific integrin-binding ligands on mesenchymal stem cell integrin expression and differentiation. Fibronectin (FN) fragments engineered to demonstrate specificity to different integrins, namely α5β1 (FNIII910) and αvβ3 (FNIII10), were conjugated to PEG functionalised with vinyl sulfone and compared to fibronectin wild type (FNIIIWT) fragment and the RGD peptide, which both provide binding sites for a range of integrins. We found that assembly of focal adhesions and formation of stress fibres were altered due to the specific fragments provided, and expression of integrin- and smooth muscle cell specific genes varied in mesenchymal stem cells cultured within hydrogels functionalised with different fragments. Moreover, these fibronectin fragments turned out to be especially helpful for improved attachment of urothelial cells on top of hydrogels. With this biomaterial system in hand, we sought to develop a co-culture model of the bladder wall that permitted mesenchymal stem cells and urothelial cells cultured within / on top of PEG hydrogels, and to explore the combinatorial effect of ligand binding and cell-cell interactions on mesenchymal stem cell fate. Interestingly, we found that co-culturing with urothelial cells influenced gene expression of mesenchymal stem cells differently, depending on the protein fragment to which they were adhering. Mesenchymal stem cells cultured within PEG-FNIIIWT hydrogels were shown to up-regulate smooth muscle cell specific genes, while down-regulation was demonstrated in PEG-RGD, PEG-FNIII910 and PEG-FNIII10 hydrogels. Again, mesenchymal stem cells cultured within PEG-FNIIIWT hydrogels were shown to up-regulate integrin expressions α5, αv, β1 and β3, while down-regulation of the same genes was demonstrated for cells cultured within PEG-FNIII9*10 hydrogels, and a more complex pattern of integrin regulation was found for cells cultured within PEG-RGD and PEG-FNIII10 hydrogels. Finally, we investigated a novel approach to growth factor delivery in a controlled release manner, by providing cell- and growth factor binding protein fragments. In this system we show that transforming growth factor beta 1 (TGF-β1) release from PEG hydrogels influence smooth muscle cell specific gene expressions differently depending on the integrin- and growth factor binding proteins available within the hydrogel. Delivery of TGF-β1 within PEG-FNIIIWT hydrogels was shown to up-regulate smooth muscle specific genes in mesenchymal stem cells, while the other conditions displayed more complex patterns of gene expression. Further experiments are required to characterise the conjugation of PEGprotein-growth factor, and the release profiles for the growth factor in the different conditions. In conclusion, this thesis investigates the effect of various chemical and physical signals on mesenchymal stem cell-to-smooth muscle cell differentiation in an in vitro model to further understand cell-cell and cell-extracellular matrix interactions and create improved artificial microenvironments for directing and / or maintaining cell differentiation. The PEG hydrogel that we designed permits the co-culture of cells as well as the incorporation of peptides and proteins such as cell adhesion- and growth factor binding ligands, and growth factors for directing the differentiation of human mesenchymal stem cells into smooth muscle cells. Here, we explore these features and their effects on cell differentiation in vitro. Hence, our PEG hydrogel system can serve both as an in vitro model for studying basic cell biology, and as a cell delivery vehicle for improved bladder tissue regeneration.

EPFL2009

Quantitative analysis of gene expression and transcriptional memory in mouse embryonic stem cells

Aleksandra Mandic

Quantitative studies of gene expression in have shown widespread variability in transcript and protein product abundance at the single-cell level. Such fluctuations in gene expression have been shown to lead to heterogeneous cellular responses and phenotypes, acting in a range of systems, from immunity, cancer to development. To better understand heterogeneity in cellular populations, we developed a novel method for the precise quantification of gene expression in single living cells. Furthermore, we focused on whether and to which extent transcriptional expression profiles are transmitted during cell division. In the first section, we described a new approach for quantitative measurements of gene expression, based on internal tagging of endogenous proteins with a reporter allowing luminescence and fluorescence time-lapse imaging. Using the currently brightest luminescent reporter, fluctuations of protein expression levels could be monitored in single living cells with high sensitivity and temporal resolution over extended time periods. Moreover, our system allowed measurement of single-cell protein degradation rates in the absence of protein synthesis inhibitors, as well as calculation of absolute synthesis rates over the cell cycle. Finally, the internal tag could be excised by inducible expression of Cre recombinase, which enabled estimation of endogenous mRNA half-lives by monitoring the decay of luminescent signal. Second, to monitor transcriptional activity and quantify memory across multiple endogenous genes in mouse embryonic stem cells, we generated cell lines in which a short-lived luciferase reporter allowed sensitive monitoring of transcriptional kinetics by luminescence imaging at high time resolution over long periods of time. By coupling live single-cell monitoring of transcriptional activity of 9 different promoters with mathematical modelling, we found that different levels of expression fluctuations shape transcriptional memory. Long fluctuations (1-10 generations) and short fluctuations (1-3 h), as well as their amplitudes, vary widely between across different genes and together shape transcriptional memory. Surprisingly, we have shown that there was a large level of gene-specific cell-to-cell synchronicity in transcriptional profiles. Overall, we uncovered that noise and timescales of slow and fast fluctuations, as well as their synchronisation over the cell cycle, defined how transcriptional dynamics were correlated within a lineage of related cells. In conclusion, this thesis described an innovative method that allows measurement of absolute protein expression levels, protein turnover, as well as mRNA half-lives for endogenously expressed genes. Our approach opens new avenues in quantitative studies of gene expression in single living cells. Furthermore, we revealed different levels of transcriptional fluctuations that shape gene specific transcriptional memory and its timescales in mouse embryonic stem cells.

EPFL2018