Human long-term deregulated circadian rhythm alters regenerative properties of skin and hair precursor cells.

In mammals, desynchronized circadian rhythm leads to various biological symptoms. In skin and hair, human epidermal stem cell function in vitro is regulated by circadian oscillations, and thus contributes to tissue aging when deregulated. In mice, circadian arrhythmia of hair follicle stem cells contributes to age-related hair follicle cycling defects. Despite the well-described impact of circadian oscillations through a feedback loop involving the clock pathway on hair and skin stem cell function in vitro, little is known about the change in characteristics or regenerative properties of hHF (human hair follicle keratinocytes), hEpi (human interfollicular epidermal keratinocytes), and hHFDP (hair follicle dermal papilla stem cells) after long-term alteration of circadian rhythm in vivo. The present study was designed to asses hHF, hEpi, and hHFDP precursors and stem cell properties in response to clock pathway alteration due to long-term deregulated circadian rhythm in vivo. A clinical study protocol was designed to include two groups of women: diurnal workers (control) and shift workers (deregulated). After informed consent, two 3-mm fresh punch biopsies were taken from the occipital region of each donor (10 donors/group). Cell culture characterization, measurement of colony area, culture medium analysis, and RT-qPCR analysis were carried out. Long-term circadian rhythm deregulation affected clock pathway protein expression and correlated with alterations in hHF, hEpi, and hHFDP properties. This study provides, for the first time in humans, evidence that in vivo deregulation of the clock pathway affects regenerative properties of human skin and hair precursor cells.

3D In vitro model of the re-epithelialization phase in the wound-healing process.

Keratinocytes of the basal layer function are to maintain tissue homoeostasis and to fulfil skin repair in response to an external aggression. In wound-healing, during re-epithelialization phase, epithelial precursor cells gradually migrate from the edges of the wound. The epidermal reconstruction model called standard model allows the vertical skin regeneration process (proliferation/differentiation) to being investigated, and keratinocyte function in preserving skin homoeostasis to being assessed. Here, we developed and characterized a 3D migration model, which introduces a step of keratinocytes migration such as the one observed in the phase of re-epithelialization in wound-healing process. We validated the added value and the discriminative potential of this model by demonstrating pro-epithelializing effects of compounds. This new model allows the role of keratinocytes in different biomechanical and environmental requests to being better understood, and brings a new tool for compound screening and the study of mechanisms involved in skin regeneration.

Identification of Drugs that Regulate Dermal Stem Cells and Enhance Skin Repair.

Here, we asked whether we could identify pharmacological agents that enhance endogenous stem cell function to promote skin repair, focusing on skin-derived precursors (SKPs), a dermal precursor cell population. Libraries of compounds already used in humans were screened for their ability to enhance the self-renewal of human and rodent SKPs. We identified and validated five such compounds, and showed that two of them, alprostadil and trimebutine maleate, enhanced the repair of full thickness skin wounds in middle-aged mice. Moreover, SKPs isolated from drug-treated skin displayed long-term increases in self-renewal when cultured in basal growth medium without drugs. Both alprostadil and trimebutine maleate likely mediated increases in SKP self-renewal by moderate hyperactivation of the MEK-ERK pathway. These findings identify candidates for potential clinical use in human skin repair, and provide support for the idea that pharmacological activation of endogenous tissue precursors represents a viable therapeutic strategy.

Direct genesis of functional rodent and human schwann cells from skin mesenchymal precursors.

Recent reports of directed reprogramming have raised questions about the stability of cell lineages. Here, we have addressed this issue, focusing upon skin-derived precursors (SKPs), a dermally derived precursor cell. We show by lineage tracing that murine SKPs from dorsal skin originate from mesenchymal and not neural crest-derived cells. These mesenchymally derived SKPs can, without genetic manipulation, generate functional Schwann cells, a neural crest cell type, and are highly similar at the transcriptional level to Schwann cells isolated from the peripheral nerve. This is not a mouse-specific phenomenon, since human SKPs that are highly similar at the transcriptome level can be made from neural crest-derived facial and mesodermally derived foreskin dermis and the foreskin SKPs can make myelinating Schwann cells. Thus, nonneural crest-derived mesenchymal precursors can differentiate into bona fide peripheral glia in the absence of genetic manipulation, suggesting that developmentally defined lineage boundaries are more flexible than widely thought.

Ageing and colony-forming efficiency of human hair follicle keratinocytes.

The decline of tissue regenerative potential of skin and hair is a hallmark of physiological ageing and may be associated with age-related changes in tissue-specific stem cells and/or their environment. Human hair follicles (hHF) contain keratinocytes having the property of stem cells such as clonogenic potential. Growth capacity of hHF keratinocytes shows that most of the colony-forming cells are classified as holoclones, meroclones or paraclones when analysed in a clonal assay (Cell, Volume 76, page 1063). Despite the well-known impact of ageing on human hair growth, little is known about changes in hHF keratinocyte clonogenic potential with age. This study aimed at assessing the clone-forming efficiency (CFE) of hHF keratinocytes from three age groups of human donors. It demonstrates that ageing affects hHF keratinocyte CFE.

Convergent genesis of an adult neural crest-like dermal stem cell from distinct developmental origins.

Skin-derived precursors (SKPs) are multipotent dermal stem cells that reside within a hair follicle niche and that share properties with embryonic neural crest precursors. Here, we have asked whether SKPs and their endogenous dermal precursors originate from the neural crest or whether, like the dermis itself, they originate from multiple developmental origins. To do this, we used two different mouse Cre lines that allow us to perform lineage tracing: Wnt1-cre, which targets cells deriving from the neural crest, and Myf5-cre, which targets cells of a somite origin. By crossing these Cre lines to reporter mice, we show that the endogenous follicle-associated dermal precursors in the face derive from the neural crest, and those in the dorsal trunk derive from the somites, as do the SKPs they generate. Despite these different developmental origins, SKPs from these two locations are functionally similar, even with regard to their ability to differentiate into Schwann cells, a cell type only thought to be generated from the neural crest. Analysis of global gene expression using microarrays confirmed that facial and dorsal SKPs exhibit a very high degree of similarity, and that they are also very similar to SKPs derived from ventral dermis, which has a lateral plate origin. However, these developmentally distinct SKPs also retain differential expression of a small number of genes that reflect their developmental origins. Thus, an adult neural crest-like dermal precursor can be generated from a non-neural crest origin, a finding with broad implications for the many neuroendocrine cells in the body.

SKPs derive from hair follicle precursors and exhibit properties of adult dermal stem cells.

Despite the remarkable regenerative capacity of mammalian skin, an adult dermal stem cell has not yet been identified. Here, we investigated whether skin-derived precursors (SKPs) might fulfill such a role. We show that SKPs derive from Sox2(+) hair follicle dermal cells and that these two cell populations are similar with regard to their transcriptome and functional properties. Both clonal SKPs and endogenous Sox2(+) cells induce hair morphogenesis, differentiate into dermal cell types, and home to a hair follicle niche upon transplantation. Moreover, hair follicle-derived SKPs self-renew, maintain their multipotency, and serially reconstitute hair follicles. Finally, grafting experiments show that follicle-associated dermal cells move out of their niche to contribute cells for dermal maintenance and wound-healing. Thus, SKPs derive from Sox2(+) follicle-associated dermal precursors and display functional properties predicted of a dermal stem cell, contributing to dermal maintenance, wound-healing, and hair follicle morphogenesis.

TAp63 prevents premature aging by promoting adult stem cell maintenance.

The cellular mechanisms that regulate the maintenance of adult tissue stem cells are still largely unknown. We show here that the p53 family member, TAp63, is essential for maintenance of epidermal and dermal precursors and that, in its absence, these precursors senesce and skin ages prematurely. Specifically, we have developed a TAp63 conditional knockout mouse and used it to ablate TAp63 in the germline (TAp63(-/-)) or in K14-expressing cells in the basal layer of the epidermis (TAp63(fl/fl);K14cre+). TAp63(-/-) mice age prematurely and develop blisters, skin ulcerations, senescence of hair follicle-associated dermal and epidermal cells, and decreased hair morphogenesis. These phenotypes are likely due to loss of TAp63 in dermal and epidermal precursors since both cell types show defective proliferation, early senescence, and genomic instability. These data indicate that TAp63 serves to maintain adult skin stem cells by regulating cellular senescence and genomic stability, thereby preventing premature tissue aging.

Homeodomain transcription factor Phox2a, via cyclic AMP-mediated activation, induces p27Kip1 transcription, coordinating neural progenitor cell cycle exit and differentiation.

Mechanisms coordinating neural progenitor cell cycle exit and differentiation are incompletely understood. The cyclin-dependent kinase inhibitor p27(Kip1) is transcriptionally induced, switching specific neural progenitors from proliferation to differentiation. However, neuronal differentiation-specific transcription factors mediating p27(Kip1) transcription have not been identified. We demonstrate the homeodomain transcription factor Phox2a, required for central nervous system (CNS)- and neural crest (NC)-derived noradrenergic neuron differentiation, coordinates cell cycle exit and differentiation by inducing p27(Kip1) transcription. Phox2a transcription and activation in the CNS-derived CAD cell line and primary NC cells is mediated by combined cyclic AMP (cAMP) and bone morphogenetic protein 2 (BMP2) signaling. In the CAD cellular model, cAMP and BMP2 signaling initially induces proliferation of the undifferentiated precursors, followed by p27(Kip1) transcription, G(1) arrest, and neuronal differentiation. Small interfering RNA silencing of either Phox2a or p27(Kip1) suppresses p27(Kip1) transcription and neuronal differentiation, suggesting a causal link between p27(Kip1) expression and differentiation. Conversely, ectopic Phox2a expression via the Tet-off expression system promotes accelerated CAD cell neuronal differentiation and p27(Kip1) transcription only in the presence of cAMP signaling. Importantly, endogenous or ectopically expressed Phox2a activated by cAMP signaling binds homeodomain cis-acting elements of the p27(Kip1) promoter in vivo and mediates p27(Kip1)-luciferase expression in CAD and NC cells. We conclude that developmental cues of cAMP signaling causally link Phox2a activation with p27(Kip1) transcription, thereby coordinating neural progenitor cell cycle exit and differentiation.

The cAMP pathway in combination with BMP2 regulates Phox2a transcription via cAMP response element binding sites.

Combined BMP2 and cAMP signaling induces the catechola-minergic lineage in neural crest (NC) cultures by increasing expression of the proneural transcription factor Phox2a, in a cAMP response element (CRE)-binding protein (CREB)-mediated mechanism. To determine whether CREB acts directly on Phox2a transcription induced by BMP2+cAMP-elevating agent IBMX, transient transfections of hPhox2a-reporter constructs were performed in avian NC cultures and murine, catecholaminergic CAD cells. Although BMP2+IBMX increased endogenous Phox2a expression, the 7.5-kb hPhox2a reporters expressing either luciferase or DsRed1-E5 fluorescent protein were unresponsive to BMP2+IBMX, but active in both cell types. Cell sorting of fluorescence-positive NC cells expressing the 7.5-kb hPhox2a fluorescent timer reporter differentiated to equal numbers of catecholaminergic cells as fluorescence-negative cells, suggesting inappropriate transcription from the transfected hPhox2a promoter. NC or CAD cells treated with histone deacetylase inhibitor trichostatin A and BMP2+IBMX display increased endogenous Phox2a transcription and prolonged CREB phosphorylation, indicating Phox2a chromatin remodeling is linked to CREB activation. Chromatin immunoprecipitations employing CREB, CREB-binding protein, and acetylated H4 antibodies identified two CRE half-sites at -5.5 kb in the murine Phox2a promoter, which is also conserved in the human promoter. Proximal to the CRE half-sites, within a 170-bp region, are E-box and CCAAT binding sites, also conserved in mouse and human genes. This 170-bp promoter region confers cAMP, BMP2, and enhanced BMP2+cAMP regulation to Phox2a-luciferase reporters. We conclude these CREs are functional, with CREB directly activating Phox2a transcription. Because the E-box binds bHLH proteins like ASH1 induced in NC cells by BMP2, we propose this novel 170-bp cis-acting element is a composite site, mediating the synergistic regulation by BMP2+cAMP on Phox2a transcription.

The cAMP pathway regulates both transcription and activity of the paired homeobox transcription factor Phox2a required for development of neural crest-derived and central nervous system-derived catecholaminergic neurons.

Pluripotent neural crest (NC) cells differentiate to diverse lineages, including the neuronal, sympathoadrenal lineage. In primary NC cultures, bone morphogenetic protein 2 (BMP2) requires moderate activation of cAMP signaling for induction of the sympathoadrenal lineage. However, the mechanism by which cAMP signaling synergizes with BMP2 to induce the sympathodrenal lineage is unknown. Herein, we demonstrate that moderate activation of cAMP signaling induces both transcription and activity of proneural transcription factor Phox2a. In NC cultures inhibition of cAMP-response element-binding protein (CREB)-mediated transcription by expression of dominant-negative CREB suppresses Phox2a transcription and sympathoadrenal lineage development. Interestingly, the constitutively active CREB(DIEDML), despite inducing Phox2a transcription, is insufficient for sympathoadrenal lineage development, requiring activation of the cAMP pathway. Because CREB(DIEDML)-mediates cAMP-dependent transcription without requiring activation by the cAMP-dependent protein kinase A (PKA), these results identify PKA activation as necessary in sympathoadrenal lineage development. Treatment of NC cultures with the PKA inhibitor H89 or 1-10 nm okadaic acid (OA), a serine/threonine PP2A-like phosphatase inhibitor, suppresses sympathoadrenal lineage development. Likewise, OA treatment of the CNS-derived catecholaminergic CAD cell line inhibits cAMP-mediated neuronal differentiation. Specifically, OA inhibits cAMP-mediated Phox2a dephosphorylation, cAMP-dependent Phox2a DNA binding in vitro, and cAMP- and Phox2a-dependent dopamine-beta-hydroxylase-luciferase reporter expression. Together, these results support cAMP-dependent Phox2a dephosphorylation is required for its activation. We conclude that moderate activation of cAMP signaling has dual inputs in catecholaminergic, sympathoadrenal lineage development; that is, regulation of both Phox2a transcription and activity. These results provide the first mechanistic understanding of how moderate activation of the cAMP pathway in synergy with BMP2 promotes sympathoadrenal lineage development.

Adenosine signaling promotes neuronal, catecholaminergic differentiation of primary neural crest cells and CNS-derived CAD cells.

In neural crest (NC) cultures cAMP signaling is an instructive signal in catecholaminergic, sympathoadrenal cell development. However, the extracellular signals activating the cAMP pathway during NC cell development have not been identified. We demonstrate that in avian NC cultures, evidenced by tyrosine hydroxylase expression and catecholamine biosynthesis, adenosine and not adrenergic signaling, together with BMP2, promotes sympathoadrenal cell development. In NC cultures, addition of the adenosine receptor agonist NECA in the presence of BMP2 promotes sympathoadrenal cell development, whereas the antagonist CGS 15943 or the adenosine degrading enzyme adenosine deaminase (ADA) suppresses TH expression. Importantly, NC cells express A2A and A2B receptors which couple with Gsalpha increasing intracellular cAMP. Employing the CNS-derived catecholaminergic CAD cell line, we also demonstrate that neuronal differentiation mediated by serum withdrawal is further enhanced by treatment with IBMX, a cAMP-elevating agent, or the adenosine receptor agonist NECA, acting via cAMP. By contrast, the adenosine receptor antagonist CGS 15943 or the adenosine degrading enzyme ADA inhibits CAD cell neuronal differentiation mediated by serum withdrawal. These results support that adenosine is a physiological signal in neuronal differentiation of the CNS-derived catecholaminergic CAD cell line and suggest that adenosine signaling is involved in NC cell development in vivo.

Review: pancreatic beta-cell neogenesis revisited.

Beta-cell neogenesis triggers the generation of new beta-cells from precursor cells. Neogenesis from duct epithelium is the most currently described and the best documented process of differentiation of precursor cells into beta-cells. It is contributes not only to beta-cell mass expansion during fetal and nonatal life but it is also involved in the maintenance of the beta-cell mass in adults. It is also required for the increase in beta-cell mass in situations of increase insulin demand (obesity, pregnancy). A large number of factors controlling the differentiation of beta-cells has been identified. They are classified into the following main categories: growth factors, cytokine and inflammatory factors, and hormones such as PTHrP and GLP-1. The fact that intestinal incretin hormone GLP-1 exerts a major trophic role on pancreatic beta-cells provides insights into the possibility to pharmacologically stimulate beta-cell neogenesis. This could have important implications for the of treatment of type 1 and type 2 diabetes. Transdifferentiation, that is, the differentiation of already differentiated cells into beta-cells, remains controversial. However, more and more studies support this concept. The cells, which can potentially "transdifferentiate" into beta-cells, can belong to the pancreas (acinar cells) and even islets, or originate from extra-pancreatic tissues such as the liver. Neogenesis from intra-islet precursors also have been proposed and subpopulations of cell precursors inside islets have been described by some authors. Nestin positive cells, which have been considered as the main candidates, appear rather as progenitors of endothelial cells rather than beta-cells and contribute to angiogenesis rather than neogenesis. To take advantage of the different differentiation processes may be a direction for future cellular therapies. Ultimately, a better understanding of the molecular mechanisms involved in beta-cell neogenesis will allow us to use any type of differentiated and/or undifferentiated cells as a source of potential cell precursors.

Role of glucose in IRS signaling in rat pancreatic islets: specific effects and interplay with insulin.

We investigated the possible interplay between insulin and glucose signaling pathways in rat pancreatic beta-cell with a special focus on the role of glucose in IRS signaling in vivo. Three groups of rats were constituted by combining simultaneous infusion during 48 h either of glucose and/or insulin, or glucose+diazoxide: Hyperglycemic-Hyperinsulinemic (HGHI), euglycemic-Hyperinsulinemic (eGHI), Hyperglycemic-euinsulinemic (HGeI). Control rats were infused with 0,9%NaCl. In HGHI and HGeI rats plasma glucose levels were maintained at 20-22 mmol/l. In eGHI rats, plasma glucose was not different from that of controls, whereas plasma insulin was much higher than in controls. In HGHI rats, IRS-2 mRNA expression, total protein and phosphorylated protein amounts were increased compared to controls. In HGeI rats, only IRS-2 mRNA expression was increased. No change was observed in eGHI rats whatever the parameter considered. In all groups, mRNA concentration of IRS-1 was similar to that of controls. The quantity of total and phosphorylated IRS-1 protein was dramatically increased in HGHI rats and to a lesser extent in eGHI rats. Neither mRNA nor IRS-1 protein expression were modified in HGeI rats. The data suggest that glucose and insulin play at once a specific and a complementary role in islet IRSs signaling. Especially, glucose stimulates IRS-2 mRNA expression whatever the insulin status and independently of the secretory process. The differential regulation of IRS-1 and IRS-2 expressions is in agreement with their supposed different involvement in the control of beta-cell growth and function.

Specific and combined effects of insulin and glucose on functional pancreatic beta-cell mass in vivo in adult rats.

We investigated the specific and associated effects of insulin and glucose on beta-cell growth and function in adult rats. By combining simultaneous infusion either of glucose and/or insulin or glucose and diazoxide, three groups of rats were constituted: hyperglycemic-hyperinsulinemic rats (high glucose-high insulin), hyperglycemic-euinsulinemic rats (high glucose), and euglycemic-hyperinsulinemic rats (high insulin). All the infusions lasted 48 h. Control rats were infused with 0.9% NaCl (saline controls). In all groups, beta-cell mass was significantly increased, compared with controls (by 70% in high glucose-high insulin rats, 65% in high glucose rats, and 50% in high insulin rats). The stimulation of neogenesis was suggested by the high number of islets budding from pancreatic ducts in high glucose-high insulin and high glucose rats and by the presence of numerous clusters of few beta-cells within the exocrine pancreas in high insulin rats. beta-Cell hypertrophy was observed only in high glucose-high insulin rats. The rate of beta-cell proliferation was similar to that of controls in high glucose-high insulin rats after a 48-h glucose infusion, dropped dramatically in high insulin rats, and dropped to a lesser extent in high glucose rats. In high glucose-high insulin and high glucose rats, beta-cell mass increase was related to a higher beta-cell responsiveness to glucose in vitro as measured by islet perifusion studies, whereas in high insulin rats, no significant enhancement of glucose induced insulin secretion could be noticed. The data show that glucose and insulin may have specific stimulating effects on beta-cell growth and function in vivo in adult rats independently of the influence they exert each other on their respective plasma concentration.