Live imaging of stem cell and progeny behaviour in physiological hair-follicle regeneration

Tissue development and regeneration depend on cell-cell interactions and signals that target stem cells and their immediate progeny. However, the cellular behaviours that lead to a properly regenerated tissue are not well understood. Using a new, non-invasive, intravital two-photon imaging approach we study physiological hair-follicle regeneration over time in live mice. By these means we have monitored the behaviour of epithelial stem cells and their progeny during physiological hair regeneration and addressed how the mesenchyme influences their behaviour. Consistent with earlier studies, stem cells are quiescent during the initial stages of hair regeneration, whereas the progeny are more actively dividing. Moreover, stem cell progeny divisions are spatially organized within follicles. In addition to cell divisions, coordinated cell movements of the progeny allow the rapid expansion of the hair follicle. Finally, we show the requirement of the mesenchyme for hair regeneration through targeted cell ablation and long-term tracking of live hair follicles. Thus, we have established an in vivo approach that has led to the direct observation of cellular mechanisms of growth regulation within the hair follicle and that has enabled us to precisely investigate functional requirements of hair-follicle components during the process of physiological regeneration. © 2012 Macmillan Publishers Limited. All rights reserved

Intestinal epithelial stem cells do not protect their genome by asymmetric chromosome segregation

The idea that stem cells of adult tissues with high turnover are protected from DNA replication-induced mutations by maintaining the same 'immortal' template DNA strands together through successive divisions has been tested in several tissues. In the epithelium of the small intestine, the provided evidence was based on the assumption that stem cells are located above Paneth cells. The results of genetic lineage-tracing experiments point instead to crypt base columnar cells intercalated between Paneth cells as bona fide stem cells. Here we show that these cells segregate most, if not all, of their chromosomes randomly, both in the intact and in the regenerating epithelium. Therefore, the 'immortal' template DNA strand hypothesis does not apply to intestinal epithelial stem cells, which must rely on other strategies to avoid accumulating mutations

SACK-Expanded Hair Follicle Stem Cells Display Asymmetric Nuclear Lgr5 Expression With Non-Random Sister Chromatid Segregation

We investigated the properties of clonally-expanded mouse hair follicle stem cells (HF-SCs) in culture. The expansion method, suppression of asymmetric cell kinetics (SACK), is non-toxic and reversible, allowing evaluation of the cells' asymmetric production of differentiating progeny cells. A tight association was discovered between non-random sister chromatid segregation, a unique property of distributed stem cells (DSCs), like HF-SCs, and a recently described biomarker, Lgr5. We found that nuclear Lgr5 expression was limited to the HF-SC sister of asymmetric self-renewal divisions that retained non-randomly co-segregated chromosomes, which contain the oldest cellular DNA strands, called immortal DNA strands. This pattern-specific Lgr5 association poses a potential highly specific new biomarker for delineation of DSCs. The expanded HF-SCs also maintained the ability to make differentiated hair follicle cells spontaneously, as well as under conditions that induced cell differentiation. In future human cell studies, this capability would improve skin grafts and hair replacement therapies

Dipoid-Specific Genome Stability Genes of S. cerevisiae: Genomic Screen Reveals Haploidization as an Escape from Persisting DNA Rearrangement Stress

Maintaining a stable genome is one of the most important tasks of every living cell and the mechanisms ensuring it are similar in all of them. The events leading to changes in DNA sequence (mutations) in diploid cells occur one to two orders of magnitude more frequently than in haploid cells. The majority of those events lead to loss of heterozygosity at the mutagenesis marker, thus diploid-specific genome stability mechanisms can be anticipated. In a new global screen for spontaneous loss of function at heterozygous forward mutagenesis marker locus, employing three different mutagenesis markers, we selected genes whose deletion causes genetic instability in diploid Saccharomyces cerevisiae cells. We have found numerous genes connected with DNA replication and repair, remodeling of chromatin, cell cycle control, stress response, and in particular the structural maintenance of chromosome complexes. We have also identified 59 uncharacterized or dubious ORFs, which show the genome instability phenotype when deleted. For one of the strongest mutators revealed in our screen, ctf18Δ/ctf18Δ the genome instability manifests as a tendency to lose the whole set of chromosomes. We postulate that this phenomenon might diminish the devastating effects of DNA rearrangements, thereby increasing the cell's chances of surviving stressful conditions. We believe that numerous new genes implicated in genome maintenance, together with newly discovered phenomenon of ploidy reduction, will help revealing novel molecular processes involved in the genome stability of diploid cells. They also provide the clues in the quest for new therapeutic targets to cure human genome instability-related diseases

A Functional Role of RB-Dependent Pathway in the Control of Quiescence in Adult Epidermal Stem Cells Revealed by Genomic Profiling

Continuous cell renewal in mouse epidermis is at the expense of a pool of pluripotent cells that lie in a well defined niche in the hair follicle known as the bulge. To identify mechanisms controlling hair follicle stem cell homeostasis, we developed a strategy to isolate adult bulge stem cells in mice and to define their transcriptional profile. We observed that a large number of transcripts are underexpressed in hair follicle stem cells when compared to non-stem cells. Importantly, the majority of these downregulated genes are involved in cell cycle. Using bioinformatics tools, we identified the E2F transcription factor family as a potential element involved in the regulation of these transcripts. To determine their functional role, we used engineered mice lacking Rb gene in epidermis, which showed increased expression of most E2F family members and increased E2F transcriptional activity. Experiments designed to analyze epidermal stem cell functionality (i.e.: hair regrowth and wound healing) imply a role of the Rb-E2F axis in the control of stem cell quiescence in epidermis

Investigation of combined electro-osmotic and pressure-driven flow in rough microchannels

The present study is carried out to investigate the influence of surface roughness in combined electro-osmotic and pressure-driven flow in microchannel. Two-dimensional theoretical model is developed to predict the behavior of velocity profiles in rough microchannel. The concept of surface roughness-viscosity model is used to account the effect of surface roughness. The pluglike velocity profile for electro-osmotic flow and the parabolic velocity profile for pressure-driven flow with delay in attaining the centerline velocity are observed. It is found that for electro-osmotic flow, the deviation in velocity profile from a flow in a smooth channel occurs near the wall, whereas in pressure-driven flow, such deviation is dominant in the core region. A superposition of pluglike and parabolic velocity profiles is found in combined electro-osmotic and pressure-driven flow. It is also observed that in the case of combined flow, the deviation in velocity profile from the smooth channel case reduces gradually with the distance from the wall

Monitoring dopants by Raman scattering in an electrochemically top-gated graphene transistor

The recent discovery of graphene has led to many advances in two-dimensional physics and devices. The graphene devices fabricated so far have relied on $SiO_2$ back gating. Electrochemical top gating is widely used for polymer transistors, and has also been successfully applied to carbon nanotubes. Here we demonstrate a top-gated graphene transistor that is able to reach doping levels of up to $5\times 10^{13} cm^{-2}$, which is much higher than those previously reported. Such high doping levels are possible because the nanometre-thick Debye layer in the solid polymer electrolyte gate provides a much higher gate capacitance than the commonly used $SiO_2$ back gate, which is usually about 300 nm thick. In situ Raman measurements monitor the doping. The G peak stiffens and sharpens for both electron and hole doping, but the 2D peak shows a different response to holes and electrons. The ratio of the intensities of the G and 2D peaks shows a strong dependence on doping, making it a sensitive parameter to monitor the doping