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.     

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.     

IL-1 and IL-1 receptor antagonist regulation during keratinocyte cell cycle and differentiation in normal and psoriatic epidermis

Abstract Changes in the levels of IL-1 (IL-1α, IL-1β, and its receptor antagonist, IL-1RA) occur upon keratinocyte differentiation in vitro and are associated in vivo with abnormal differentiated and hyperproliferative states of psoriatic keratinocytes. A flow cytometric procedure, capable of detecting changes in the intracellular levels of IL-1, was used to determine whether intracellular IL-1/IL-1RA levels in psoriatic and normal keratinocytes alter during in vivo differentiation and the cell cycle. Increases in the IL-1RA levels and IL-1α levels were observed as both normal and psoriatic keratinocytes differentiated from basal stem cells (β₁ integrin⁺, small size) into transient amplifying cells (TAC; β₁ integrin⁺, large size). Upon further differentiation (β₁ integrin^–, large size) both IL-1RA and IL-1α levels dropped. However, while psoriatic IL-1β levels increased as cells differentiated into TACs, little change occurred in the IL-1β levels of normal keratinocytes during differentiation. Changes in IL-1/IL-1RA levels were also detected as keratinocytes progressed through the cell cycle. Within the basal stem cell population of both normal and psoriatic keratinocytes, the IL-1α and IL-1RA levels increased between G0/G1 and S but not between S and G2/M. However, psoriatic basal keratinocyte IL-1β levels differed from those of normal keratinocytes by showing no increase between S and G2/M. The IL-1/IL-1RA levels of normal TAC increased throughout the cell cycle. However, in psoriatic TAC, a slight decrease in IL-1α and IL-1RA levels was observed between G0/G1 and S followed by a delayed increase between S and G2/M. IL-1β levels in psoriatic TAC varied little throughout the cell cycle. Thus, we were able to detect precisely the regulation of IL-1/IL-1RA intracellular levels during the keratinocyte cell cycle and differentiation, showing notably decreased IL-1β upregulation in psoriatic keratinocytes progressing through the cell cycle. Received: 15 July 1996     

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.     

Expression of Ulex europaeus agglutinin I lectin-binding sites in squamous cell carcinomas and their absence in basal cell carcinomas. Indicator of tumor type and differentiation.

Lectins bind tightly to carbohydrate moieties on cell surfaces. Alterations in lectin binding have been reported to accompany epidermal cell differentiation, marking alterations in membrane sugars during this process. The presence of UEA I (Ulex europaeus agglutinin I) L-fucose-specific lectin-binding sites has been used as a marker for terminally differentiated (committed) keratinocytes. In this article, we report the presence of UEA-I-binding sites on squamous keratinocytes of well-differentiated squamous cell carcinomas, with patchy loss of UEA I positivity on poorly differentiated cells of squamous cell carcinomas, suggesting a possible use for this technique in the rapid assessment of less differentiated areas within the squamous cell tumor. The absence of UEA-I-binding sites on basal cell carcinomas may be related to an inability of cells comprising this tumor to convert the L-D-pyranosyl moiety on basal cells to the L-fucose moiety, resulting in an inability of basal cell carcinoma cell to undergo terminal differentiation into a committed keratinocyte.     

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.     

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.     

Analysis of adenovirus early and late gene expression in cultured epidermal keratinocytes

Previous studies have shown that expression of adenovirus type 2 (Ad2) is restricted in epidermal keratinocytes in what appears to be a differentiation specific manner. We have analyzed the relationship between keratinocyte differentiation and Ad2 early and late gene expression. Cultured epidermal keratinocytes infected with Ad2 were fractionated in density gradients of Ficoll 400 to enrich for populations of nondifferentiated cells and differentiated cells. Analysis of these populations revealed that both populations supported early Ad2 gene expression but restricted Ad2 late gene expression. The restriction to late gene expression differed in the two cell populations: Nondifferentiated keratinocytes did not support production of high levels of Ad2 capsid proteins, whereas differentiated keratinocytes supported synthesis of Ad2 capsid proteins but restricted Ad2 expression at a later step that normally leads to production of high titers of progeny virus. The changing restriction to Ad2 expression during keratinocyte differentiation may have resulted from changes in cellular components that play a role in cell differentiation.     

Flow cytometric sorting and keratin analysis of human epidermal basal cells

A prerequisite condition for the elucidation of the expression of keratin subunits in human basal cells is obtaining highly purified basal cell fractions. A method has therefore been developed for the isolation of the basal cell sub-population from normal human epidermis at very high purity. Epidermal cell suspensions obtained by trypsinization at 4°C were first enriched in basal cells by selective attachment to collagen. The cells were then stained in suspension with KLI monoclonal antikeratin antibody which had been shown to be reactive with suprabasal cells. The suprabasal cells contaminating the suspensions were then eliminated by selective flow cytometry sorting and basal keratinocyte populations of 99.5% purity were obtained. Analysis of the keratin composition of these cells showed that the large majority of basal cells contained two keratins, one from each keratin subfamily: type I acidic, 50 Kd [keratin no 14 as defined by Moll et al. (1)] and type II basic, 58 Kd [no 5]. Nevertheless, a small subpopulation of basal cells (2%) was shown to express, in addition to the 50/58 Kd subunits, the 56.5/65–67 Kd keratins (nos 10, 1–2) which are regarded as markers for cells committed to terminal differentiation.     

Directing stem cells into the keratinocyte lineage in vitro

A major area of research in regenerative medicine is the potential application of stem cells in skin grafting and tissue engineering. This would require well defined and efficient protocols for directing the commitment and differentiation of stem cells into the keratinocyte lineage, together with their selective purification and proliferation in vitro. The development of such protocols would reduce the likelihood of spontaneous differentiation of stem cells into divergent lineages upon transplantation, as well as reduce the risk of teratoma formation in the case of embryonic stem cells. Additionally, such protocols could provide useful in vitro models for studying skin tissue biology, as well as facilitate the genetic manipulation of stem cells for therapeutic applications. The development of pharmacokinetic and cytotoxicity/genotoxicity screening tests for skin-related biomaterials and drugs could also utilize protocols developed for the commitment and differentiation of stem cells into the keratinocyte lineage. Hence, this review critically examines the various strategies that could be employed to direct the commitment and differentiation of stem cells into the keratinocyte lineage in vitro.     

Syndecan-1, a cell-surface proteoglycan, changes in size and abundance when keratinocytes stratify.

In epidermis, keratinocytes in the basal cell layer differentiate, lose their attachment to the underlying extracellular matrix, and form extensive intercellular adhesions as they stratify. The alterations in cell-matrix and cell-cell adhesion required for keratinocyte stratification result from changes in the expression of numerous adhesion molecules. Syndecan-1, a member of a family of cell-surface proteoglycans, is known to bind cells to interstitial matrix. Syndecan-1 localizes to specific layers of mouse epidermal keratinocytes; its expression is modest in the basal layer, heavy in the suprabasal layers, but absent from the most superficial, terminally differentiated layers. This layer-specific difference suggests that syndecan-1 expression changes with keratinocyte differentiation. To assess this hypothesis, syndecan-1 expression was evaluated before and after calcium-induced stratification and differentiation. Cells growing as an unstratified monolayer express a higher molecular mass form of syndecan-1 than do stratified cells (modal relative mass of 160 kD versus 110 kD). This structural difference is due to larger and more heparan sulfate chains on syndecan-1 from monolayer cells. In addition, the amount of cell-surface syndecan-1 changes with stratification; stratified cultures show approximately 2.5 times more syndecan-1 per cell than do unstratified cultures, but do not significantly change the level of syndecan-1-specific mRNA. Thus, the structure and amount of syndecan-1 may be regulated to meet the changing adhesive requirements of stratifying keratinocytes.     

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.     

Phospholipase Cγ1 is required for normal irritant contact dermatitis responses and sebaceous gland homeostasis

Differentiation and proliferation of keratinocyte are controlled by various signalling pathways. The epidermal growth factor receptor (EGFR) is known to be an important regulator of multiple epidermal functions. Inhibition of EGFR signalling disturbs keratinocyte proliferation, differentiation and migration. Previous studies have revealed that one of the EGFR downstream signalling molecules, phospholipase Cγ1 (PLCγ1), regulates differentiation, proliferation and migration of keratinocytes in in vitro cell culture system. However, the role of PLCγ1 in the regulation of keratinocyte functions in animal epidermis remains unexplored. In this study, we generated keratinocyte‐specific PLCγ1 knockout (KO) mice (PLCγ1 cKO mice). Contrary to our expectations, loss of PLCγ1 did not affect differentiation, proliferation and migration of interfollicular keratinocytes. We further examined the role of PLCγ1 in irritant contact dermatitis (ICD), in which epidermal cells play a pivotal role. Upon irritant stimulation, PLCγ1 cKO mice showed exaggerated ICD responses. Further study revealed that epidermal loss of PLCγ1 induced sebaceous gland hyperplasia, indicating that PLCγ1 regulates homeostasis of one of the epidermal appendages. Taken together, our results indicate that, although PLCγ1 is dispensable in interfollicular keratinocyte for normal differentiation, proliferation and migration, it is required for normal ICD responses. Our results also indicate that PLCγ1 regulates homeostasis of sebaceous glands.     

Flotillas of lipid rafts in transit amplifying cell-like keratinocytes

Lipid rafts are dynamic membrane microdomains enriched in cholesterol and sphingolipids and are involved in the regulation of a variety of cellular processes, such as proliferation, apoptosis and cell motility. We have previously described that large lipid raft aggregates are readily detectable on cultured keratinocyte cell line HaCaT by staining with the fluorescein-tagged cholera toxin (CTx-FITC). In this paper we adopted this method for the detection of lipid rafts in human epidermis and keratinocytes in culture. Double labelling of showed the non-overlapping clusters of basal cells in human epidermis: CD29dimCTx-FITCbright cells in the deep rate ridges and CD29brightCTx-FITCdim cells at the tips of dermal papillae. A similar patchy, non-overlapping staining pattern was observed in cultured keratinocytes in vitro. CTx-FITCbright cells are mitotically active whereas a large proportion of CTx-FITCdim cells are quiescent. We conclude that the epidermal stem-like cells, previously shown to occupy the tips of dermal papillae and to exhibit high density of membrane beta1 integrin have a low content of lipid rafts. In contrast, the putative transit amplifying cells in deep rate ridges show enrichment in lipid rafts. Thus, lipid rafts may be a factor controlling the mitotic activity of epidermal keratinocytes and possibly the differentiation of stem cells into the transit amplifying cells.     

Keratinocyte survival,differentiation, and death: many roads lead to mitogen-activated protein kinase

The epidermis is a dynamic and continually renewing surface that provides and maintains a life-sustaining interface with the environment. The epidermal keratinocyte, the major cell type of the epidermis, undergoes a complex and carefully choreographed program of differentiation. This process requires a balance between keratinocyte proliferation, differentiation, and apoptosis. This overview will concentrate on cascades that regulate the balance between keratinocyte cell proliferation and survival, and apoptosis and cell differentiation, with a particular emphasis on the role of the mitogen-activated protein kinase cascades. A summary of the literature suggests that extracellular regulated kinases function to promote keratinocyte proliferation and survival, whereas p38 mitogen-activated protein kinase functions to promote differentiation and apoptosis.     

Properties of the carcinoma-associated antigen MH 99/KS 1/4 in normal and transformed human keratinocytes: regulation of synthesis, molecular cross-linking and ultrastructural localization

A 38-kDa cell-surface glycoprotein defined by monoclonal antibody MH 99 is markedly increased in many epithelial tumours. In normal human skin, it is a characteristic marker for germ-cell phenotypic tissues. Although the gene encoding the MH 99 antigen has recently been cloned, and several histological and biochemical studies have been performed, the biological function of this interesting antigen still remains unknown. In the present study, we examined the synthesis of MH 99 in keratinocyte populations showing different in vitro differentiation capacity. Normal keratinocytes, spontaneously immortalized keratinocytes (cell line HaCaT), three SV-40-transformed keratinocyte lines (130, 425, and HaSV), and two squamous cell carcinoma lines (SCL-1 and SCL-2), were compared. Radioimmuno-precipitation revealed the highest levels of synthesis in cell populations with the least differentiation. This was paralleled by an increase of MH 99 synthesis in normal keratinocytes cultured in low concentrations of Ca²⁺ and by an increase of MH 99 synthesis during subculture of normal keratinocytes. Both phenomena were paralleled by an opposite behaviour of a differentiation marker. Molecular cross-linking and subsequent immunoprecipitation led to a decrease of the MH 99 signal, but an increase of a high molecular weight protein signal was seen. After cleavage of the crosslinker, the MH 99 signal reappeared, whereas the signal of the large protein remained unchanged. Thus, the MH 99 antigen may be associated with a high molecular weight protein on the cell surface, supporting the suggestion of a receptor-like function. Phosphorylation of the molecule could not be detected. Immunoelectron microscopy revealed homogeneous distribution on the cell surface, but cells of the same culture exhibited clear differences in their MH 99 expression. A concept for MH 99 regulation in normal and transformed human keratinocyte populations in vitro is proposed, showing that the synthesis of MH 99 is inversely correlated with cell differentiation. The association with a high molecular weight protein supports the suggestion that the MH 99 antigen interacts with other molecules.     

Knockdown of PKD1 in normal human epidermal keratinocytes increases mRNA expression of keratin 10 and involucrin: early markers of keratinocyte differentiation

Subconfluent normal human keratinocytes exhibit autonomous (autocrine growth factor driven) proliferation and express the specific markers for keratinocyte proliferation K5 (keratin 5) and K14 (keratin 14). Utilizing this model the effects of PKD1 (Protein kinase D1) knockdown on activation of differentiation was studied. siRNA approach was applied to achieve specific knockdown of PKD1 and the mRNA levels of different keratinocyte markers—K14 and PCNA (markers of basal proliferating keratinocytes), involucrin and K10 (early differentiation markers) were analyzed. Treatment of cultured keratinocytes with siRNA for PKD1 resulted in reduction of mRNA levels of PKD1, altered cell phenotype and promotion of keratinocyte differentiation, demonstrated by increased expression of involucrin and K10 mRNAs. No significant changes in K14 mRNA expression levels were detected, but the expression of PCNA mRNA was markedly diminished. This study was the first to show that mRNA expression of PKD1 in subconfluent normal human keratinocytes is very low, the PKD1 mRNA levels were more than 8-fold lower than the same ones in hTert keratinocytes. These findings suggest antidifferentiative role of PKD1 in normal human keratinocytes, contrary to the prodiferentiative role of PKD1 in human hTert keratinocytes. We came to the conclusion that there are differences between transduction pathways involving PKD1 in primary human keratinocyte cultures and these in immortalized hTert keratinocytes.