Bone morphogenetic protein 5 regulates the number of keratinocyte stem cells from the skin of mice

Understanding keratinocyte stem cell regulation is important in understanding the pathogenesis of wound healing and nonmelanoma skin cancer. We previously used a sensitive and quantitative assay for in vitro keratinocyte colony formation and mapped the keratinocyte stem cell locus (Ksc1) on mouse chromosome 9. Examination of the candidate genes in this locus disclosed a sequence variant in the gene for bone morphogenetic protein 5 (Bmp5). In this report, we used a naturally occurring mouse with a null mutation in this gene to probe stem cell properties in mouse epidermis. We found that the mutant keratinocytes had a significant reduction in the size and number of clonogenic keratinocytes. The mutant mice had a 50% reduction in the number of label-retaining cells when compared with their littermates. Addition of exogenous Bmp5 protein increased the number and size of keratinocyte colonies in the mutant as well as their wild-type littermates. Surprisingly, the mutant mice showed at least a 2-fold increase in skin tumor susceptibility over their littermates. We conclude that a naturally occurring mutation in Bmp5 affects keratinocyte stem cell proliferation, and skin tumor susceptibility, and is a candidate stem cell regulatory gene in the Ksc1 locus.     

Isolation and characterization of putative epidermal stem cells derived from Cashmere goat fetus

This study was undertaken to select putative epidermal stem cells from cultured keratinocytes in Cashmere goat fetus and to characterize them in stem cell nature. The keratinocytes were separated enzymatically from fetuses of 12-16 weeks of gestation and co-cultured with mitotically inactivated fetal skin fibroblasts. Putative epidermal stem cells were selected by rapid adherence on collagen type IV substrate and maintained in three different medium conditions: high Ca(2+) concentration, low Ca(2+) concentration, and low Ca(2+) concentration with 50% conditioned medium. The results indicated that epidermal basal cells grew clonally on the feeder layer and were maintained for approximately 48 population doublings without showing signs of replicative senescence. Clonal analysis revealed the presence of three clonal types: holoclones, meroclones and paraclones. Selected keratinocytes on collagen IV substrate could be propagated serially in three medium conditions and the population showed high colony formation efficiency, the same morphology with a high nuclear to cytoplasmic ratio and positive expression of p63, Keratin 19, Keratin 15 and CD71, which are believed to be possible specific markers for keratinocyte stem cells. This study reports a method to isolate a selected keratinocyte population with the characteristics of stem cells.     

Epidermal stem cells do not communicate through gap junctions

Although enrichment of putative epidermal stem cells has been achieved, a need for additional markers that can enable isolation of live keratinocytes is crucial for characterization of these cells. Earlier work has shown that connexin proteins are absent from basal cells in the limbal epithelium, a region of the corneal epithelium enriched in corneal stem cells. Accordingly, we investigated whether connexin 43, a gap junction protein present in the basal layer of normal human epidermis, can serve as a negative marker for keratinocyte stem cells. In humans, cells with immunohistochemically undetectable levels of connexin 43 are found in the epidermal basal layer of neonatal foreskin and in the follicular bulge region. About 10% of the basal keratinocytes are connexin 43 negative, as determined by flow cytometry. These cells are uniformly small and low in granularity. Restricted gap junction communication was confirmed by the failure of low molecular weight dyes to transfer between cells. Experiments carried out in mouse epidermis demonstrated that most of the slowly cycling cells, detected as label-retaining cells, do not express connexin 43. Thus, presumptive keratinocyte stem cells can be identified and separated based on connexin 43 expression.     

Label-retaining cells (presumptive stem cells) of mice vibrissae do not express gap junction protein connexin 43

We investigated whether connexin 43, a gap junction protein present in human epidermis and mouse hair follicle, can serve as a negative marker for keratinocyte stem cells. Experiments carried out in mouse pelage and vibrissae hair follicles demonstrated that most of the slowly cycling cells, detected as label-retaining cells, do not express connexin 43. In humans, cells with immunohistochemically undetectable levels of connexin 43 are found in the epidermal basal layer of neonatal foreskin, and in the follicular bulge region. About 10% of the basal keratinocytes are connexin 43 negative, as determined by flow cytometry. These cells are uniformly small and low in granularity suggesting that presumptive keratinocyte stem cells can be identified and separated based on connexin 43 expression.     

Murine epidermal label-retaining cells isolated by flow cytometry do not express the stem cell markers CD34, Sca-1, or Flk-1.

Keratinocyte stem cells are present in the murine epidermis, based on both in vitro and in vivo evidence, and better characterization of these cells remains an active goal. Because keratinocyte stem cells are believed to cycle slowly, a good method for identification is based on their ability to retain nucleoside analog, such as bromodeoxyuridine. Adult stem cells have been identified in other tissues, including hematopoietic, neural, and skeletal muscle, and stem cell surface markers have been characterized. We wanted to determine if cell-surface markers present on both hematopoietic and skeletal muscle stem cells (CD34, Sca-1, and Flk-1) were also present on keratinocyte stem cells, and could be used to identify them. The cell-surface expression of cells that retained bromodeoxyuridine label for at least 21 d was compared with that of nonlabel-retaining cells. Double-labeling for flow cytometric analysis was employed, and label-retaining cells were found to lack expression of the tested markers. Beta1 integrin levels were also evaluated, and although high expression was found on label-retaining cells, it was not specific for these cells.     

Conversion from human haematopoietic stem cells to keratinocytes requires keratinocyte secretory factors

Background. Recent studies have reported that bone‐marrow‐derived stem cells (BMSCs), including haematopoietic stem cells (HSCs) and mesenchymal stromal cells, differentiate in order to regenerate various cellular lineages. Based on these findings, it is known that BMSCs can be used clinically to treat various disorders, such as myocardial infarction and neurotraumatic injuries. However, the mechanism of HSC conversion into organ cells is incompletely understood. The mechanism is suspected to involve direct cell–cell interaction between BMSCs, damaged organ cells, and paracrine‐regulated soluble factors from the organ, but to date, there have been no investigations into which of these are essential for keratinocyte differentiation from HSCs. Aim. To elucidate the mechanism and necessary conditions for HSC differentiation into keratinocytes in vitro. Methods. We cultured human (h)HSCs under various conditions to try to elucidate the mechanism and necessary conditions for hHSCs to differentiate into keratinocytes. Result. hHSCs cocultured with mouse keratinocytes induced expression of human keratin 14 and transglutaminase I. Only 0.1% of the differentiated keratinocytes possessed multiple nuclei indicating cell fusion. Coculture of hHSCs with fixed murine keratinocytes (predicted to stabilize cellular components) failed to induce conversion into keratinocytes. Conversely, keratinocyte‐conditioned medium from both human and mouse keratinocytes was found to mediate hHSC conversion into keratinocytes. Conclusions. Human HSCs are capable of differentiation into keratinocytes, and cell fusion is extremely rare. This differentiating is mediated by the plasma environment rather than by direct cell–cell interactions.     

Differentiation of mouse induced pluripotent stem cells into a multipotent keratinocyte lineage

Recent breakthroughs in the generation of induced pluripotent stem cells (iPSCs) have provided a novel renewable source of cells with embryonic stem cell-like properties, which may potentially be used for gene therapy and tissue engineering. Although iPSCs have been differentiated into various cell types, iPSC-derived keratinocytes have not yet been obtained. In this study, we report the in vitro differentiation of mouse iPSCs into a keratinocyte lineage through sequential applications of retinoic acid and bone-morphogenetic protein-4 and growth on collagen IV-coated plates. We show that iPSCs can be differentiated into functional keratinocytes capable of regenerating a fully differentiated epidermis, hair follicles, and sebaceous glands in an in vivo environment. Keratinocytes derived from iPSCs displayed characteristics similar to those of primary keratinocytes with respect to gene and protein expression, as well as their ability to differentiate in vitro and to reconstitute normal skin and its appendages in an in vivo assay. At present, no effective therapeutic treatments are available for many genetic skin diseases. The development of methods for the efficient differentiation of iPSCs into a keratinocyte lineage will enable us to determine whether genetically corrected autologous iPSCs can be used to generate a permanent corrective therapy for these diseases.     

Isolating a pure population of epidermal stem cells for use in tissue engineering

Continuously renewing tissues, such as the epidermis, are maintained by stem cells that slowly proliferate and remain in the tissue for life. Although it has been known for decades that epithelial stem cells can be identified as label-retaining cells (LRCs) by long term retention of a nuclear label, isolating a pure population of stem cells has been problematic. Using a Hoechst and propidium iodide dye combination and specifically defined gating, we sorted mouse epidermal basal cells into three fractions, which we have now identified as stem, transient amplifying (TA), and non-proliferative basal cells. More than 90% of freshly isolated stem cells showed a G0/G1 cell cycle profile, while greater than 20% of the TA cells were actively dividing. Both stem and TA cells retained proliferative capacity, but the stem cells formed larger, more expandable colonies in culture. Both populations could be transduced with a retroviral vector and used to bioengineer an epidermis. However, only the epidermis from the stem cell population continued to grow and express the reporter gene for 6 months in organotypic culture. The epidermis from the transient amplifying cell fraction completely differentiated by 2 months. This novel sorting method yields pure viable epithelial stem cells that can be used to bioengineer a tissue and to test permanent recombinant gene expression.     

Cellular senescence induced loss of stem cell proportion in the skin in vitro

BACKGROUND: It is known that cellular senescence could affect culture results. A previous study on K19 found that the loss of stem cell proportion is the reason for difficulties experienced when culturing aged keratinocytes. But the situation is unclear, because K19 is not generally accepted as an epidermal stem cell marker. OBJECTIVE: The aim of this study was to investigate the effects of cellular senescence caused by chronological aging or by repeated subcultures. METHODS: The effects of cellular senescence were investigated using monolayer cultures of keratinocytes and reconstructed epidermis. We prepared keratinocytes from donors of different ages and by repeated subcultures. Flow cytometric analysis was performed using alpha6 integrin and CD71 antibodies, and candidate keratinocyte stem cell proportions were separated according to reactivities to these antibodies. Living skin equivalents (LSEs) were reconstructed using keratinocytes from child, adult and elderly donors. RESULTS: Flow cytometric analysis showed a decrease in the candidate stem cell proportion in an age- or culture passage-dependent manner. LSE experiments showed that a reconstructed epidermis using child's keratinocytes was well formed compared to epidermis reconstructed using an elderly donor's keratinocytes. Different expression of proliferation markers was also observed according to donor age. CONCLUSION: Our results showed that cellular senescence by chronological aging or repeated sub-culture induced the loss of candidate stem cell proportion in keratinocyte cultures. This seems to be the reason why it is difficult to culture keratinocytes from the elderly or by repeatedly culturing keratinocytes in vitro.     

Optimization of a transplant model to assess skin reconstitution from stem cell-enriched primary human keratinocyte populations

Given that an important functional attribute of stem cells in vivo is their ability to sustain tissue regeneration, we set out to establish a simple and easy technique to assess this property from candidate populations of human keratinocyte stem cells in an in vivo setting. Keratinocytes were inoculated into devitalized rat tracheas and transplanted subcutaneously into SCID mice, and the epithelial lining regenerated characterized to establish the validity of this heterotypic model. Furthermore, the rate and quality of epidermal tissue reconstitution obtained from freshly isolated unfractionated vs. keratinocyte stem cell-enriched populations was tested as a function of (a) cell numbers inoculated; and (b) the inclusion of irradiated support keratinocytes and dermal cells. Rapid and sustained epidermal tissue regeneration from small numbers of freshly isolated human keratinocyte stem cells validates the utilization of this simple and reliable model system to assay for enrichment of epidermal tissue-reconstituting cells.     

Characterization of hair follicles induced in implanted, cultured rat keratinocyte sheets

Cultured rat keratinocyte sheets form hair follicles in combination with rat vibrissa dermal papillae when they are transplanted subcutaneously in syngeneic rats and athymic mice. In the present study, the histologic details of these induced follicles were analyzed by preparing cultured sheets mixed with normal rat keratinocytes and green fluorescent protein (GFP)-transgenic rat keratinocytes. Histologic examination demonstrated that some induced follicles maintained their size and morphology for at least 18 weeks, whereas others decreased in size and others totally differentiated into cornified structures between 3 and 6 weeks. The percentage of the grafts with GFP-positive cells decreased during the same period. This finding suggests that some GFP-positive cells were transient-amplifying cells that turned into terminally differentiated cells and were lost during this period. Some large follicles and some small follicles maintained their hair-producing ability and the proliferative activity in their hair matrix for 18 weeks. In addition, one 6-week-old follicle contained label-retaining cells in the outer root sheath. Seven of 25 follicles induced from chimera epithelium contained both GFP-positive cells and GFP-negative cells. These results suggest that stem cells are present in the induced follicle and the induced follicle consists of polyclonally derived cells. The presence of early anagen-like large follicles at week 6 and 9 and a telogen-like small follicle at week 18 also suggests that hair-growth cycle phases proceeded in the induced follicles. In conclusion, the follicles induced in the cultured keratinocyte sheets maintained hair-producing ability and proliferative activity for at least 18 weeks. This and the presence of label-retaining cells suggest that there are stem cells in the induced follicles, which seem to have a hair-growth cycle.     

A novel role of fibrin in epidermal healing: plasminogen-mediated migration and selective detachment of differentiated keratinocytes

Recent studies have shown that fibrin promotes epidermal regeneration in vitro and maintains the stem cell population after transplantation of keratinocytes in vivo. As epidermal keratinocytes do not express integrin alpha(v)beta3, the receptor for fibrin and fibrinogen, the mechanism through which fibrin affects epidermal cells remains elusive. To investigate the role of fibrin in epidermal wound healing, we developed an in vitro model in which fibrin was added to the top of wounded keratinocyte monolayers grown on collagen. With this matrix topology, keratinocytes migrate between the collagen on their basal side and fibrin on their apical side mimicking migration of the epidermis in vivo. Using this model, we found that fibrin promoted keratinocyte migration in low and high calcium concentrations by exposing the cells to plasminogen. The migration rate depended strongly on the concentration of fibrinogen and the rate of plasmin-mediated fibrin degradation. Surprisingly, fibrin and fibrinogen caused significant detachment of keratinocytes which was prevented by the addition of calcium. Further examination using flow cytometry revealed that the detached cells were larger, more granular, and had very low levels of beta1 integrin, which are all signs of differentiated keratinocytes. Our results suggest a novel dual role of fibrin in epidermal healing. First, fibrin promotes keratinocyte migration indirectly by exposing plasminogen to migrating cells, and second, fibrin selectively disrupts adhesion of differentiated keratinocytes. Our data are novel and may have important implications in understanding wound healing and in the use of fibrin as a biomaterial for protein and gene delivery.     

Properties of skin stem cells and their potential clinical applications in modern dermatology

Stem cells play an important role in medical science, and scientists are investing large sums in order to perform sophisticated studies designed to establish potential clinical applications of stem cells. Growing experience has enabled researchers to determine the precise nature of stem cell division. Although the properties of this particular population of cells have been known and used for some time, mainly with regards to bone marrow-derived mesenchymal stem cell transplantation, we now face a significant challenge in implementing the practical use of skin-derived precursors, making it possible to avoid the necessity for patients to undergo invasive procedures in order to obtain stem cells from bone marrow. Multiple trials have so far been performed, bringing hope for the treatment of disorders previously considered untreatable. Patients suffering from a number of dermatological diseases, including malignant melanoma, systemic lupus erythematosus, vitiligo, alopecia or junctional epidermolysis bullosa, may benefit from treatment based on stem cells. The aim of this review is to summarize available data on stem cells and their potential application in the treatment of dermatological disorders. Thework described is based on data published up to the end of September 2016.     

Independent inheritance of genes regulating two subpopulations of mouse clonogenic keratinocyte stem cells

Mouse keratinocyte stem cells originate from the bulge of hair follicle, and, according to definition, possess a clonogenic activity in vitro. We have investigated seven inbred (C57BL/6, C3H, DBA/2, BALB/c, FVB) and outbred (SENCAR, CD-1) mouse strains and found that three genetically distinct subsets of mouse strains differ significantly in the frequency of clonogenic activity in vitro. The analysis of keratinocyte colonies in two reciprocal backcross [C57BL/6 x (BALB/c x C57BL/6); BALB/c x (BALB/c x C57BL/6)] and intercross [(BALB/c x C57BL/ 6)F2] of BALB/c and C57BL/6 mice allowed us to identify two subpopulations of clonogenic keratinocytes able to produce small (less than 2 mm2) and large (more than 2 mm2) colonies. We conducted linkage analysis and found that small colonies associated with mouse chromosomes 1, 6, 7, 8, and 9; but large colonies--with the chromosome 4. We defined locus on the chromosome 9 that associated with small colonies as keratinocyte stem cell locus 1 (Ksc1), and locus on the mouse chromosome 4 associated with large colonies-keratinocyte stem cell locus 2 (Ksc2). Ksc1 and loci on chromosomes 6 and 7 are close if not equal to loci associated with sensitivity to skin carcinogenesis. We conclude that two subpopulations of stem cells able to produce small and large colonies regulated by different genes and genes regulating small colonies might be responsible for sensitivity to skin carcinogenesis.     

诱导多潜能干细胞在皮肤病治疗中的应用

诱导多潜能干细胞拥有胚胎干细胞所有特征,包括多能性和生成各种体细胞.用皮肤细胞产生诱导多潜能干细胞,不仅起始细胞易获取,而且这些诱导多潜能干细胞更容易定向分化为角质形成细胞、黑素细胞和成纤维细胞等多种功能性皮肤细胞.患者自体来源的诱导多潜能干细胞是细胞疗法理想的细胞库,用诱导多潜能干细胞分化后的细胞治疗皮肤病,不仅细胞量充足,且可避免伦理问题和免疫排斥反应.利用回复突变体嵌合体,结合诱导多潜能干细胞技术,能获得充分的患者特异性功能性回复体细胞而用于治疗遗传性皮肤病.该技术可避免常规基因治疗中出现的免疫排斥和插入诱变.     

Adhesive properties of human basal epidermal cells: an analysis of keratinocyte stem cells, transit amplifying cells, and postmitotic differentiating cells

The basal layer of human epidermis is a heterogeneous population of proliferative and differentiating cells that can be divided into at least three functionally discrete compartments: keratinocyte stem cells, transit amplifying cells, and postmitotic differentiating cells. Basal cells adhere to the underlying basement membrane via integrins, and although decreased adhesion is a key event in epidermal differentiation, the specific role of particular integrins is poorly understood. We report here on the comparative expression and function of the beta1 versus alpha6beta4 integrins in keratinocyte stem cells, transit amplifying cells, and postmitotic differentiating cells of neonatal human foreskin epidermis. Adhesion assays demonstrate that both keratinocyte stem cells and transit amplifying cells comprise rapidly adhering cells that exhibit high levels of functional beta1 and alpha6beta4 integrins. Interestingly, a proportion of basal cells that have begun to differentiate in vivo within the basal layer as determined by their expression of the differentiation-specific markers K10 and involucrin also retain high levels of activated beta1 integrin, but downregulate alpha6beta4 expression selectively (termed alpha6dimbeta1bri). These cells also retain their adhesive capacity, indicating that induction of differentiation in vivo does not correlate with decreased beta1 integrin expression or function. We have previously reported on the use of alpha6 integrin in conjunction with a proliferation associated marker (10G7 ag) to separate keratinocyte stem cells (phenotype alpha6bri10G7dim) from other basal cells (Li et al. Proc Natl Acad Sci 95:3902-3907 1998). A comparison of the long-term proliferative potential of beta1bri10G7dim cells with alpha6bri10G7dim showed that selection of alpha6bri10G7dim allows the isolation of a purer fraction of keratinocyte stem cells.     

Co-culture of melanocytes with adipose-derived stem cells as a potential substitute for co-culture with keratinocytes

Cell-to-cell interactions between melanocytes and keratinocytes increase the proliferation and migration of melanocytes. In fact, mixed keratinocyte and melanocyte cultures have been used for autologous cell transplantation for treatment of vitiligo. However, this may require taking an amount of skin tissue large enough to leave scars. In this study, the in vitro effect of adipose-derived stem cells (ADSCs) on proliferation, differentiation and migration of melanocytes was compared with that of keratinocytes using immunohistochemistry and a Boyden chamber migration assay. The proliferation and migration of melanocytes was significantly stimulated by co-culture with ADSCs compared with melanocyte monocultures, al-though the effect of ADSCs was less powerful than that of keratinocytes. This may be related to increases in stem cell factor and basic fibroblast growth factor, growth factors for melanocytes, produced by the ADSCs. The ratios of melanocytes stained with antibodies against Trp-2, E-cadherin and N-cadherin were significantly increased by co-culturing with ADSCs compared with co-culturing with keratinocytes as well as melanocyte monocultures. The proportion of less-pigmented melanocytes was also increased and sustained for a longer duration in the presence of ADSCs. Our data show that co-culturing with ADSCs results in increased melanocyte proliferation and migration while reducing differentiation, and could provide a means to treat disorders such as vitiligo.     

Investigating human keratinocyte stem cell identity

Studies of the regulatory networks controlling intrinsic properties and fate of adult stem cells are in a large part performed in animal models. Epidermis is one of the most accessible human tissues for researchers, which is a critical parameter for conducting programs dedicated to this knowledge in human stem cell systems. Keratinocyte stem cells constitute a particularly valuable model, because of this practical aspect, but more importantly because their existence is for decades validated by the clinical demonstration of their impressive capacity for epidermis regeneration. For the fundamentalist, human keratinocyte stem cells represent a unique system to dissect the genetic and epigenetic controls of "stemness" and self-renewal. For this purpose, a highly limiting point is our current inability of obtaining a cellular material corresponding to keratinocyte stem cells with homogeneous phenotypic and functional characteristics. The search for tools suitable for the prospective selection of keratinocyte stem cells will benefit from studies conducted at the broad level of the global stem cell field, as well as from more specifically targeted approaches. Advances in that direction are tightly linked to the development of functional assays allowing reliable assessment and modeling of the different stem cell-associated functional characteristics.     

A membrane protein preferentially expressed by a subpopulation of immature lymphoid cells, epidermal basal keratinocytes, and other epithelial cells

A murine monoclonal antibody, designated EL-1, was raised by immunization with a human malignant T cell line. It reacted specifically with a membrane antigen expressed on T and B lymphoblastoid cell lines, a subpopulation of normal thymocytes and bone marrow lymphocytes, lymphocytes from a subset of patients with non-B, non-T cell acute lymphoblastic leukemia or T cell acute lymphoblastic leukemia and epithelial stem cells. The latter reactivity was especially striking in the skin, where only basal epidermal keratinocytes and epidermal appendages, including eccrine sweat glands, sebaceous glands and hair follicles, stained positively. A human epidermoid carcinoma cell line was also stained by EL-1. Suprabasilar keratinocytes and acellular keratin did not stain. However, in vitro proliferating fetal lung fibroblasts stained positively. Membrane immunoprecipitation analysis demonstrated that the antigen recognized by antibody EL-1 is a single protein of molecular weight 105 kilodaltons which did not change with exhaustive chemical reduction. Metabolic radiolabeling studies demonstrated that this protein is synthesized by the cell and not merely taken up from the culture medium. This antibody can be useful in studying keratinocyte differentiation in epidermal malignancies and normal skin.     

Biological characteristics and gene expression pattern of bone marrow mesenchymal stem cells in patients with psoriasis

Mesenchymal stem cells (MSCs) have immunoregulatory and proangiogenic effects and are suggested to be involved in the pathological processes of immune‐related diseases, including psoriasis. Biological characteristics of bone marrow MSCs (BMSCs) from patients with autoimmune diseases, such as systemic lupus erythematosus or rheumatoid arthritis, but not psoriasis, have been characterized. We compared the gene expression profile and biological characteristics of BMSCs from patients with psoriasis and healthy controls. Although the phenotype, differentiation potential and ability to support CD34⁺ cell proliferation were similar to those of normal BMSCs, psoriatic BMSCs showed aberrant proliferative activity, increased apoptosis rate and a characteristic gene expression profile. These aberrations may develop after the abnormal immune response in psoriasis and result in BMSC dysfunction. The functionally deficient BMSCs may then fail to suppress overactive immune cells, thereby contributing to the pathogenesis of psoriasis.