Hearts and bones': the ups and downs of plasticity' in stem cell biology

More than a decade ago, plasticity suddenly became a fashionable topic with overemphasized implications for regenerative medicine. The concept of plasticity is supported by old transplantation work, at least for embryonic cells, and metaplasia is a classic example of plasticity observed in patients. Nevertheless, the publication of a series of papers showing rare conversion of a given cell type into another unrelated cell raised the possibility of using any unaffected tissue to create at will new cells to replace a different failing tissue or organ. This resulted in disingenuous interpretations and a reason not to fund anymore research on embryonic stem cells (ESc). Moreover, many papers on plasticity were difficult to reproduce and thus questioned; raising issues about plasticity as a technical artefact or a consequence of rare spontaneous cells fusion. More recently, reprogramming adult differentiated cells to a pluripotent state (iPS) became possible, and later, one type of differentiated cell could be directly reprogrammed into another (e.g. fibroblasts into neurons) without reverting to pluripotency. Although the latter results from different and more robust experimental protocols, these phenomena also exemplify plasticity. In this review, we want to place plasticity in a historical perspective still taking into account ethical and political implications.

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

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

Quantitative analysis of SOX2 and OCT4 expression in pluripotency and differentiation

Daniel Strebinger

Embryonic stem (ES) cells have the capacity to give rise to all cell types of the adult organism and can be used as a model to study early cell fate decisions as they closely recapitulate in vivo events. The M and G1 phases have been suggested to be the key time windows for pluripotent cells to engage toward differentiation and SOX2 and OCT4 have been shown to orchestrate the dissolution of pluripotency and induction of cell fate commitment. Furthermore, it is known that the levels of some pluripotency transcription factors fluctuate over time, leading to the notion that ES cells experience dynamic heterogeneity, where for example low levels of NANOG can be found in cells prone to differentiation, whereas cells with high NANOG expression are in a robust pluripotent state. However, it remains unclear if and how (i) the action of transcription factors in specific cell cycle phases and (ii) protein level variations of factors that show mild differences between single cells impact cell fate decisions. This is in part due to the lack of quantitative single-cell meth-ods allowing dissecting the origin and functional consequences of gene expression heterogeneity. In this work, we established a method based on internal tagging of endogenous proteins with an excisable reporter to perform absolute molecule quantification in single living cells. This enabled us to investigate fluctuations of endogenous protein expression levels, the heterogeneity of degradation rates between individual cells and determine absolute amounts of proteins synthesized over the cell cycle. Additionally, inducible excision of the tag enabled us to estimate endogenous mRNA half-lives. Next, by using live-cell microscopy of fusion proteins, we show that SOX2 and OCT4 stay bound to mitotic chromosomes through their DNA binding domains. We then characterized the function of SOX2 at the M-G1 transition, showing that its absence specifically in this phase impairs pluripotency maintenance and abolishes its ability to induce neuroectodermal commitment. This suggests, that transcription factor activity at the M-G1 transition is essential for cell fate maintenance and differentiation. Lastly, we show that mild endogenous fluctuations in the levels of key transcription factors in ES cells bias cell fate decisions. We found that high OCT4 levels at the onset of differentiation increase the probability of single cells to commit to both neuroectoderm and mesendoderm. Further, we showed that high SOX2 levels result in a higher number of cells acquiring a neuroectodermal cell fate. This not only shows that the differentiation potential of embryonic stem cells is highly sensitive to small variations in intracellular concentrations of pluripotency transcription factors but further suggests that endogenous fluctuations of these are a major source of heterogeneity in cell fate decisions. In conclusion, the presented work identifies that the SOX2 activity in the M-G1 transition is important for cell fate maintenance and differentiation. Furthermore, the heterogeneity of protein levels governs cell fate decisions, suggesting the existence of different short-lived cell states in populations of presumed homogenous cells. We think that the minor differences in the levels of transcription factors and the cell cycle specific sensitivity of cells to transcription factor activity could be important mechanisms guiding maintenance and differentiation potential in various stem cell types.

EPFL2018