Isolation and identification of stem cells from adult cashmere goat skin

Background  Continuously renewing epithelia are maintained by stem cells that slowly proliferate and remain in the tissues for life. It has been known for decades that mouse epithelial stem cells can be selected by adherence to specific integrins.
Methods  The adherence of cashmere goat epidermal cells to collagen type IV for 10 min was used to obtain enriched epidermal stem cells. The characteristics of the rapidly adherent epidermal cells were determined.
Results  The rapidly adherent epidermal cells exhibited the stem cell characteristics of immaturity, were quiescent, showed a high colony formation efficiency, and expressed candidate surface markers for epidermal stem cells (keratin 15, keratin 19, p63, CD34, and β1-integrin).
Conclusions  The rapidly adherent epidermal cells represented the epidermal stem cell population.     

Epithelial stem cells in the skin: definition, markers, localization and functions

In recent years, cutaneous epithelial stem cells have attained a genuine celebrity status. They are considered the key resource for epidermal and skin appendage regeneration, and are proposed as a preferential target of cutaneous gene therapy. Follicular epithelial stem cells may also give rise to a large variety of epithelial tumors, and cutaneous epithelial stem cells likely are crucial targets for physical or chemical agents (including carcinogens) that damage the skin and its appendages. However, as this Controversies feature illustrates, few experts can agree on how exactly to define and identify these elusive cells, or on where precisely in the skin they are localized. Given their potential importance in skin biology, pathology and future dermatological therapy, it is, therefore, timely to carefully reconsider the basic questions: What exactly is a stem cell, and how can we reliably identify epithelial stem cells? How many different kinds are there, and how do they differ functionally? Where exactly in the skin epithelium is each of the putative stem cell subpopulations located, and can we selectively manipulate any of them?     

Epidermal and hair follicle progenitor cells express melanoma-associated chondroitin sulfate proteoglycan core protein

Basal keratinocytes in the epidermis and hair follicle are biologically heterogeneous but must include a stable subpopulation of epidermal stem cells. In animal models these can be identified by their retention of radioactive label due to their slow cycle (label-retaining cells) but human studies largely depend on in vitro characterization of colony forming efficiency and clonogenicity. Differential integrin expression has been used to detect cells of increased proliferative potential but further stem cell markers are urgently required for in vivo and in vitro characterization. Using LHM2, a monoclonal antibody reacting with a high molecular weight melanoma-associated proteoglycan core protein, a subset of basal keratinocytes in both the interfollicular epidermis and the hair follicle has been identified. Coexpression of melanoma-associated chondroitin sulfate proteoglycan with keratins 15 and 19 as well as beta 1 and alpha 6 integrins has been examined in adult and fetal human skin from hair bearing, nonhair bearing, and palmoplantar regions. Although melanoma-associated chondroitin sulfate proteoglycan coexpression with a subset of beta 1 integrin bright basal keratinocytes within the epidermis suggests that melanoma-associated chondroitin sulfate proteoglycan colocalizes with epidermal stem cells, melanoma-associated chondroitin sulfate proteoglycan expression within the hair follicle was more complex and multiple subpopulations of basal outer root sheath keratinocytes are described. These data suggest that epithelial compartmentalization of the outer root sheath is more complex than interfollicular epidermis and further supports the hypothesis that more than one hair follicle stem cell compartment may exist.     

Stem cell therapies for recessive dystrophic epidermolysis bullosa

Human epidermis is composed of a stratified squamous epithelium that provides a mechanical barrier against the external environment and which is renewed every 3–4 weeks by resident stem cells in the epidermis. However, in the inherited skin fragility disorder, recessive dystrophic epidermolysis bullosa (RDEB), there is recurrent trauma‐induced subepidermal blistering that disrupts epidermal homeostasis and is likely to deplete the epidermal stem cell pool. This review article discusses the nature of epidermal stem cells and other stem cell populations in the skin, as well as other possible extracutaneous sources of stem cells, that might have physiological or therapeutic relevance to cell therapy approaches for RDEB. Strategies to identify, create and use cells with multipotent or pluripotent properties are explored and current clinical experience of stem cell therapy in RDEB is reviewed. There is currently no single optimal therapy for patients with RDEB, but cell therapy technologies are evolving and hold great potential for modifying disease severity and improving quality of life for people living with RDEB.     

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.     

Montagna symposium on epidermal stem cells oligonucleotide-directed gene correction in epidermis

Oligonucleotide-directed gene alteration produces a targeted DNA sequence change in the genome of mammalian cells. The advantage of this approach is that expression of the corrected gene is regulated in the same way as a normal gene. Reliable, sensitive, and standardized assays played a critical role in the measurement of gene correction frequency among different cell types and in evaluating the structure-activity relationship of oligonucleotides. Mechanistic studies using these assays have become critical for understanding the gene repair process and setting realistic expectations on the capability of this technology. The epidermis is an ideal tissue where oligonucleotides can be administered locally and the treated sites can be monitored easily. But given the low frequency of gene correction, general selection procedures and amplification of corrected cells via epidermal stem cells are ultimately needed to make the gene repair technology practical. Recent data suggest that the in vivo application of oligonucleotides may be capable of gene correction in epidermal stem cells and the subsequent expansion of the corrected cells may result in an apparent high-level and long-lasting gene repair. Advances in oligonucleotide delivery and targeting of epidermal stem cells will be required for potential application of oligonucleotides toward treatment of genodermatoses.     

Role of bulge cells in wound healing: possible implications for hidradenitis suppurativa

The hair follicle bulge harbors a cluster of quiescent epithelial stem cells, which generate the new lower hair follicle during follicle cycling. The role of bulge cells for maintenance of the hair follicle and epidermis during normal homeostasis and after wounding remains controversial. To address these questions, we targeted the suicide gene, thymidine kinase (TK), or CrePR recombinase to hair follicle bulge cells using a K15 promoter. Administration of ganciclovir to K15‐TK mice caused their death due to gastrointestinal injury. We then treated immunodeficient mice carrying K15‐TK skin grafts with ganciclovir. This resulted in injury of bulge cells within several days. Loss of hair and permanent injury to hair follicles ensued. Dermal scarring was also present. The epidermis without follicles survived for several months. To further assess the contribution of bulge cells to epidermis, we used double transgenic K15‐crePR1, R26R mice. After 5 days of treatment with RU‐486 (time 0), bulge cells and a small fraction of epidermal cells expressed LacZ under control of the ROSA promoter. To determine whether bulge cells moved into the epidermis under normal homeostatic conditions, we counted labeled epidermal basal cells at times 0, 30 days, and 180 days. We found that the percentage of labeled epidermal basal cells (approximately 1%) remained constant, indicating an absence of cell movement from the bulge to the epidermis. After punch wounding, bulge progeny were detected in a radial pattern in the re‐epithelialized area. Radial streaks of bulge cell progeny emanated from the hair follicles at the wound edge. Bulge cell progeny proliferated and expressed normal epidermal differentiation markers such as keratin 10 and loricrin. Overall, these results indicate that follicular stem cells in the bulge are necessary for hair follicle survival and that these cells functionally contribute to epidermal regeneration in response to wounding; however, the epidermis self‐renews autonomously of the bulge under normal conditions. The role of bulge cells in hidradenitis suppurativa (HS) is not known; however, pathological studies suggest aberrant proliferation of the hair follicle may accompany HS and inappropriate stem cell activation and differentiation could be a component of this disorder. The K15‐crePR1 transgenic mouse will serve as a powerful tool for evaluating the role of hair follicle stem cells in mouse models of HS. Studying hair follicle stem cells with recently defined markers for these cells will be useful for evaluating stem cell behavior in HS lesions.     

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.     

Side population keratinocytes resembling bone marrow side population stem cells are distinct from label-retaining keratinocyte stem cells

Very primitive hematopoietic stem cells have been identified as side population cells based on their ability to efflux a fluorescent vital dye, Hoechst 33342. In this study we show that keratinocytes with the same side population phenotype are also present in the human epidermis. Although side population keratinocytes have the same dye-effluxing phenotype as bone marrow side population cells and can be blocked by verapamil, they do not express increased levels of the ABCG2 transporter that is believed to be responsible for the bone marrow side population phenotype. Because bone marrow side population cells have stem cell characteristics, we sought to determine if side population keratinocytes represent a keratinocyte stem cell population by comparing side population keratinocytes with a traditional keratinocyte stem cell candidate, label-retaining keratinocytes. Flow cytometric analyses demonstrated that side population keratinocytes have a different cell surface phenotype (low beta1 integrin and low alpha6 integrin expression) than label-retaining keratinocytes and represent a unique population of keratinocytes distinctly different from the traditional keratinocyte stem cell candidate. Future in vivo studies will be required to analyze the function of side population keratinocytes in epidermal homeostasis and to determine if side population keratinocytes have characteristics of keratinocyte stem cells.     

Hair follicle stem cells

The workshop on Hair Follicle Stem Cells brought together investigators who have used a variety of approaches to try to understand the biology of follicular epithelial stem cells, and the role that these cells play in regulating the hair cycle. One of the main concepts to emerge from this workshop is that follicular epithelial stem cells are multipotent, capable of giving rise not only to all the cell types of the hair, but also to the epidermis and the sebaceous gland. Furthermore, such multipotent stem cells may represent the ultimate epidermal stem cell. Another example of epithelial stem cell and transit amplifying cell plasticity, was the demonstration that adult corneal epithelium, under the influence of embryonic skin dermis could form an epidermis as well as hair follicles. With regards to the location of follicular epithelial stem cells, immunohistochemical and ultrastructural data was presented, indicating that cells with stem cell attributes were localized to the prominent bulge region of developing human fetal hair follicles. Finally, a new notion was put forth concerning the roles that the bulge-located stem cells and the hair germ cells played with respect to the hair cycle.     

A novel niche for skin derived precursors in non-follicular skin

BackgroundSkin derived precursors (SKP) comprise a subset of specialized dermal cells that can be distinguished from fibroblast by their capacity for spheroidal growth. Recent investigations have shown that hair follicles constitute a niche for this cell type, but their localization and their definite function in non-follicular skin remains largely unknown.ObjectiveTo identify the dermal niche of non-follicular SKPs and to analyze whether functional aspects correlate with this localization.MethodsSKPs were isolated from separate anatomical regions of human abdominal skin. Fluorescence activated cell sorting then was used to obtain a pure population of non-follicular SKPs. Functional characterization of these cells was performed applying differentiation and proliferation assays. Information on specific in vivo functions was derived from histological evaluation of quantity and localization patterns.ResultsSphere forming capacity and differentiation assays show that SKPs reside in the papillary part of the dermis. Further delineation revealed that the dermal capillaries represent a niche for these cells which subsequently could be isolated by FACS utilizing a perivascular marker. Whereas functional properties described for follicular SKPs could also be detected in the perivascular SKP population, histological analyses additionally point to a cross-talk with epidermal stem cells and a reduction during chronological aging.ConclusionOur data show that SKPs isolated from non-follicular skin originate from a perivascular niche. Compared to their follicular counterparts, no functional differences could be observed upon cultivation, but ex vivo analyses also point to unique functions and a contribution to the phenotype of aged skin.     

Rodent epidermal Langerhans cells demonstrate greater histochemical specificity for ADP than for ATP and AMP

Langerhans cells (LCs) in mammalian epidermis possess the ectoenzyme Ca++/Mg++-dependent adenosine triphosphatase (ATPase), which has served as a useful histochemical marker for these dendritic cells in a variety of tissue preparations. Since ATPase represents only one of several potential cell surface polyphosphatases, we investigated the capacities of 3 related adenine nucleotide substrates to identify rodent epidermal LCs. Cell surface ATPase activity was not inhibited in the presence of ouabain and was observed to be strictly divalent cation-dependent, with complete interchangeability between Ca++ and Mg++. Optimal staining in the presence of either cation occurred at a 20 mM concentration. Substrate concentration dependence was also observed, with optimal staining at 0.33 mM adenosine 5'-triphosphate (ATP). On an equimolar basis, however, adenosine 5'-diphosphate (ADP) was superior to ATP for the identification of LCs both in whole mounts of epidermis and in suspensions of disaggregated epidermal cells. The substrate adenosine 5'-monophosphate (AMP) stained follicular epithelial cells in both rodent species but failed to identify epidermal LCs in the mouse and only weakly stained these dendritic cells in rat epidermis. We conclude from these studies that ADP demonstrates greater specificity for LC surface polyphosphatase activity than ATP and that the inadvertent inclusion of AMP during identification procedures for epidermal cell suspensions will falsely identify cells other than LCs.     

Epidermale Stammzellen

Current understanding of the biology of epidermal stem cells opens a totally new perspective in the function of the epidermis and adjacent epithelial structures. A number of pathogenetic as well as clinical‐therapeutic approaches against a variety of dermatoses may become possible with knowledge about keratinocyte proliferation, differentiation and regeneration. The reservoir of epidermal stem cells is located in the interfollicular epidermis, the hair follicle area and the germinal hair follicle matrix. Endogenous stem cell clones exist here, giving rise to transient amplifying cells and postmitotic cells. The stem cell clones are organized in clusters and display high expression of adhesion proteins, which guarantee their stability in a specific environment consisting of different cell types and extracellular substrates in the stratum basale. Differentiation is determined by a specific cascade of chemical signals from the stem cell environment and from the genetic program of the cell. The clinical relevance of stem cells lies primarily in their therapeutic potential with reconstruction of epithelia by reimplantation of autologous stem cells or gene therapeutic applications such as targeted transfection. However, the benefit‐to‐risk ratio cannot yet be accurately estimated.     

Review of hair follicle dermal cells

Hair follicle stem cells in the epithelial bulge are responsible for the continual regeneration of the hair follicle during cycling. The bulge cells reside in a niche composed of dermal cells. The dermal compartment of the hair follicle consists of the dermal papilla and dermal sheath. Interactions between hair follicle epithelial and dermal cells are necessary for hair follicle morphogenesis during development and in hair reconstitution assays. Dermal papilla and dermal sheath cells express specific markers and possess distinctive morphology and behavior in culture. These cells can induce hair follicle differentiation in epithelial cells and are required in hair reconstitution assays either in the form of intact tissue, dissociated freshly prepared cells or cultured cells. This review will focus on hair follicle dermal cells since most therapeutic efforts to date have concentrated on this aspect of the hair follicle, with the idea that enriching hair-inductive dermal cell populations and expanding their number by culture while maintaining their properties, will establish an efficient hair reconstitution assay that could eventually have therapeutic implications.     

Gene therapy approaches for epidermolysis bullosa

Human epidermis consists of a stratified epithelium mainly composed of keratinocytes and relies on a stem cell compartment to undergo constant regeneration. Genetic mutations affecting the capacity of basal keratinocytes to adhere firmly to the epidermal basement membrane lead to severe, and very often lethal, blistering disorders known as epidermolysis bullosa. Gene therapy represents a promising potential treatment for these devastating inherited disorders. Human epidermal stem cells can be cultivated ex vivo and stably transduced with integrating gene transfer vectors, allowing genetic and, more important, phenotypic correction of the adhesion properties of keratinocytes. Here we will review some of the issues that need to be addressed to make gene therapy a realistic treatment for these disorders, such as (1) which cells should be targeted, (2) which approach (in vivo or ex vivo) should be chosen, and (3) which gene transfer vector (retrovirus, lentivirus, or integrating nonviral strategies) should be used for stable gene correction. In the last 10 years, many reports have shown that gene transfer approaches to target epidermal stem cells are feasible and able to restore the adhesion properties of primary keratinocytes from patients with epidermolysis bullosa. In addition, tremendous progress has been achieved in culturing epidermal stem cells and generating sheets of stratified epithelium for permanent coverage of full-thickness burns. Gene modification of stem cells in combination with advanced tissue-engineering techniques could therefore represent a realistic option for patients with epidermolysis bullosa.     

Basal cell carcinoma: cell of origin, cancer stem cell hypothesis and stem cell markers

Cancer stem cells have recently been described in several high-grade neoplasms. It is still unclear if they also occur in cutaneous malignancies. Cancer stem cells are not identical with somatic stem cells. The presence of tumour stem cells in a neoplasm does not in itself equal that the tumour derives from a somatic stem cell. A cell originally lacking stem cell characteristics could also acquire those features during the course of carcinogenesis and then becomes the clonal founder cell of a tumour. Basal cell carcinoma (BCC) is the most common cutaneous malignancy. A plethora of various stem cell markers has been applied to study its cellular origin. Intriguingly, the anatomical origin of BCC is still uncertain. This review will discuss the various stem cell markers used in BCC and the cellular origin of this tumour, and touches briefly on the possibility of cancer stem cells in BCC. If BCC or other skin cancers harbour tumour stem cells, these cells could be specifically targeted, making use of specific cell surface molecules such as receptor proteins. Novel drugs directed against those receptor proteins could replace currently available shotgun approaches including imiquimod.     

As epidermal stem cells age they do not substantially change their characteristics

In this study, we ask the basic question: do stem cells age? We demonstrated that epidermal stem cells isolated from neonatal mice had the capacity to form multiple cell lineages during development. Here we demonstrate the cell lineages are clonal, and that epidermal stem cells isolated from the footpad epithelium of old mice have similar capabilities. Using Hoechst dye exclusion and cell size, we isolated viable homogenous populations of epidermal stem and transit-amplifying (TA) cells from GFP-transgenic mice, and injected these cells into 3.5-d blastocysts. Only the stem-injected blastocysts produced mice with GFP+ cells in their tissues. Furthermore, aged and young stem cells showed similar gene and protein expression profiles that showed some differences from the TA cell profiles. These data suggest that there may be a fundamental difference between somatic stem and TA cells, and that an epidermal stem cell placed in a developmental environment can alter its fate determination no matter what its age.     

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.     

Host immune responses in ex vivo approaches to cutaneous gene therapy targeted to keratinocytes

Epidermal gene therapy may benefit a variety of inherited skin disorders and certain systemic diseases. Both in vivo and ex vivo approaches of gene transfer have been used to target human epidermal stem cells and achieve long-term transgene expression in immunodeficient mouse/human chimera models. Immunological responses however, especially in situations where a neoantigen is expressed, are likely to curtail expression and thereby limit the therapy. In vivo gene transfer to skin has been shown to induce transgene-specific immune responses. Ex vivo gene transfer approaches, where keratinocytes are transduced in culture and transplanted back to patient, however, may avoid signals provided to the immune system by in vivo administration of vectors. In the current study, we have developed a stable epidermal graft platform in immunocompetent mice to analyze host responses in ex vivo epidermal gene therapy. Using green fluorescent protein (GFP) as a neoantigen and an ex vivo retrovirus-mediated gene transfer to mouse primary epidermal cultures depleted of antigen-presenting cells (APCs), we show induction of GFP-specific immune responses leading to the clearance of transduced cells. Similar approach in immunocompetent mice tolerant to GFP resulted in permanent engraftment of transduced cells and continued GFP expression. Activation of transgene-specific immune responses in ex vivo gene transfer targeted to keratinocytes require cross-presentation of transgene product to APCs, a process that is most amenable to immune modulation. This model may be used to explore strategies to divert transgene-specific immune responses to less destructive or tolerogenic ones.