Cellule souche pluripotente induite

Les cellules souches pluripotentes induites (CSPi) (en anglais Induced pluripotent stem cells soit iPS ou iPSCs) sont des cellules souches pluripotentes générées en laboratoire à partir de cellules somatiques. Ces cellules souches induites ont le potentiel de se différencier en n'importe quelle cellule du corps humain et ont donc des applications très variées en thérapie et en recherche biomédicale. Elles sont considérées comme l'une des avancées majeures de la biotechnologie puisqu'elles permettent de constituer une alternative à l'utilisation de cellules souches embryonnaires humaines, permettant ainsi de dépasser les problèmes éthiques liés à ces dernières. Historique Jusque dans les années 2000, les cellules souches pluripotentes humaines utilisées en recherche étaient d’origine embryonnaire. Mais malgré les nombreuses applications potentielles des cellules souches embryonnaires, leur utilisation soulève des problèmes éthiques car leur obtention repose sur la destruc

Non-integrating episomal plasmid-based reprogramming of human amniotic fluid stem cells into induced pluripotent stem cells in chemically defined conditions

Lilia Salimova

Amniotic fluid stem cells (AFSC) represent an attractive potential cell source for fetal and pediatric cell-based therapies. However, upgrading them to pluripotency confers refractoriness toward senescence, higher proliferation rate and unlimited differentiation potential. AFSC were observed to rapidly and efficiently reacquire pluripotency which together with their easy recovery makes them an attractive cell source for reprogramming. The reprogramming process as well as the resulting iPSC epigenome could potentially benefit from the unspecialized nature of AFSC. iPSC derived from AFSC also have potential in disease modeling, such as Down syndrome or -thalassemia. Previous experiments involving AFSC reprogramming have largely relied on integrative vector transgene delivery and undefined serum-containing, feeder-dependent culture. Here, we describe non-integrative oriP/EBNA-1 episomal plasmid-based reprogramming of AFSC into iPSC and culture in fully chemically defined xeno-free conditions represented by vitronectin coating and E8 medium, a system that we found uniquely suited for this purpose. The derived AF-iPSC lines uniformly expressed a set of pluripotency markers Oct3/4, Nanog, Sox2, SSEA-1, SSEA-4, TRA-1-60, TRA-1-81 in a pattern typical for human primed PSC. Additionally, the cells formed teratomas, and were deemed pluripotent by PluriTest, a global expression microarray-based in-silico pluripotency assay. However, we found that the PluriTest scores were borderline, indicating a unique pluripotent signature in the defined condition. In the light of potential future clinical translation of iPSC technology, non-integrating reprogramming and chemically defined culture are more acceptable.

Taylor & Francis Inc2016

Differentiation of human embryonic stem cells to hepatocytes

Noam Beckmann

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

2010

Temperature impacts human epidermal stem cell behaviour through thermoTRPs and mTOR signalling

Johannes Alexander Mosig

Adult stem cells are characterized by their unique ability to self-renew and give rise to a progeny of specialized/differentiated cells. Therefore, they have a remarkable potential for regenerative medicine. However, the use of adult stem cells is restrained by several obstacles such as limited in vitro amplification and uncontrolled differentiation. Adult stem cells reside in tightly regulated microenvironments termed “niches”. The niche is composed of nursing cells, extracellular matrix and a variety of signalling factors that dynamically regulate stem cell behaviour. Consequently, recapitulating the niche in vitro is fundamental in manipulating adult stem cells. In recent years, the physicochemical microenvironment of the niche (e.g. temperature, pH, oxygen tension) has proven to be as important as classical signalling pathways for the regulation of stem cell fate. Therefore, the aim of this thesis is to understand the influence of temperature variations on human epidermal stem cell behaviour. This effort has been accomplished in three main directions. First, we have contributed to the development of two different telemetric temperature sensors that precisely measure and record temperature in vivo and in vitro. These sensors have allowed us to demonstrate that epidermal keratinocytes are continuously subjected to small physiological temperature variations (3 ± 2oC) in vivo. Then, we have engineered a device that allows a tight and dynamic control of temperature while simultaneously providing real-time imaging of cultured human epidermal stem cells. It is shown that temperature variations of 1oC are not trivial, since they significantly affect human epidermal stem cell growth and proliferation. Second, we have shown that human keratinocytes sense temperature by means of temperature-sensitive calcium channels termed thermoTRPs (Transient Receptor Potential). Consequently, we have monitored intracellular calcium concentrations to evaluate the reaction of epidermal stem cells to thermal or chemical stimulations. The strongest calcium influxes have been observed after temperature drops (from 37 to 32oC) suggesting that human keratinocytes are particularly sensitive to temperature decreases. Furthermore, we have demonstrated the importance of these channels for keratinocyte viability by silencing a thermoTRP channel (i.e. TRPV3) using lentiviral delivery of shRNA. Third, we have demonstrated that thermal signals affecting keratinocytes are integrated through the mTOR (mammalian Target Of Rapamycin) signalling pathway, since cool stimuli (32oC) decreased mTOR complex 1 (mTORC1) kinase activity. Culture at 32oC and 100nm rapamycin treatment affected growth and proliferation of human keratinocyte stem cells similarly, further stressing the inhibitory effects of cool temperatures on mTORC1. We also have observed that inhibition of mTORC1 induced nuclear translocation of mTOR, suggesting a putative transcriptional role for mTOR. To conclude, we have established that prolonged inhibition of mTORC1 favours an apparent maintenance of the stem cell phenotype by reducing clonal conversion, without however stopping stem cell aging. Altogether, our results demonstrate that thermoTRPs are wired to the mTOR signalling in human epidermal stem cells. Furthermore, our work thoroughly supports the hypothesis that temperature is a significant actor of the epidermal stem cell niche as it can directly affect stem cell behaviour.

EPFL2013

Temperature impacts human epidermal stem cell behaviour through thermoTRPs and mTOR signalling

Johannes Alexander Mosig

EPFL2013