Single-Cell Transcriptomics Reveals that Differentiation and Spatial Signatures Shape Epidermal and Hair Follicle Heterogeneity

The murine epidermis with its hair follicles represents an invaluable model system for tissue regeneration and stem cell research. Here we used single-cell RNA-sequencing to reveal how cellular heterogeneity of murine telogen epidermis is tuned at the transcriptional level. Unbiased clustering of 1,422 single-cell transcriptomes revealed 25 distinct populations of interfollicular and follicular epidermal cells. Our data allowed the reconstruction of gene expression programs during epidermal differentiation and along the proximal-distal axis of the hair follicle at unprecedented resolution. Moreover, transcriptional heterogeneity of the epidermis can essentially be explained along these two axes, and we show that heterogeneity in stem cell compartments generally reflects this model: stem cell populations are segregated by spatial signatures but share a common basal-epidermal gene module. This study provides an unbiased and systematic view of transcriptional organization of adult epidermis and highlights how cellular heterogeneity can be orchestrated in vivo to assure tissue homeostasis.

Cellular Heterogeneity During Embryonic Stem Cell Differentiation to Epiblast Stem Cells Is Revealed by the ShcD/RaLP Adaptor Protein

Daniel Constam, Anja Dietze, Antonio Simeone

The Shc family of adaptor proteins are crucial mediators of a plethora of receptors such as the tyrosine kinase receptors, cytokine receptors, and integrins that drive signaling pathways governing proliferation, differentiation, and migration. Here, we report the role of the newly identified family member, ShcD/RaLP, whose expression in vitro and in vivo suggests a function in embryonic stem cell (ESC) to epiblast stem cells (EpiSCs) transition. The transition from the naive (ESC) to the primed (EpiSC) pluripotent state is the initial important step for ESCs to commit to differentiation and the mechanisms underlying this process are still largely unknown. Using a novel approach to simultaneously assess pluripotency, apoptosis, and proliferation by multiparameter flow cytometry, we show that ESC to EpiSC transition is a process involving a tight coordination between the modulation of the Oct4 expression, cell cycle progression, and cell death. We also describe, by high-content immunofluorescence analysis and time-lapse microscopy, the emergence of cells expressing caudal-related homeobox 2 (Cdx2) transcription factor during ESC to EpiSC transition. The use of the ShcD knockout ESCs allowed the unmasking of this process as they presented deregulated Oct4 modulation and an enrichment in Oct4-negative Cdx2-positive cells with increased MAPK/extracellular-regulated kinases 1/2 activation, within the differentiating population. Collectively, our data reveal ShcD as an important modulator in the switch of key pathway(s) involved in determining EpiSC identity. STEM CELLS2012;30:24232436

Wiley-Blackwell2012

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

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.