Differentiation of human embryonic stem cells to hepatocytes

Many liver diseases involve hepatocyte malfunction. In most cases, transplantation of corrected hepatocytes would correct for disease symptoms. Liver donor shortage and hepatocyte inability to proliferate in vitro accentuate this problem. Embryonic stem and induced pluripotent stem cells proliferate indefinitely and have the capacity to differentiate into every cell type of the body. They could thus represent a solution to this organ donor shortage. In this project, we aim to differentiate ES and iPS cells to hepatocytes. First, we consider an approach to select the ES/iPS cell line showing the best potential to generate liver lineage using teratomas as a selection tool. Then we use lentiviral vectors to transduce this selected cell line with essential liver-‐specific development factors to help differentiation efficiency. Next, we developed an in vitro differentiation protocol based on existing protocols and developmental clues of liver development to generate a pool of hepatocyte precursors. Later on, we injected these differentiated cells in mice liver of Fah-‐, Rag2-‐ and Il-‐2rγ-‐deficient mice to check for engraftment and liver marker expression. Our findings show, after checking for hepatocyte-‐ specific markers in the differentiated cells, that our previous selection for the potentially best cell line seems correct. We also find that the transcription factors involved in liver development appear to have a negative effect in vitro on liver differentiation. Applying our protocol to regular ES cells results on the production of late maturity hepatocyte markers, showing correct differentiation and maturation of newly formed hepatocytes. Finally, mouse in vivo liver transplantation of these differentiated cells gives us human albumin expression detectable in blood serum of said mice

Expansion of a VE-Cadherin+CD45+ population in the yolk sac upon Etv2 overexpression in the mouse embryo - Effects of BMP and Wnt signaling inhibition on the differentiation of a Flk1+ extraembryonic mesoderm in vitro model

Matteo Pluchinotta

The difficulty to find compatible donors for bone marrow transplantation makes the need for an alternative source of HSCs urgent. HSC derived from patient-specific iPS cells are ideal candidates for this purpose. Nevertheless, although HSCs are the best characterized adult stem cells, the in vitro generation and expansion of bone fide HSCs still has not been achieved without transgene insertion. Successful derivation of HSCs from ES cells or iPS cells will require a comprehensive understanding of inductive signals and downstream effectors involved in the normal arising of HSCs during embryonic development. BMP and Wnt signaling pathways play a very important role in this differentiation process. Here, we report how BMP and Wnt signaling inhibition cooperatively alter the differentiation of Flk1+PDGFRα- cells after reculture in serum-free, feeder layer-free conditions. This data will be useful in determining the factors that have to be added to a chemically defined culture medium aimed at derivation of bone fide HSC in vitro. In the second part of this master’s project, we studied the spatial and temporal localization requirements of Etv2 expression, a key regulator of the earliest events in the development of the hematopoietic and vascular systems. To gain more insight on this matter, we conditionally knocked-in Etv2 in the early endothelial compartment, using a Tie2Cre system. This leaded to an embryonically lethal phenotype by E11.5, and most interestingly, to the expansion of a VE-Cadherin+CD45+ population in the E10.5 yolk sac. We believe that these cells represent a increased pool of immature pre-definitive hematopoietic stem cells, thus underlining further the importance of Etv2 in the differentiation process towards hematopoietic, and endothelial lineages in the mouse embryo.

2011

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

Understanding engraftment of cultured epidermal stem cells

Nicolas Grasset

The epidermis and its appendages protect our body from environmental hazards. Cells generated in the basal layer continuously replace the terminally differentiated keratinocytes that are shed off the epidermal surface. Long-term renewal depends on specific tissue cells, called stem cells. The epidermis can efficiently repair itself when wounded, thanks to the proliferation, migration and differentiation of the stem cells and their progeny. In extensive full-thickness burns, restoration of the epidermal barrier can only be achieved by means of autologous skin grafts. Cell therapy using cultured epithelium autografts (CEA) has been part of the therapeutic arsenal since the early eighties (Gallico et al., 1984; O'Connor et al., 1981) and it has saved the life of many patients worldwide. Paradoxically, little is known on the behavior of the transplanted stem cells and engraftment has remained variable. Unpublished data from our laboratory have shown that the number of stem cells decrease rapidly in transplanted CEA in humans. This further highlights the necessity to thoroughly comprehend the cellular and molecular mechanisms of stem cell engraftment. Several fundamental questions must be addressed, for example: 1) By which mechanisms do stem cells adjust to the stress of transplantation? 2) How many stem cells are required for long-term renewal of the regenerated epidermis? 3) Can one manipulate the niche? To thoroughly investigate the mechanisms of engraftment, it is critical to develop a predictable animal model since it is difficult to experiment on human. We have chosen the pig as a large animal model because clinical procedures used for CEA transplantation cannot be recapitulated in laboratory animals. Firstly, we have demonstrated that pig and human skin share many features. In particular, the skin of the pig contains keratinocyte stem cells that can be characterized by clonal analysis using the same criteria as in the human (holoclone, meroclone, paraclone). Secondly, we have reproducibly produced CEA, including some made from the progeny of a single EGFP-labeled keratinocyte stem cell. Thirdly, we have recapitulated in the pig all surgical procedures used to transplanting CEA in humans. Fourthly, CEA engraftment has been systematically investigated by histology, immunochemistry and clonal analysis. Fifthly, we have demonstrated that the number of stem cells rapidly decreases following CEA transplantation and that there is little apoptosis in transplanted CEA, which suggests that stem cells are rather lost through terminal differentiation. Collectively our results demonstrate that the pig is the best available model system to investigate the mechanisms that govern stem cell engraftment. Superior engraftment may be achieved by improving the grafting bed, by designing smart matrices to better mimic the niche, or by manipulating stem cell behavior. A better comprehension of stem cell engraftment will certainly benefit patients suffering from large burns and those with disabling skin diseases (e.g. dystrophic epidermolysis bullosa). It will also profit stem cell therapy at large since optimal engraftment is also needed for hematopoïetic, muscle and neural stem cells.

EPFL2008