Pritinder Kaur

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?

A role for pericytes as microenvironmental regulators of human skin tissue regeneration

The cellular and molecular microenvironment of epithelial stem and progenitor cells is poorly characterized despite well-documented roles in homeostatic tissue renewal, wound healing, and cancer progression. Here, we demonstrate that, in organotypic cocultures, dermal pericytes substantially enhanced the intrinsically low tissue-regenerative capacity of human epidermal cells that have committed to differentiate and that this enhancement was independent of angiogenesis. We used microarray analysis to identify genes expressed by human dermal pericytes that could potentially promote epidermal regeneration. Using this approach, we identified as a candidate the gene LAMA5, which encodes laminin alpha5, a subunit of the ECM component laminin-511/521 (LM-511/521). LAMA5 was of particular interest as we had previously shown that it promotes skin regeneration both in vitro and in vivo. Analysis using immunogold localization revealed that pericytes synthesized and secreted LAMA5 in human skin. Consistent with this observation, coculture with pericytes enhanced LM-511/521 deposition in the dermal-epidermal junction of organotypic cultures. We further showed that skin pericytes could also act as mesenchymal stem cells, exhibiting the capacity to differentiate into bone, fat, and cartilage lineages in vitro. This study suggests that pericytes represent a potent stem cell population in the skin that is capable of modifying the ECM microenvironment and promoting epidermal tissue renewal from non-stem cells, a previously unsuspected role for pericytes.

Extensive tissue-regenerative capacity of neonatal human keratinocyte stem cells and their progeny

Given our recent discovery that it is possible to separate human epidermal stem cells of the skin from their more committed progeny (i.e., transit-amplifying cells and early differentiating cells) using FACS techniques, we sought to determine the comparative tissue regeneration ability of these keratinocyte progenitors. We demonstrate that the ability to regenerate a fully stratified epidermis with appropriate spatial and temporal expression of differentiation markers in a short-term in vitro organotypic culture system is an intrinsic characteristic of both epidermal stem and transit-amplifying cells, although the stem cell fraction is most capable of achieving homeostasis. Early differentiating keratinocytes exhibited limited short-term tissue regeneration under specific experimental conditions in this assay, although significant improvement was obtained by manipulating microenvironmental factors, that is, coculture with minimally passaged dermal cells or exogenous supply of the ECM protein laminin-10/11. Importantly, transplantation of all classes of keratinocyte progenitors into an in vivo setting demonstrated that tissue regeneration can be elicited from stem, transit-amplifying, and early differentiating keratinocytes for up to 10 weeks. These data illustrate that significant proliferative and tissue-regenerative capacity resides not only in keratinocyte stem cells as expected, but also in their more committed progeny, including early differentiating cells.

Immortalization of Primary Human Epithelial Cells by Cloned Cervical Carcinoma DNA Containing Human Papillomavirus Type 16 E6/E7 Open Reading Frames

Primary human epithelial cells were transfected with a subgenomic fragment of DNA cloned from a cervical carcinoma, containing the putative transforming genes E6 and E7 from the human papillomavirus type 16 prototype. Several immortalized cell lines were generated, all retaining tumour DNA and expressing the viral genes, suggesting that either one or both of these genes is sufficient for the immortalization of primary human genital keratinocytes.

Cell migration velocities in the crypts of the small intestine after cytotoxic insult are not dependent on mitotic activity.

Abstract. The role of mitotic activity in the normal process of intestinal epithelial cell migration was investigated. the movement of [3H]TdR‐labelled cells in the crypt‐villus column was used to study migration both in the crypts and on the villi. Radiation alone or in conjunction with other cytotoxic agents (hydroxyurea, cyclophosphamide and isopropyl‐methane sulphonate) was used to eliminate cell division activity and to decrease crypt cellularity. This was done in order to determine the role of ‘mitotic pressure’ in driving cell migration.

Establishment of 3D organotypic cultures using human neonatal epidermal cells

This protocol describes an ex vivo three-dimensional coculture system optimized to study the skin regenerative ability of primary human keratinocytes grown at the air–liquid interface on collagen matrices embedded with human dermal fibroblasts. An option for enrichment of keratinocyte stem cells and their progeny using fluorescence-activated cell sorting is also provided. Initially, dermal equivalents, comprising human passaged fibroblasts seeded in a collagen matrix, are grown on porous filters (3 μm) placed in transwells. After 1 week, primary human keratinocytes are seeded on this base. One week later, an air-lift transition is performed, leading to the differentiation of the keratinocytes, which are macroscopically visible as artificial skin after a couple of days. The cultures can be harvested 1 week after the air-lift and processed for immunohistochemistry or gene expression analysis. The overall procedure can be completed in 3 weeks, including the preparation of the dermal equivalent and the seeding of the primary keratinocytes.

An in vivo comparative study of sonic, desert and Indian hedgehog reveals that hedgehog pathway activity regulates epidermal stem cell homeostasis

Despite the well-characterised role of sonic hedgehog (Shh) in promoting interfollicular basal cell proliferation and hair follicle downgrowth, the role of hedgehog signalling during epidermal stem cell fate remains largely uncharacterised. In order to determine whether the three vertebrate hedgehog molecules play a role in regulating epidermal renewal we overexpressed sonic (Shh), desert (Dhh) and Indian (Ihh) hedgehog in the basal cells of mouse skin under the control of the human keratin 14 promoter. We observed no overt epidermal morphogenesis phenotype in response to Ihh overexpression, however Dhh overexpression resulted in a range of embryonic and adult skin manifestations indistinguishable from Shh overexpression. Two distinct novel phenotypes were observed amongst Shh and Dhh transgenics, one exhibiting epidermal progenitor cell hyperplasia with the other displaying a complete loss of epidermal tissue renewal indicating deregulation of stem cell activity. These data suggest that correct temporal regulation of hedgehog activity is a key factor in ensuring epidermal stem cell maintenance. In addition, we observed Shh and Dhh transgenic skin from both phenotypes developed lesions reminiscent of human basal cell carcinoma (BCC), indicating that BCCs can be generated despite the loss of much of the proliferative (basal) compartment. These data suggest the intriguing possibility that BCC can arise outside the stem cell population. Thus the elucidation of Shh (and Dhh) target gene activation in the skin will likely identify those genes responsible for increasing the proliferative potential of epidermal basal cells and the mechanisms involved in regulating epidermal stem cell fate.

Intestinal cell proliferation. I. A comprehensive model of steady-state proliferation in the crypt.

Abstract. Cell replacement in the crypt of the murine small intestine has been studied and modelled mathematically under steady‐state conditions. A great deal of information is available for this system, e.g. cell cycle times, S phase durations, the rate of daily cell production, the Paneth cell distribution etc. the purpose of the present work was to consider simultaneously as much of these data as possible and to formulate a model based upon the behaviour of individual cells which adequately accounted for them. A simple mathematical representation of the crypt has been developed. This consists of sixteen stem cells per crypt (Tc= 16 hr, Ts= 9 hr), and four subsequent transit cell divisions (Tc= 11 to 12 hr, Ts= 8 hr) before maturation. Experimental data considered to test the modelling were LI and data on the number of vertical runs of similarly labelled cells. All data were obtained from the ileum after 25 μCi [3H]TdR given at 09.00 hours. A number of alternative assumptions have been considered and either accepted or rejected. Two alternative model concepts of cell displacement explain the data equally well. One is dependent upon strong local cell generation age determinance while the other could accommodate any weak local cell displacement process in conjunction with an environmental cut‐off determinant at the middle of the crypt. Both models provide new interpretations of the data, e.g. certain rates of lateral cell exchange between neighbouring columns (250 to 350 per crypt per day out of a total of 420 cell divisions per day) can be concluded from run data, while LI data provide information about the mechanisms involved in maintaining a position‐related age order in the crypt.

Side population in adult murine epidermis exhibits phenotypic and functional characteristics of keratinocyte stem cells.

Based on functional studies in the bone marrow, it has been suggested that the ability to efflux Hoechst 33342 may represent a universal stem cell trait. In this phenotypic and functional characterization of the Hoechst side population (SP) in adult murine epidermis, we demonstrate that these cells are a rare subset of the keratinocyte stem cell-enriched α6briCD71dim fraction comprising SSClow/K14+/CD34−/Oil red O−/c-kit−/CD45− keratinocytes. Epidermal SPs have the smallest cell and nuclear size but exhibit the highest nuclear-to-cytoplasmic ratio of any fraction examined, consistent with a primitive cell type. Although SPs demonstrated poor cumulative in vitro proliferative output, they exhibited sustained epidermal tissue-regenerative activity in vivo compared with unfractionated and non-SP cells. Collectively, these results indicate that the epidermal SP contains the most potent keratinocyte stem cell population in skin epithelium.

Laminin 10/11: an alternative adhesive ligand for epidermal keratinocytes with a functional role in promoting proliferation and migration

Abstract: We have investigated the expression and function of the isoforms of laminin bearing the α5 chain, i.e. laminin‐10/11 in neonatal and adult human skin. By immunostaining human skin derived from a variety of anatomic sites, we found that the laminin‐α5 chain is expressed abundantly in the basement membrane underlying the interfollicular epidermis and the blood vessels in the dermis. Interestingly, while the expression level of the well‐studied laminin‐5 isoform did not change significantly with age, laminin‐10/11 (α5 chain) appeared to decrease in the basement membrane underlying the epidermis, in adult skin. In contrast, the levels of laminin‐10/11 in the basement membrane underlying blood vessels remained unchanged in neonatal vs. adult skin. Importantly, in vitro cell adhesion assays demonstrated that laminin‐10/11 is a potent adhesive substrate for both neonatal and adult keratinocytes and that this adhesion is mediated by the α3β1 and α6β4 integrins. Adhesion assays performed with fractionated basal keratinocytes showed that stem cells, transit amplifying cells and early differentiating cells all adhere to purified laminin‐10/11 via these receptors. Further, laminin‐10/11 provided a proliferative signal for neonatal foreskin keratinocytes, adult breast skin keratinocytes, and even a human papillomavirus type‐18 transformed tumorigenic keratinocyte cell line in vitro. Finally, laminin‐10/11 was shown to stimulate keratinocyte migration in an in vitro wound healing assay. These results provide strong evidence for a functional role for laminin‐10/11 in epidermal proliferation during homeostasis, wound healing and neoplasia.