Melanocyte stem cells: a melanocyte reservoir in hair follicles for hair and skin pigmentation

Most mammals are coated with pigmented hair. Melanocytes in each hair follicle produce melanin pigments for the hair during each hair cycle. The key to understanding the mechanism of cyclic melanin production is the melanocyte stem cell (MelSC) population, previously known as 'amelanotic melanocytes'. The MelSCs directly adhere to hair follicle stem cells, the niche cells for MelSCs and reside in the hair follicle bulge-subbulge area, the lower permanent portion of the hair follicle, to serve as a melanocyte reservoir for skin and hair pigmentation. MelSCs form a stem cell system within individual hair follicles and provide a 'hair pigmentary unit' for each cycle of hair pigmentation. This review focuses on the identification of MelSCs and their characteristics and explains the importance of the MelSC population in the mechanisms of hair pigmentation, hair greying, and skin repigmentation.     

Nerve-derived sonic hedgehog defines a niche for hair follicle stem cells capable of becoming epidermal stem cells

In adult skin, stem cells in the hair follicle bulge cyclically regenerate the follicle, whereas a distinct stem cell population maintains the epidermis. The degree to which all bulge cells have equal regenerative potential is not known. We found that Sonic hedgehog (Shh) from neurons signals to a population of cells in the telogen bulge marked by the Hedgehog response gene Gli1. Gli1-expressing bulge cells function as multipotent stem cells in their native environment and repeatedly regenerate the anagen follicle. Shh-responding perineural bulge cells incorporate into healing skin wounds where, notably, they can change their lineage into epidermal stem cells. The perineural niche (including Shh) is dispensable for follicle contributions to acute wound healing and skin homeostasis, but is necessary to maintain bulge cells capable of becoming epidermal stem cells. Thus, nerves cultivate a microenvironment where Shh creates a molecularly and phenotypically distinct population of hair follicle stem cells.     

In search of markers for the stem cells of the corneal epithelium

The anterior one-fifth of the human eye is called the cornea. It consists of several specialized cell types that work together to give the cornea its unique optical properties. As a result of its smooth surface and clarity, light entering the cornea focuses on the neural retina allowing images to come into focus in the optical centres of the brain. When the cornea is not smooth or clear, vision is impaired. The surface of the cornea consists of a stratified squamous epithelium that must be continuously renewed. The cells that make up this outer covering come from an adult stem cell population located at the corneal periphery at a site called the corneal limbus. While engaging in the search for surface markers for corneal epithelial stem cells, vision scientists have obtained a better understanding of the healthy ocular surface. In this review, we summarize the current state of knowledge of the ocular surface and its adult stem cells, and analyse data as they now exist regarding putative corneal epithelial stem cell markers.     

Induced pluripotent stem cells from hair follicles as a cellular model for neurodevelopmental disorders

Disease-specific induced pluripotent stem cells (iPSC) allow unprecedented experimental platforms for basic research as well as high-throughput screening. This may be particularly relevant for neuropsychiatric disorders, in which the affected neuronal cells are not accessible. Keratinocytes isolated from hair follicles are an ideal source of patients' cells for reprogramming, due to their non-invasive accessibility and their common neuroectodermal origin with neurons, which can be important for potential epigenetic memory. From a small number of plucked human hair follicles obtained from two healthy donors we reprogrammed keratinocytes to pluripotent iPSC. We further differentiated these hair follicle-derived iPSC to neural progenitors, forebrain neurons and functional dopaminergic neurons.This study shows that human hair follicle-derived iPSC can be differentiated into various neural lineages, suggesting this experimental system as a promising in vitro model to study normal and pathological neural developments, avoiding the invasiveness of commonly used skin biopsies.     

Morphogenesis and renewal of hair follicles from adult multipotent stem cells

The upper region of the outer root sheath of vibrissal follicles of adult mice contains multipotent stem cells that respond to morphogenetic signals to generate multiple hair follicles, sebaceous glands, and epidermis, i.e., all the lineages of the hairy skin. At the time when hair production ceases and when the lower region of the follicle undergoes major structural changes, the lower region contains a significant number of clonogenic keratinocytes, and can then respond to morphogenetic signals. This demonstrates that multipotent stem cells migrate to the root of the follicle to produce whisker growth. Moreover, our results indicate that the clonogenic keratinocytes are closely related, if not identical, to the multipotent stem cells, and that the regulation of whisker growth necessitates a precise control of stem cell trafficking.     

Reconstruction of corneal epithelium with cryopreserved corneal limbal stem cells in a goat model

We describe a procedure to construct an artificial corneal epithelium from cryopreserved limbal stem cells (LSCs) for corneal transplantation. The LSCs were separated from limbal tissue of male goats. The primary LSCs were identified by flow cytometry and were expanded. They were examined for stem cell-relevant properties and cryopreserved in liquid nitrogen. Cryopreserved LSCs were thawed and then transplanted onto human amniotic membrane, framed on a nitrocellulose sheet, to construct corneal epithelium sheets. The artificial corneal epithelium was transplanted into the right eye of pathological models of total limbal stem cell deficiency (LSCD). Then, the effects of reconstruction were evaluated by clinical observation and histological examination. Polymerase chain reaction analysis was used to detect the SRY gene. The data showed that transplantation of cryopreserved LSCs, like fresh LSCs, successfully reconstructed damaged goat corneal surface gradually, but the SRY gene expression from male goat cells could only be detected in the first 2 months after transplantation. The therapeutic effect of the transplantation may be associated with the inhibition of inflammation-related angiogenesis after transplantation of cryopreserved LSCs. This study provides the first line of evidence that cryopreserved LSCs can be used for reconstruction of damaged corneas, presenting a remarkable potential source for transplantation in the treatment of corneal disorders.     

Quantitative proliferation dynamics and random chromosome segregation of hair follicle stem cells

Regulation of stem cell (SC) proliferation is central to tissue homoeostasis, injury repair, and cancer development. Accumulation of replication errors in SCs is limited by either infrequent division and/or by chromosome sorting to retain preferentially the oldest 'immortal' DNA strand. The frequency of SC divisions and the chromosome-sorting phenomenon are difficult to examine accurately with existing methods. To address this question, we developed a strategy to count divisions of hair follicle (HF) SCs over time, and provide the first quantitative proliferation history of a tissue SC during its normal homoeostasis. We uncovered an unexpectedly high cellular turnover in the SC compartment in one round of activation. Our study provides quantitative data in support of the long-standing infrequent SC division model, and shows that HF SCs do not retain the older DNA strands or sort their chromosome. This new ability to count divisions in vivo has relevance for obtaining basic knowledge of tissue kinetics.     

骨髓间充质干细胞向角膜上皮细胞分化及其免疫调节作用

目的：探讨大鼠骨髓间充质干细胞（rBMMSCs）转分化为角膜上皮的潜能,并在体外共培养体系中研究rBMMSCs对促炎细胞因子干扰素-γ（IFN-γ）和肿瘤坏死因子-α（TNF-α）刺激下的人角膜上皮细胞（hCECs）的免疫调节作用。方法采用聚蔗糖梯密度离心法获得rBMMSCs,并通过上皮细胞培养微环境来诱导rBMMSCs分化为上皮样细胞。通过免疫组织化学方法鉴定CD29、CD34、CK5＆8和ZO-1等标记物在rBMMSCs及诱导的上皮样细胞中的表达。流式细胞术用来分析CD29/CD34的表达及细胞分化过程中表达量的变化。hCECs单独培养或与rBMMSCs共培养,并采用IFN-γ/TNF-α刺激24或48 h。通过流式细胞术来分析细胞间黏附分子-1（ICAM-1）于IFN-γ/TNF-α刺激前后在hCECs上的表达,并通过黏附分析实验验证rBMMSC条件培养基对单核细胞黏附于IFN-γ/TNF-α刺激后的hCECs的作用。多组间比较采用单因素方差分析（ANOVA）,两组间比较采用双侧t检验。结果成功分离rBMMSCs,细胞表达CD29,但不表达CD34。在上皮细胞培养条件中培养5 d,大约4﹪的rBMMSCs可分化为上皮样细胞。此类细胞失去了CD29的标志,转为表达CK5＆8和ZO-1。IFN-γ/TNF-α能显著上调hCECs中ICAM-1的表达,在IFN-γ/TNF-α处理24 h和48 h后,ICAM-1分别呈现10倍和8倍的升高,分别达到4524±554.2和3107±329.6（P=0.0025,0.0014）。但与MSC共同培养时,上调作用被显著抑制,ICAM-1平均值为1356±325.6（24 h）与1323±106.6（48 h）（P=0.0079,0.0024）。MSC条件培养基可显著抑制单核细胞对hCECs的黏附作用,黏附细胞数从（10.01±3.01）×10^3/ml细胞降至（2.21±0.19）×10^3/ml细胞（P=0.0271）。结论rBMMSCs可转分化为角膜上皮样细胞,并抑制由促炎细胞因子诱导的ICAM-1在hCECs上的表达,同时对促炎细胞因子诱导的单核细胞的黏附性具有抑制作用,提示BMMSCs具有在角膜炎症疾病和损伤修复中的治疗潜能。     

Reversible commitment of neural and epidermal progenitor cells during embryogenesis of Drosophila melanogaster

Summary Cell-cell interactions are involved in mediating developmental fate. An example is the decision of the neuroectodermal cells of Drosophila to develop as neural or epidermal progenitors, where cellular interactions participate in the process of acquisition of either cell fate. The results of heterochronic cell transplantations we describe here suggest that both neuroblasts and epidermoblasts are not irreversibly committed to a particular developmental fate. Rather, they retain the ability to interact with neighbouring cells and, under our experimental conditions, are capable of switching their fate during a relatively long period of time, i.e. until the end of embryonic stage 11.     

Reepithelialization of a full-thickness burn from stem cells of hair follicles micrografted into a tissue-engineered dermal template (Integra)

We report a head and neck full-thickness burn injury that was reconstructed with a tissue-engineered dermal template and then early implantation of microdissected hair follicles through the silicone epidermis 12 days after the burn injury. The treatment resulted in complete reepithelialization and a hair-bearing scalp without the need for a split-thickness skin graft. Restoration of the stem cell population, hair growth, and earlier reepithelialization were achieved using this novel micrografting technique, and histologic examination confirmed maturation of a normal skin type over the subsequent 2 years.     

Transdifferentiation of human adipose-derived stem cells into urothelial cells: potential for urinary tract tissue engineering

Autologous urothelial cells (UCs) provide a cell source for urinary tissue engineering because they can be used safely due to their lack of immunogenicity. However, these cells cannot be harvested under the following circumstances: malignancy, infection and organ loss, etc. Human adipose-derived stem cells (HADSCs) possess the traits of high differentiation potential and ease of isolation, representing a promising resource for tissue engineering and regenerative medicine. Nevertheless, HADSCs have been poorly investigated in urology and the optimal approaches to induce HADSCs into urothelium are still under investigation. In this study, we hypothesized that the change of microenvironment by a conditioned medium was essential for the transdifferentiation of HADSCs into UCs. We then used a conditioned medium derived from urothelium to alternate the microenvironment of HADSCs. After 14 days of culture in a conditioned medium, about 25–50% HADSCs changed their morphology into polygonal epithelium-like shapes. In addition, these cells expressed up-regulating of urothelial lineage-specific markers (uroplakin 2and cytokeratin-18) and down-regulating of mesenchymal marker (vimentin) in RNA and protein level, respectively, which confirmed that HADSCs were induced into urothelial lineage cells. We also measured the growth factors in the conditioned medium in order to analyze the molecular mechanisms regulating transdifferentiation. We observed that the expression levels of PDGF-BB and VEGF were significantly higher than those of the control group after 14 days induction, suggesting they were abundantly secreted into the medium during the culturing period. In conclusion, HADSCs showed in vitro the upregulation of markers for differentiation towards urothelial cells by culturing in an urothelial-conditioned medium, which provides an alternative cell source for potential use in urinary tract tissue engineering.     

Troika of the mouse blastocyst: lineage segregation and stem cells

The initial period of mammalian embryonic development is primarily devoted to cell commitment to the pluripotent lineage, as well as to the formation of extraembryonic tissues essential for embryo survival in utero. This phase of development is also characterized by extensive morphological transitions. Cells within the preimplantation embryo exhibit extraordinary cell plasticity and adaptation in response to experimental manipulation, highlighting the use of a regulative developmental strategy rather than a predetermined one resulting from the non-uniform distribution of maternal information in the cytoplasm. Consequently, early mammalian development represents a useful model to study how the three primary cell lineages; the epiblast, primitive endoderm (also referred to as the hypoblast) and trophoblast, emerge from a totipotent single cell, the zygote. In this review, we will discuss how the isolation and genetic manipulation of murine stem cells representing each of these three lineages has contributed to our understanding of the molecular basis of early developmental events.     

Origins and selection of epidermal progenitors and stem cells: a challenge for tissue engineering

The use of epidermal stem cells and their progeny for tissue engineering and cell therapy represents a source of hope and major interest in view of applications such as replacing the loss of functionality in failing tissues or obtaining physiologic skin equivalents for skin grafting. The use of such cells necessitates the isolation and purification of rare populations of keratinocytes and then increasing their numbers by mass culture. This is not currently possible since part of the specific phenotype of these cells is lost once the cells are placed in culture. Furthermore, few techniques are available to unequivocally detect the presence of skin stem cells and/or their progeny in culture and thus quantify them. Two different sources of stem cells are currently being studied for skin research and clinical applications: skin progenitors either obtained from embryonic stem cells (ESC) or from selection from adult skin tissue. It has been shown that "keratinocyte-like" cells can be derived from ESC; however, the culturing processes must still be optimized to allow for the mass culture of homogeneous populations at a controlled stage of differentiation. The functional characterization of such populations must also be more thoroughly achieved. In order to use stem cells from adult tissues, improvements must be made in order to obtain a satisfactory degree of purification and characterization of this rare population. Distinguishing stem cells from progenitor cells at the molecular level also remains a challenge. Furthermore, stem cell research inevitably requires cultivating these cells outside their physiological environment or niche. It will thus be necessary to better understand the impact of this specific environmental niche on the preservation of the cellular phenotypes of interest.     

Dynamics between stem cells, niche, and progeny in the hair follicle

Here, we exploit the hair follicle to define the point at which stem cells (SCs) become irreversibly committed along a differentiation lineage. Employing histone and nucleotide double-pulse-chase and lineage tracing, we show that the early SC descendents en route to becoming transit-amplifying cells retain stemness and slow-cycling properties and home back to the bulge niche when hair growth stops. These become the primary SCs for the next hair cycle, whereas initial bulge SCs become reserves for injury. Proliferating descendents further en route irreversibly lose their stemness, although they retain many SC markers and survive, unlike their transit-amplifying progeny. Remarkably, these progeny also home back to the bulge. Combining purification and gene expression analysis with differential ablation and functional experiments, we define critical functions for these non-SC niche residents and unveil the intriguing concept that an irreversibly committed cell in an SC lineage can become an essential contributor to the niche microenvironment.