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.     

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.     

In vitro differences between keratinocyte stem cells and transit-amplifying cells of the human hair follicle

Epithelial stem cells within the human hair follicle are critical for hair development, hair cycling, wound healing, and tumorigenesis. We and others have previously shown that the hair follicle bulge area contains keratinocyte stem cells, whereas the hair matrix represents the proliferating and differentiating transit-amplifying (TA) cell compartment. In order to better characterize the phenotypic differences between human keratinocyte stem cells and their daughter TA cells, we compared the in vitro properties of cell adhesion, cell migration, clonogenicity, and in vitro life span. Epithelial outgrowths from the hair matrix appeared within 2 d of explant, whereas stem cell outgrowths appeared between 7 and 10 d after explant. Both populations form colonies; however, stem cells from telogen follicles formed more total colonies, and more colonies greater than 3 mm. Upon subculture, stem cells formed colonies until passage 6 and terminally differentiated at passage 7, whereas TA cells only formed colonies until passage 2. Stem cells express more beta1 integrin and adhere more rapidly to collagen IV. Most strikingly, TA cells showed a 7-fold greater mobility on migration assays than stem cells (0.704 vs 0.102 microm per min). These results help define the human hair follicle stem cell and TA cell phenotypes and correlate with the in vivo properties of these compartments.     

Exploration of the functional hierarchy of the basal layer of human epidermis at the single‐cell level using parallel clonal microcultures of keratinocytes

Please cite this paper as: Exploration of the functional hierarchy of the basal layer of human epidermis at the single‐cell level using parallel clonal microcultures of keratinocytes. Experimental Dermatology 2010. Abstract: The basal layer of human epidermis contains both stem cells and keratinocyte progenitors. Because of this cellular heterogeneity, the development of methods suitable for investigations at a clonal level is dramatically needed. Here, we describe a new method that allows multi‐parallel clonal cultures of basal keratinocytes. Immediately after extraction from tissue samples, cells are sorted by flow cytometry based on their high integrin‐α6 expression and plated individually in microculture wells. This automated cell deposition process enables large‐scale characterization of primary clonogenic capacities. The resulting clonal growth profile provided a precise assessment of basal keratinocyte hierarchy, as the size distribution of 14‐day‐old clones ranged from abortive to highly proliferative clones containing 1.7 × 10⁵ keratinocytes (17.4 cell doublings). Importantly, these 14‐day‐old primary clones could be used to generate three‐dimensional reconstructed epidermis with the progeny of a single cell. In long‐term cultures, a fraction of highly proliferative clones could sustain extensive expansion of >100 population doublings over 14 weeks and exhibited long‐term epidermis reconstruction potency, thus fulfilling candidate stem cell functional criteria. In summary, parallel clonal microcultures provide a relevant model for single‐cell studies on interfollicular keratinocytes, which could be also used in other epithelial models, including hair follicle and cornea. The data obtained using this system support the hierarchical model of basal keratinocyte organization in human interfollicular epidermis.     

A dynamic model of keratinocyte stem cell renewal and differentiation: role of the p21WAF1/Cip1 and Notch1 signaling pathways

We present here a dynamic model of functional equilibrium between keratinocyte stem cells, transit amplifying populations and cells that are reversibly versus irreversibly committed to differentiation. According to this model, the size of keratinocyte stem cell populations can be controlled at multiple levels, including relative late steps in the sequence of events leading to terminal differentiation and by the influences of a heterogeneous extra-cellular environment. We discuss how work in our laboratory, on the interconnection between the cyclin/CDK inhibitor p21WAF1/Cip1 and the Notch1 signaling pathways, provides strong support to this dynamic model of stem cell versus committed and/or differentiated keratinocyte populations.     

Damage at the root of cell renewal--UV sensitivity of human epidermal stem cells

Background

The epidermis harbors adult stem cells that reside in the basal layer and ensure the continuous maintenance of tissue homeostasis. Various studies imply that stem cells generally possess specific defense mechanisms against several forms of exogenous stress factors. As sun exposition is the most prevalent impact on human skin, this feature would be of particular importance in terms of sensitivity to UV-induced DNA damage.

Objective

To investigate whether human epidermal stem cells are susceptible to UV-induced DNA damage and subsequent functional impairment.

Methods

A method to isolate human epidermal stem cells from suction blister epidermis was established and validated. Volunteers were treated with solar-simulated irradiation on test areas of the forearm and stem cells were isolated from suction blister material of this region. DNA damage was analyzed by staining for cyclobutane thymidine dimers. The functional consequences of UV-induced damages were assessed by colony forming efficiency assays and gene expression analyses.

Results

Compared to an unirradiated control, stem cells isolated from areas that were exposed to solar-simulated radiation showed significantly more DNA lesions. Although the number of stem cells was not reduced by this treatment, a functional impairment of stem cells could be shown by reduced colony forming efficiency and altered gene expression of stem cell markers.

Conclusions

Despite their essential role in skin maintenance, epidermal stem cells are sensitive to physiological doses of UV irradiation in vivo.     

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.     

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.     

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.     

Keratinocyte stem cells: a commentary

For many years it has been widely accepted that stem cells play a crucial role in adult tissue maintenance. The concept that the renewing tissues of the body contain a small subcompartment of self-maintaining stem cells, upon which the entire tissue is dependent, is also now accepted as applicable to all renewing tissues. Gene therapy and tissue engineering are driving considerable interest in the clinical application of such hierarchically organized cellular compartments. Recent initial observations have provided a tantalizing insight into the large pluripotency of these cells. Indeed, scientists are now beginning to talk about the possible totipotency of some adult tissue stem cells. Such work is currently phenomenologic, but analysis of data derived from genomics and proteomics, identifying the crucial control signals involved, will soon provide a further impetus to stem cell biology with far reaching applications. The epidermis with its relatively simple structure, ease of accessibility, and the ability to grow its cells in vitro is one obvious target tissue for testing stem cell manipulation theories. It is crucial, however, that the normal keratinocyte stem cell is thoroughly characterized prior to attempting to manipulate its pluripotency. This commentary assesses the data generated to date and critically discusses the conclusions that have been drawn. Our current level of understanding, or lack of understanding, of the keratinocyte stem cell is reviewed.     

Keratinocyte stem cells, label-retaining cells and possible genome protection mechanisms

Stem cells are the crucial cells upon which the entire tissue is dependent. Here we define and discuss what is meant by and known about keratinocyte stem cells. One way in which these cells have been studied is by their ability to retain radioactivity labelled thymidine for long periods of time (label retaining cells, LRCs). The underlying mechanism has been assumed in the past to be slow cycling but a more likely explanation is the selective segregation of old and new DNA strands (Cairns's hypothesis). Experiments in the small intestine indicate that the stem cells here are selectively sorting their DNA and becoming LRCs. A possible role for p53 in stem cell biology is presented.     

Keratinocyte stem cell assays: an evolving science

Although the existence of epithelial stem cells in the skin has been known for some decades from cell kinetic studies performed in vivo, attempts to prospectively isolate these cells for further biological characterization have been made possible relatively recently facilitated by the availability of antibodies that detect cell surface markers on epidermal cells. Elegant gene marking studies in vivo have provided confirmation of the patterns of epithelial tissue replacement predicted by classical cell turnover studies. But, the identification of candidate epidermal stem cells ex vivo remains an area of great controversy, requiring the re-evaluation of current experimental approaches that rely of necessity on predicted epidermal stem cell behavior in culture. Here we review the diverse experimental approaches utilized to identify keratinocyte stem cells and their underlying assumptions. We conclude that hair follicles and interfollicular epidermis each have their own self-renewing stem cell populations, contributing to distinct regions of the epithelium during homeostasis, although this is perturbed during wound healing. The need for the development of more rigorous assays for stem cell activity is highlighted given our recent observations using current assays and the discovery of new surface markers that identify putative epidermal stem cells.     

Effects of extracellular calcium on the growth-differentiation switch in immortalized keratinocyte HaCaT cells compared with normal human keratinocytes

Abstract:  The keratinocyte growth and differentiation switch, tightly regulated by several mechanisms, is generally associated with decreased proliferation, cell cycle arrest in G0/G1 phase and expression of epidermal differentiation markers, such as keratin 1 (K1), keratin 10 (K10) and involucrin. In vitro , the spontaneously immortalized human keratinocyte cell line HaCaT is often used as a model to study keratinocyte functions. Comparative differentiation studies between HaCaT cells and normal human keratinocytes (NHK) over an extended time-period have rarely been reported. Therefore, we studied their switch from a proliferating to a differentiated state over 13 days. As culture conditions involved changes in cellular responses, cells were cultured in a specific medium for keratinocyte growth and differentiation was induced by increasing extracellular calcium concentration from 0.09 to 1.2 m m . In NHK, addition of calcium-induced morphological changes and concomitant decreased proliferation. For HaCaT cells, calcium addition resulted in morphological changes, but in an unexpected manner, cells were more proliferative than when cultured at low calcium levels. HaCaT cell hyperproliferation correlated with cell cycle analysis, showing an accumulation in S/G2-M phases. Furthermore, RT-PCR and western blot analysis revealed a delay in the expression of the differentiation markers K1, K10 and involucrin in HaCaT cells compared with NHK. In conclusion, even though calcium-induced differentiation was not associated with a decreased cell proliferation, HaCaT cells conserved properties characteristic of differentiation.     

An extended epidermal response heals cutaneous wounds in the absence of a hair follicle stem cell contribution

Hair follicles have been observed to provide a major cellular contribution to epidermal healing, with emigration of stem-derived cells from the follicles aiding in wound reepithelialization. However, the functional requirements for this hair follicle input are unknown. Here we have characterized the keratinocyte stem cell status of mutant mice that lack all hair follicle development on their tail, and analyzed the consequent alterations in epidermal wound healing rate and mechanisms. In analyzing stem cell behavior in embryonic skin we found that clonogenic keratinocytes are relatively frequent in the ectoderm prior to hair follicle formation. However, their frequency in the interfollicular epidermis drops sharply by birth, at which time the majority of stem cells are present within the hair follicles. We find that in the absence of hair follicles cutaneous wounds heal with an acute delay in reepithelialization. This delay is followed by expansion of the region of activated epidermis, beyond that seen in normal haired skin, followed by appropriate wound closure. JID Journal Club article: for questions, answers, and open discussion about this article please go to http://network.nature.com/group/jidclub.     

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.     

Stress urinary incontinence in women and cell therapy: What can we expect from the future?

Stress urinary incontinence(SUI)is a common disorder that affects a large number of women and their quality of life.The aim of SUI therapy is to restore the existing urethral function via physical therapy,biofeedback,pelvic floor rehabilitation,pharmacological therapy,bulking agents and surgical approaches.Currently,the gold standard for the management of SUI is the tensionfree vaginal sling,which provides structural support to the female urethra.However,even minimally invasive surgical procedure such as"slings"carries risks for the patients,lost efficacy over the time and has long-term complications.For this reason,new therapeutic modalities are needed.Cell therapy has been emerged as an alternative to be used on the treatment of different diseases.The use of stem cells as a therapeutic option for SUI is an attractive alternative because,theoretically,injected cells could restore functional muscle cells and aid in sphincter closure in women with sphincterassociated incontinence.This study aims to review the current literature regarding evidences for using stem cell therapy on stress urinary incontinence in women.     

Autocrine growth stimulation of human keratinocytes by epidermal cell-derived thymocyte-activating factor: implications for skin aging

Summary The monocyte-derived cytokine interleukin-1 (IL-1) has growth-promoting activity for a variety of cell types, including lymphocytes and fibroblasts. We have previously shown that the epidermal cell-derived thymocyte-activating factor (ETAF) strongly resembles IL-1 in terms of biological, biochemical, and molecular biological properties. Because some lymphokines are known ot alter epidermal cell growth and differentiation and because cultured epidermal keratinocytes are capable of autocrine growth stimulation in vitro through conditioning of their culture medium, we sought to evaluate the effect of ETAF on keratinocyte growth. While there was marked donor variability in the responsiveness of keratinocytes to ETAF, partially purified preparations of ETAF showed substantial ability to stimulate the growth of keratinocytes, particularly those of newborn donors. In addition, in conditioned media there appeared to be activities distinct from ETAF that also promoted keratinocyte growth. Keratinocytes in serum-free medium secreted large amounts of ETAF, as reported previously, and keratinocyte cultures derived from newborn donors secreted significantly more than did those derived from adult donors. These results are consistent with an autocrine growth regulatory role of ETAF in human epidermis and with an age-associated loss of this phenomenon.     

The bone marrow is akin to skin: HCELL and the biology of hematopoietic stem cell homing

The recent findings that adult stem cells are capable of generating new blood vessels and parenchymal cells within tissues they have colonized has raised immense optimism that these cells may provide functional recovery of damaged organs. The use of adult stem cells for regenerative therapy poses the challenging task of getting these cells into the requisite sites with minimum morbidity and maximum efficiency. Ideally, tissue-specific colonization could be achieved by introducing the stem cells intravascularly and exploiting the native physiologic processes governing cell trafficking. Critical to the success of this approach is the use of stem cells bearing appropriate membrane molecules that mediate homing from vascular to tissue compartments. Hematopoietic stem cells (HSC) express a novel glycoform of CD44 known as hematopoietic cell E-/L-selectin ligand (HCELL). This molecule is the most potent E-selectin ligand natively expressed on any human cell. This article reviews our current understanding of the molecular basis of HSC homing and will describe the fundamental "roll" of HCELL in opening the avenues for efficient HSC trafficking to the bone marrow, the skin and other extramedullary sites.