Nicholas Hole

Hair follicle dermal cells differentiate into adipogenic and osteogenic lineages

Abstract: The adult hair follicle dermal papilla (DP) and dermal sheath (DS) cells are developmentally active cell populations with a proven role in adult hair follicle‐cycling activity and unique inductive powers. In stem cell biology, the hair follicle epithelium has recently been the subject of a great deal of investigation, but up to now, the follicle dermis has been largely overlooked as a source of stem cells. Following the sporadic appearance of muscle, lipid and bone‐type cells in discretely isolated follicle DP and DS cell primary cultures, we demonstrated that cultured papilla and sheath cell lines were capable of being directed to lipid and bone differentiation. Subsequently, for the first time, we produced clonal DP and DS lines that had extended proliferative capabilities. Dye exclusion has been reported to be an identifying feature of stem cells; therefore, clonal papilla and sheath lines with differing capacity to exclude rhodamine123 were cultured in medium known to induce adipocyte and osteocyte differentiation. Both DS‐ and DP‐derived clones showed the capacity to make lipid and to produce calcified material; however, different clones had varied behaviour and there was no obvious correlation between their stem cell capabilities and dye exclusion or selected gene expression markers. As a highly accessible source, capable of being discretely isolated, the follicle has important potential as a stem cell source for tissue engineering and cell therapy purposes. It will also be interesting to compare follicle dermal stem cell properties with the broader stem cell capabilities discovered in skin dermis and investigate whether, as we believe, the follicle is a key dermal stem cell niche. Finally, the discovery of stem cells in the dermis may have implications for certain pathologies in which abnormal differentiation occurs in the skin.

Phenotypic Characterization of Murine Primitive Hematopoietic Progenitor Cells Isolated on Basis of Aldehyde Dehydrogenase Activity

There are several different technical approaches to the isolation of hematopoietic stem cells (HSCs) with long‐term repopulating ability, but these have problems in terms of yield, complexity, or cell viability. Simpler strategies for HSC isolation are needed. We have enriched primitive hematopoietic progenitors from murine bone marrow of mice from different genetic backgrounds by lineage depletion followed by selection of cells with high aldehyde dehydrogenase activity using the Aldefluor reagent (BD Biosciences, Oxford, U.K.). Lin−ALDHbright cells comprised 26.8 ± 1.0% of the total Lin− population of C57BL6 mice, and 23.5 ± 1.0% of the Lin− population of BALB/c mice expressed certain cell‐surface markers typical of primitive hematopoietic progenitors. In vitro hematopoietic progenitor function was substantially higher in the Lin−ALDHbright population compared with the Lin−ALDHlow cells. These cells have higher telomerase activity and the lowest percentage of cells in S phase. These data strongly suggest that progenitor enrichment from Lin−cells on the basis of ALDH is a valid method whose simplicity of application makes it advantageous over conventional separations.

Hair follicle dermal cells repopulate the mouse haematopoietic system

Skin and hair follicle stem cell biology is the focus of increasing interest, not least because the adult hair follicle has well defined dermal and epithelial populations that display distinct developmental properties. Recent evidence suggests that a number of adult cell populations have much broader stem cell capabilities than previously thought. To examine whether this applied to the hair follicle, and with a view to developing the follicle as a stem cell model system we investigated whether adult hair follicles were capable of demonstrating haematopoietic stem cell activity. To investigate haematopoietic activity in hair follicles we first used in vitro haematopoietic colony assays. This demonstrated that rodent hair follicle end bulbs as well as micro-dissected dermal papilla and dermal sheath cells actively produced cells of erythroid and myeloid lineages but that follicle epithelial cells did not. As a more stringent test, we then transplanted cultured dermal papilla or dermal sheath cells from transgenically marked donor mice into lethally irradiated recipient mice and observed multi-lineage haematopoietic reconstitution when assayed at intervals of up to one year. Colony assays from bone marrow of primary recipients revealed that over 70% of clonogenic precursors were derived from donor hair follicle cells. When bone marrow from primary mice was harvested and used to repopulate secondary myeloablated recipients, multi-lineage haematopoietic engraftment was observed. Our data show that dermal but not epidermal compartments of the adult hair follicle have much broader stem cell activities than previously described. Although the treatment for many forms of blood disorder, such as leukemia, often requires transplantation of haematopoietic stem cells (HSC), their availability can be rate limiting. Given its easy accessibility, our identification of the hair follicle as a source of extramedullary haematopoietic stem cell activity makes it an attractive potential source for blood stem cell therapeutics and highlights its value as a model system in adult stem cell biology.

Overexpression of telomerase confers growth advantage, stress resistance, and enhanced differentiation of ESCs toward the hematopoietic lineage

Embryonic stem cells (ESCs) are capable of extended self‐renewal and maintenance of pluripotency even after many population doublings. This is supported by high levels of telomerase activity and enhanced antioxidant protection in ESCs, both of which are downregulated during differentiation. To examine the role of telomerase for ESC self‐renewal and differentiation, we overexpressed the reverse transcriptase subunit (Tert) of murine telomerase in ESCs. Increased telomerase activity enhances the self‐renewal ability of the Tert‐overexpressing ESCs, improves their resistance to apoptosis, and increases their proliferation. The differentiated progeny of wild‐type ESCs express little Tert and show shortening of telomeric overhangs. In contrast, the progeny of Tert‐overexpressing ESCs maintain high telomerase activity, as well as the length of G‐rich overhangs. In addition, these cells accumulate lower concentrations of peroxides than wild‐type cells, implying greater resistance to oxidative stress. Finally, differentiation toward hematopoietic lineages is more efficient as a result of the continued expression of Tert. Microarray analysis revealed that overexpression of Tert altered expression of a variety of genes required for extended self‐renewal and lifespan. Our results suggest that telomerase functions as a “survival enzyme” in ESCs and its differentiated progeny by protecting the telomere cap and by influencing the expression patterns of stress response and defense genes. This results in improved proliferation of ESCs and more efficient differentiation, and these results might have profound consequences for stem cell–replacement therapies.

Generation of mTert-GFP mice as a model to identify and study tissue progenitor cells

Stem cells hold great promise for regenerative medicine, but remain elusive in many tissues in part because universal markers of “stemness” have not been identified. The ribonucleoprotein complex telomerase catalyzes the extension of chromosome ends, and its expression is associated with failure of cells to undergo cellular senescence. Because such resistance to senescence is a common characteristic of many stem cells, we hypothesized that telomerase expression may provide a selective biomarker for stem cells in multiple tissues. In fact, telomerase expression has been demonstrated within hematopoietic stem cells. We therefore generated mouse telomerase reverse transcriptase (mTert)-GFP-transgenic mice and assayed the ability of mTert-driven GFP to mark tissue stem cells in testis, bone marrow (BM), and intestine. mTert-GFP mice were generated by using a two-step embryonic stem cell-based strategy, which enabled primary and secondary screening of stably transfected clones before blastocyst injection, greatly increasing the probability of obtaining mTert reporter mice with physiologically appropriate regulation of GFP expression. Analysis of adult mice showed that GFP is expressed in differentiating male germ cells, is enriched among BM-derived hematopoietic stem cells, and specifically marks long-term BrdU-retaining intestinal crypt cells. In addition, telomerase-expressing GFP+ BM cells showed long-term, serial, multilineage BM reconstitution, fulfilling the functional definition of hematopoietic stem cells. Together, these data provide direct evidence that mTert-GFP expression marks progenitor cells in blood and small intestine, validating these mice as a useful tool for the prospective identification, isolation, and functional characterization of progenitor/stem cells from multiple tissues.

Characterisation of Wnt gene expression during the differentiation of murine embryonic stem cells in vitro: role of Wnt3 in enhancing haematopoietic differentiation

The first haematopoietic stem cells in mammalian and non-mammalian vertebrates are derived from mesoderm, therefore genes that are important in mesoderm patterning and formation might also play an essential role in haematopoietic stem cell commitment and differentiation. Several members of the Wnt gene family are expressed in very specific patterns in embryonic mesoderm and have previously been shown to act as haematopoietic growth factors. In order to investigate in detail the role that such secreted proteins play in the biology of early haematopoietic commitment we have used in vitro differentiation of murine embryonal stem (ES) as a model system. Using reverse-transcriptase polymerase chain reaction analysis we identified several candidate Wnt genes whose expression pattern was consistent with a role in generation, maintenance and/or differentiation of early haematopoietic progenitor cells including three genes previously shown to have a role in haematopoiesis (Wnt5a, Wnt2b and Wnt10b). The most interesting candidate was Wnt3, because of its strong and regulated expression during in vitro differentiation of murine ES cells as well as its early embryonic expression in mesoderm. Overexpression of Wnt3 was sufficient to cause a consistent increase in the number of embryoid bodies committing to haematopoiesis further strengthening the evidence that this protein can enhance haematopoietic commitment during in vitro differentiation of ES cells. In addition, overexpression of Wnt3 caused a marked upregulation of Brachyury expression, thus providing some evidence that Brachyury may be one of the target genes for the Wnt3 signalling pathway.

Telomerase expression in the mammalian heart

While the mammalian heart has low, but functionally significant, levels of telomerase expression, the cellular population responsible remains incompletely characterized. This study aimed to identify the cell types responsible for cardiac telomerase activity in neonatal, adult, and cryoinjured adult hearts using transgenic mice expressing green fluorescent protein (GFP), driven by the promoter for murine telomerase reverse transcriptase (mTert), which is a necessary and rate‐limiting component of telomerase. A rare population of mTert‐GFP‐expressing cells was identified that possessed all detectable cardiac telomerase RNA and telomerase activity. It was heterogeneous and included cells coexpressing markers of cardiomyocytic, endothelial, and mesenchymal lineages, putative cardiac stem cell markers, and, interestingly, cardiomyocytes with a differentiated phenotype. Quantification using both flow cytometry and immunofluorescence identified a significant decline in mTert‐GFP cells in adult animals compared to neonates (~9‐ and ~20‐fold, respectively). Cardiac injury resulted in a ~6.45‐fold expansion of this population (P<0.005) compared with sham‐operated controls. This study identifies the cells responsible for cardiac telomerase activity, demonstrates a significant diminution with age but a marked response to injury, and, given the relationship between telomerase activity and stem cell populations, suggests that they represent a potential target for further investigation of cardiac regenerative potential.—Richardson, G. D., Breault, D., Horrocks, G., Cormack, S., Hole, N., Owens, W. A. Telomerase expression in the mammalian heart. FASEB J. 26, 4832–4840 (2012). www.fasebj.org

Silencing of the expression of pluripotent driven-reporter genes stably transfected into human pluripotent cells

AIMS & METHODS Marking of human embryonic stem (ES) and embryonal carcinoma (EC) cells with pluripotent promoter-driven reporter gene cassettes provides an important tool for studies related to maintenance of pluripotency, cell differentiation and cell selection. OCT4, TERF1 and telomerase reverse transcriptase component (TERT) are considered as pluripotent marker genes since they are expressed in both human ES and EC cells and significantly downregulated during the differentiation process. Our aim was to use core promoter regions from such pluripotent genes to drive expression of reporter genes that would be suitable for human ES cell selection amongst differentiated cells. RESULTS Human ES and EC cells were stably transfected with a number of TERT, OCT4 and TERF1 promoter-driven EGFP or NTR gene cassettes. Gradual loss of reporter gene expression was observed from 24 h post-transfection during transient transfection studies, while almost complete loss of reporter expression was observed upon stable transfections. The loss of reporter gene expression was partly reversed by addition of a histone deacetylase inhibitor and a demethylating agent, suggesting that in vitro methylation of these exogenous constructs and the epigenetic architecture around the site of integration are likely to play a major role in their transcriptional activity. Inclusion of gene-regulatory elements in addition to the core promoters has been shown to minimize such effects and should be considered as an important strategy in such studies. CONCLUSIONS Together our data suggest that human ES and EC cells are able to silence pluripotent promoter-driven reporter genes with high efficiency. Whether differentiated cells derived from human ES and EC cells retain this activity is unknown and need to be investigated before large-scale comparative reporter-based transfection studies can be used as a tool in human embryonic stem cell biology.

A role for nucleoprotein Zap3 in the reduction of telomerase activity during embryonic stem cell differentiation

Telomerase, the enzyme which maintains the ends of linear chromosomes in eukaryotic cells is found in murine embryonic stem cells; however, its activity is downregulated during in vitro differentiation. Previous work has indicated that this is due to the transcriptional downregulation of murine reverse transcriptase unit (mTert) of telomerase. To investigate the factors that cause the transcriptional repression of mTert we defined a 300 bp region which is essential for its transcription and performed site directed mutagenesis and electrophoretic mobility shift assays. This analysis indicated that Sp1, Sp3 and c-Myc bind to the GC-boxes and E-boxes, respectively, within the promoter and help activate the transcription of mTert gene. We also identified a novel binding sequence, found repeated within the mTert core region, which when mutated caused increased mTert expression. Yeast one hybrid screening combined with electrophoretic mobility shift assays indicated that the nuclear protein Zap3 binds to this site and its overexpression leads to the downregulation of mTert during differentiation. This suggests that regulation of mTert transcription is a complex process which depends on a quantitative balance between transcription factors that cause activation or repression of this gene. Overexpression of Zap3 in murine embryonic stem cells results in reduction in telomerase activity and telomere length as well as reduced proliferative capacity and limited ability to contribute to the development of haematopoietic cells upon differentiation.

Differentiating Embryonal Stem Cells Are a Rich Source of Haemopoietic Gene Products and Suggest Erythroid Preconditioning of Primitive Haemopoietic Stem Cells

The difficulties associated with studying molecular mechanisms important in hemopoietic stem cell (HSC) function such as the problems of purifying homogeneous stem cell populations, have prompted us to adapt the murine ES cell system as anin vitro model of HSC generation and function. We now report that careful analysis of the time course of HSC generation in differentiating ES cells allows them to be used as a source of known and novel hemopoietic gene products. We have generated a subtracted library using cDNA from ES cells collected just prior to and just following the emergence of HSCs. Analysis of this library shows it to be a rich source of known hemopoietic and hemopoietic related gene products with 44% of identifiable cDNAs falling into these camps. We have demonstrated the value of this system as a source of novel genes of relevance to HSC function by characterizing a novel membrane protein encoding cDNA that is preferentially expressed in primitive hemopoietic cells. Intriguingly, further analysis of the known components of the subtracted library is suggestive of erythroid preconditioning of the ES cell-derived HSC. We have used dot-blot andin situ analysis to indicate that this erythroid preconditioning is probably restricted to primitive but not definitive HSC.