Transient expression of OCT4 is sufficient to allow human keratinocytes to change their differentiation pathway.

In this study, we describe a simple system in which human keratinocytes can be redirected to an alternative differentiation pathway. We transiently transfected freshly isolated human skin keratinocytes with the single transcription factor OCT4. Within 2 days these cells displayed expression of endogenous embryonic genes and showed reduced genomic methylation. More importantly, these cells could be specifically converted into neuronal and contractile mesenchymal cell types. Redirected differentiation was confirmed by expression of neuronal and mesenchymal cell mRNA and protein, and through a functional assay in which the newly differentiated mesenchymal cells contracted collagen gels as efficiently as authentic myofibroblasts. Thus, to generate patient-specific cells for therapeutic purposes, it may not be necessary to completely reprogram somatic cells into induced pluripotent stem cells before altering their differentiation and grafting them into new tissues.

Skin keratinocytes pre-treated with embryonic stem cell-conditioned medium or BMP4 can be directed to an alternative cell lineage.

OBJECTIVES: In this study, we have investigated whether secreted factors from embryonic stem cells (ESCs) could reprogramme keratinocytes and increase their potential to be directed into alternative cell lineages. MATERIALS AND METHODS: Contact and non-contact co-cultures of skin keratinocytes and murine ESCs were used initially to confirm any reprogramming ability of ESC-conditioned medium (CM). Immunofluoresence was used to assess nuclear expression of octamer-4 (Oct-4), as well as to confirm neuronal protein expression in neuroectodermally directed keratinocytes. Transcript expression changes were evaluated using semiquantitative reverse transcription-polymerase chain reaction. Western blotting, accompanied by densitometry analysis, was used to evaluate protein expression following morphology changes. RESULTS: We found that keratinocytes treated with ESC-CM changed their morphology and were stimulated to express the pluripotency regulator, Oct-4, and its target transcripts, Sox-2, Nanog, Utf1 and Rex-1. We demonstrate that at least one of the reprogramming factors is bone morphogenetic factor-4 (BMP4). Pre-treated keratinocytes could be specifically directed to differentiate into cells of the neuronal lineage. The majority of responsive keratinocytes were the epidermal stem cell population, with a small percentage of transit-amplifying cells also being affected. CONCLUSIONS: Our results suggest that ESC-CM contains a number of factors, including BMP4, which are capable of reprogramming mouse skin keratinocytes to make them more developmentally potent, as evidenced by their ability to be re-differentiated into cells of the neuronal lineage. Our findings also imply a continuum of differentiation within the basal keratinocyte population. An increase in developmental potential combined with directed differentiation could increase the therapeutic relevancy of somatic cells.

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

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

Expression of MK6a dominant-negative and C-terminal mutant transgenes in mice has distinct phenotypic consequences in the epidermis and hair follicle.

Mouse keratin 6a (MK6a) is constitutively expressed in a single cell layer of the outer root sheath (ORS) of hair follicles, but its synthesis can be induced in interfollicular epidermis including the basal cell layer in response to perturbing stimuli. A basally inducible human K6 (HK6) isoform has not been described, and it is not clear which of the known HK6 isoforms is expressed in the ORS. In this study we show that expression of a dominant-negative MK6a construct (Delta2B-P) in the interfollicular epidermis caused severe blistering and neonatal lethality, suggesting that mutations in a yet to be identified basally expressed HK6 isoform might result in a severe blistering phenotype. Surviving Delta2B-P animals showed transgene expression only in isolated epidermal cells and not in all cells of the ORS, but nevertheless developed severe alopecia. Expression of two different C-terminal mutant transgenes also caused alopecia while a third C-terminal mutant had no phenotypic conse- quences. Electron microscopy revealed that Delta2B-P expression resulted in the collapse of keratin filaments, while destruction of hair follicles in the two phenotypic C-terminal mutant lines occurred in the absence of filament abnormalities. The latter finding indicates that the innermost ORS cells are uniquely sensitive to expression of even slightly altered K6 proteins, suggesting that mutations affecting an HK6 isoform expressed in this cell layer could result in alopecia in humans as well.

The mouse keratin 6 isoforms are differentially expressed in the hair follicle, footpad, tongue and activated epidermis.

Keratin 6 (K6) is expressed constitutively in a variety of internal stratified epithelia as well as in palmoplantar epidermis and in specialized cells of the hair follicle. K6 expression can also be induced by hyperproliferative conditions as in wound healing or by conditions that perturb normal keratinocyte function. The functional significance of the expression of K6 on keratinocyte biology under these disparate conditions is not known. Here we report on the characterization of two isoforms of mouse K6 that are encoded by separate genes. The two genes (denoted K6a and K6b) are linked, have the same orientation and are actively transcribed. Sequence analysis revealed, that although they encode almost identical products, they have distinctly different regulatory regions, suggesting that the two K6 genes would be differentially expressed. In an attempt to define the expression characteristics of the K6 isoforms, we produced transgenic mice with each gene after modifying the C-terminal sequences to enable detection of the transgenic proteins with specific antibodies. The constitutive expression of the K6a transgene paralleled that of the endogenous genes in all K6 expressing tissues, except in the tongue. The K6b transgene was also expressed in these tissues but, in contrast to K6a, was only expressed in suprabasal cells. Both K6 transgenes were also induced in the interfollicular epidermis in response to phorbol esters, with K6a induced in all layers of the treated epidermis, while K6b was expressed only in suprabasal cells. These studies suggest that the K6 isoforms have overlapping yet distinct expression profiles.

Transduction of a preselected population of human epidermal stem cells: consequences for gene therapy.

Continuously renewing tissues, such as the epidermis, are populated by a hierarchy of dividing transient amplifying cells, which are maintained by stem cells. Transient amplifying cells divide to maintain the tissue, but they are limited to a finite number of cell divisions before they differentiate and are sloughed. Only the stem cells remain for the life of the tissue. Thus, it is critical to target stem cells when designing gene therapy regimes for genetically inherited diseases, such as epidermolysis bullosa simplex (EBS). Unfortunately, isolating pure epithelial stem cells has been problematic. In this study, we used rapid adherence to collagen type IV to successfully enrich for epidermal stem cells from adult human skin. These preselected stem cells were slow to proliferate, but they ultimately formed large colonies. When recombined with the dermal substrate AlloDerm, the stem cells re-formed a stratified squamous epidermis within 1 week after raising the AlloDerm to the air-liquid interface. These organotypic cultures grew continuously and, even after 6 weeks in culture, they maintained a proliferative basal layer. When transduced with a retroviral LacZ vector, preselected stem cells formed beta-galactosidase-positive clones in submerged and organotypic cultures. Transduced cells showed persistent expression through 12 weeks in organotypic culture, demonstrating the feasibility of using preselected stem cells for gene therapy. Currently, we are developing two models of EBS to test a gene therapy approach, which is based on the premise that EBS stem cells with a mutant keratin (K)14 gene corrected to wild type will have a growth advantage over noncorrected EBS stem cells.

Long-term culture of murine epidermal keratinocytes.

The production of transgenic and null mice with skin abnormalities makes it increasingly important to establish cultures of mouse epidermal keratinocytes for in vitro studies. This requires that each cell line be derived from a single mouse and that the cells be carried for multiple passages. Freezing the cells would also be advantageous by allowing comparison of keratinocytes from several mouse lines at the same time. Mouse keratinocytes, however, have been exceedingly difficult to grow as primary cultures, and subculturing these cells has been virtually impossible until now. We describe a gentle dissociation method and a highly supplemented fibroblast conditioned medium that allows us to grow and subculture total mouse keratinocytes for up to 19 subcultures, allowing an increase in cell number of greater than 10 logs. Epidermal keratinocytes from newborn mice were grown on collagen IV coated dishes in murine fibroblast conditioned medium with 0.06 mM calcium and added growth factors. The cells could be passaged, frozen as viable stocks, and induced to differentiate. Morphologically the cultured keratinocytes demonstrated a pattern characteristic of basal cells. Stratified cultures which made mouse keratin 1 and profilaggrin through passage 10 were induced by purging the monolayer cultures of growth factors, then adding medium with 0.15 mM calcium; expression of mouse keratin 1 and profilaggrin was lost by passage 15. The methods explained in detail here should be of great interest to investigators who are now trying to analyze skin phenotypes and expression of markers of epidermal differentiation of their transgenic or knockout mice.

Characterization of loricrin regulation in vitro and in transgenic mice.

We have previously shown that the promoter of a 6.5 kb mouse loricrin clone contains a functional AP-1 element and directs tissue-specific, but not differentiation-specific, expression. We now report the isolation of a 14-kb genomic clone containing an additional 7 kb of genomic sequence. The additional sequences limit expression of a reporter construct to differentiated keratinocytes in culture. The expression of the 6.5-kb and 14-kb loricrin constructs were also analyzed in transgenic mice. Significantly, loricrin was found in all layers of the epidermis of the 6.5-kb transgenics, including basal and spinous cells. The expression of the 14-kb clone was indistinguishable from that of the endogenous gene, confirming that the additional sequences contain negative regulatory elements that restrict loricrin expression to the granular layer in vivo. In addition, we show the AP-1 element localized in the loricrin proximal promoter is necessary but not sufficient for expression of the loricrin gene in vivo in transgenic mice. Finally, to gain further insight into how AP-1 family members regulate expression of the loricrin gene, we co-transfected the loricrin reporter constructs with expression plasmids for various fos and jun family members and demonstrated that c-Fos/Jun-B heterodimers could mimic the differentiation-specific induction of loricrin.

Telomerase is not an epidermal stem cell marker and is downregulated by calcium.

The ribonucleoprotein complex telomerase, which was found to be active in germ line, immortal, and tumor cells, and in cells from continuously renewing normal tissues such as epidermis or bone marrow, is thought to be correlated with an indefinite life span. Therefore, it has been postulated that in the normal tissues, telomerase activity may be restricted to stem cells, the possible precursors of tumor cells. Here, we demonstrate that a 56% enriched population of epidermal stem cells exhibited less telomerase activity than the more actively proliferating transit amplifying cells, which are destined to differentiate after a finite number of cell divisions. Thus telomerase is not a stem cell marker. In human epidermis we found a heterogeneous expression of the telomerase RNA component (hTR) within the basal layer, with clusters of hTR-positive cells showing variable activities. Histone-3 expressing S-phase basal cells were distributed evenly, illustrating that hTR upregulation may not strictly be correlated with proliferation. We further show for human epidermal cells that differentiation-dependent downregulation of telomerase correlates with Ca++-induced cell differentiation and that increasing the amount of Ca++ but not Mg++ or Zn++ reduced telomerase activity in a dose-dependent manner in a cell-free system (differentiation-independent). Furthermore, addition of ethyleneglycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid completely reversed this Ca++-induced inhibition. These data indicate that Ca++ is not only an important regulator of epidermal differentiation but also a key regulator of telomerase.

Selection and extended growth of murine epidermal stem cells in culture.

Continuously renewing epithelia contain small undifferentiated stem cells capable of self-renewal and maintenance of the differentiating cell population. In murine epidermis stem cells have been identified as label-retaining cells (LRCs) by long-term retention of tritiated thymidine or BrdU. It has been suggested that epidermal stem cells adhere to basement membranes through differential expression of specific integrins. To determine whether we could use a specific integrin to enrich for murine epidermal stem cells, we tested adherence of LRCs to several substrates. Regardless of the substrate used, approximately 10% of total basal cells and 100% of LRCs adhered in 10 min. In our medium specifically formulated for murine keratinocytes, rapidly adherent stem cells formed large colonies and could be used to form a structurally complete epidermis in organotypic culture. They showed a fivefold greater transient transfection efficiency than total basal cells, and when individual adherent cells were transduced with a retroviral vector, they formed large clones. Although these stem cells grew more slowly than the total basal cell population, they could be subcultured more times. Our results indicate that murine epidermal stem cells can be selected by rapid attachment to a substrate, but not by one specific integrin, and that they can be expanded in culture if the appropriate conditions are maintained.

Inhibition of retinoid signaling in transgenic mice alters lipid processing and disrupts epidermal barrier function.

To explore the role of retinoids in epidermal development, we recently targeted expression of a dominant-negative, retinoic acid receptor mutant (RAR alpha403) in the epidermis of transgenic mice and observed an unexpected loss of barrier function. In this paper, we demonstrate that transgenic mice expressing the RAR alpha403 transgene show attenuated responsiveness to topical application of all-trans retinoic acid, in agreement with our previous in vitro data. We also show that the vitamin D3 receptor is unaffected in its ability to transactivate in the presence of the dominant-negative RAR alpha403 transgene, indicating that the RAR alpha403 is unlikely to be functioning through a global sequestration of retinoid X receptors. Additionally, we show that the disruption of epidermal barrier function results in a dramatic 4 C drop in mean body surface temperature, probably accounting for the extremely high incidence of neonatal mortality in severely phenotypic pups. Some severely affected pups do survive and show a pronounced hyperkeratosis at postpartum day 4, consistent with previously documented effects of vitamin A deficiency. Biochemical analysis of the severely phenotypic neonates indicates elevated phospholipids and glycosylceramides in the stratum comeum, which results from altered lipid processing. Taken together with previous studies, these data provide strong evidence linking the retinoid-signaling pathway with modulation of lipid processing required for formation of epidermal barrier function.

Expression of a dominant-negative type II transforming growth factor beta (TGF-beta) receptor in the epidermis of transgenic mice blocks TGF-beta-mediated growth inhibition.

To determine whether a functional type II receptor of transforming growth factor beta (TGF-beta) is required to mediate the growth inhibitory effect of TGF-beta on the skin in vivo, we have generated transgenic mice that overexpress a dominant negative-type II TGF-beta receptor (delta beta RII) in the epidermis. The delta beta RII mice exhibited a thickened and wrinkled skin, and histologically the epidermis was markedly hyperplastic and hyperkeratotic. In vivo labeling with BrdUrd showed a 2.5-fold increase in the labeling index over controls, with labeled nuclei occurring in both basal and suprabasal cells of transgenic epidermis. In heterozygotes, this skin phenotype gradually diminished, and by 10-14 days after birth the transgenic mice were indistinguishable from their normal siblings. However, when F1 mice were mated to homozygosity, perinatal lethality occurred due to the severe hyperkeratotic phenotype, which restricted movement. Cultured primary keratinocytes from delta beta RII mice also exhibited an increased rate of growth in comparison with nontransgenic controls, and were resistant to TGF-beta-induced growth inhibition. These data document the role of the type II TGF-beta receptor in mediating TGF-beta-induced growth inhibition of the epidermis in vivo and in maintenance of epidermal homeostasis.

Immunological gene therapy approaches for malignant melanoma. 2. Preclinical studies and clinical strategies.

Immuno-gene therapy approaches for the treatment of malignant melanoma are categorized into two major subgroups according to an active or passive immunological principle. Active immuno-gene therapy is subdivided into melanoma cell vaccines, DNA-based vaccinations and the treatment of pre-existing tumor tissue by cell-mediated or direct transfer of cytokine and/or cell surface signal genes. Passive immuno-gene therapy, employing an adoptive treatment with in vitro activated and expanded anti-tumor effector cells, involves two major application fields for gene transfer techniques, first the genetic modification of the effector cells, and second the in vivo amplification of pre-effector cells by procedures also used in active immuno-gene therapy. Corresponding preclinical studies are reviewed. The clinical studies inaugurated during the last few years are mostly still ongoing and focus on treatment safety and tolerability rather than efficacy. A recent trend is emerging to explore recombinant adenovirus and vaccinia virus vectors particularly with regard to in vivo gene transfer applications. Overall, immuno-gene therapy of melanoma is still in a highly experimental stage of development but may become a safe, efficacious and practical adjuvant treatment modality in the future.

Immunological gene therapy approaches for malignant melanoma. 1. Tumor-immunological background.

Gene therapy approaches pursuing immunological strategies for the treatment of malignant melanoma play major roles in the current efforts to explore the potential benefits of gene transfer technologies for medicine. This may be explained by the nearly complete resistance of advanced metastatic melanoma towards conventional non-surgical treatment modalities, and the particular immunogenicity of melanoma in connection with a presumed immuno-gene therapeutic 'field effect'. The latter relates to the potency of the immune system to amplify gene transfer effects that are limited due to the imperfection of the currently available gene delivery systems. The ongoing clinical trials focus predominantly on treatment safety and tolerability rather than efficacy. The corresponding tumor-immunological background is reviewed, focusing on a treatment concept centred on tumor-reactive, cytotoxic CD8+ T effector cells.

A transgenic mouse model that recapitulates the clinical features of both neonatal and adult forms of the skin disease epidermolytic hyperkeratosis.

Keratins are the major structural proteins of keratinocytes, which are the most abundant cell type in the mammalian epidermis. Mutations in epidermal keratin genes have been shown to cause severe blistering skin abnormalities. One such disease, epidermolytic hyperkeratosis (EHK), also known as bullous congenital ichthyosiform erythroderma, occurs as a result of mutations in highly conserved regions of keratins K1 and K10. Patients with EHK first exhibit erythroderma with severe blistering, which later is replaced by thick patches of scaly skin. To assess the effect of a mutated K1 gene on skin biology and to produce an animal model for EHK, we removed 60 residues from the 2B segment of HK1 and observed the effects of its expression in the epidermis of transgenic mice. Phenotypes of the resultant mice closely resembled those observed in the human disease, first with epidermal blisters, then later with hyperkeratotic lesions. In neonatal mice homozygous for the transgene, the skin was thicker, with an increased labeling index, and the spinous cells showed a collapse of the keratin filament network around the nuclei, suggesting that a critical concentration of the mutant HK1, over the endogenous MK1, was required to disrupt the structural integrity of the spinous cells. Additionally, footpad epithelium, which is devoid of hair follicles, showed blistering in the spinous layer, suggesting that hair follicles can stabilize or protect the epidermis from trauma. Blisters were not evident in adult mice, but instead they showed a thick, scaly hyperkeratotic skin with increased mitosis, resulting in an increased number of corneocytes and granular cells. Irregularly shaped keratohyalin granules were also observed. To date, this is the only transgenic model to show the typical morphology found in the adult form of EHK.

Loricrin expression is coordinated with other epidermal proteins and the appearance of lipid lamellar granules in development.

In mouse, epidermal development proceeds from a single basal cell layer covered by a specialized single cell layer called the periderm at E14 to a fully differentiated stratified squamous epithelium at E18. To determine when loricrin, a major cell envelope component, is expressed during development, we examined fetal skin from mice of gestational ages E13 through E19 and compared the temporal pattern of loricrin expression with that of other differentiation markers. We found that loricrin mRNA and protein were expressed by E16, following the expression of keratins K1 and K10 and preceding the expression of profilaggrin. Interestingly, both loricrin and profilaggrin were initially expressed focally in areas corresponding to more advanced morphologic stages of maturation. Because the cornified envelope is a composite structure consisting of both protein and lipid components, we also monitored the appearance of lipid lamellar granules during epidermal development. These granules were first evident at E16 and the extrusion of lipids from the granules into the intercellular space occurred at E17, prior to the cross linking of loricrin into the cell envelope. Our results document that loricrin is expressed and accumulates at the cell periphery subsequent to the extrusion of lipids, but prior to processing of profilaggrin. We suggest that the sequential regulation of these events is critical for formation of epidermal barrier function during development.

Functional analysis of activins during mammalian development.

Activins are dimeric (beta A beta A; beta B beta B; beta A beta B) members of the transforming growth factor-beta superfamily. They are widely expressed during murine development, are highly conserved during vertebrate evolution, and may be involved in mesoderm induction and neurulation in Xenopus laevis and Oryzias latipes. To investigate the function of mammalian activins in vivo, we generated mice with mutations either in activin-beta A or in both activin-beta A and activin-beta B. Activin-beta A-deficient mice develop to term but die within 24 h of birth. They lack whiskers and lower incisors and have defects in their secondary palates, including cleft palate, demonstrating that activin-beta A must have a role during craniofacial development. Mice lacking both activin subunits show the defects of both individual mutants but no additional defects, indicating that there is no functional redundancy between these proteins during embryogenesis. In contrast to observations in lower vertebrates, zygotic expression of activins is not essential for mesoderm formation in mice.

Targeting expression of a dominant-negative retinoic acid receptor mutant in the epidermis of transgenic mice results in loss of barrier function.

To study the effects of retinoic acid on the skin in vivo, we have subverted the activity of endogenous receptors by targeting expression of a dominant negative mutant of retinoic acid receptor alpha (RAR alpha) to the epidermis of transgenic mice. At birth, mice expressing the mutant RAR alpha transgene exhibited a marked phenotype of a red, shiny skin that was somewhat sticky to touch. Severely affected neonates died within 24 hr. Histological changes in the epidermis were subtle with the phenotypic stratum corneum appearing slightly thinner and more loosely packed than in controls. Electron microscopic studies revealed that lipid multilamellar structures were not present between cells in the stratum corneum of phenotypic mice. When assayed for transepidermal water loss, phenotypic skin lost water at a rate three times faster than controls, suggesting that neonatal lethality resulted from loss of epidermal barrier function. The absence of a functional lipid barrier in transgenic mice first became evident at E17 when lipids were extruded initially into the intercellular space. We have identified a potential pathway linking inhibition of retinoid signaling with disruption of the lipid barrier that involves peroxisome proliferator-activated receptors. This study documents the role of the retinoid signaling pathway in formation and maintenance of a functional epidermis and provides the first evidence that this is mediated in part by modulation of lipid metabolism.

TGF alpha and v-fos cooperation in transgenic mouse epidermis induces aberrant keratinocyte differentiation and stable, autonomous papillomas.

To assess the synergistic effect of growth and transcription factor deregulation on carcinogenesis in vivo, mating experiments were performed between transgenic mice expressing human TGF alpha or v-fos exclusively in the epidermis by means of a human keratin K1-based targeting vector (HK1.fos, HK1.TGF alpha and HK1.fos/alpha). While HK1.TGF alpha mice exhibited mild epidermal hyperplasia resulting in a wrinkled appearance, this hyperplasia was significantly increased in HK1.fos/alpha mice which also exhibited a novel opalescent and peeling skin phenotype. HK1.fos/alpha keratinocyte differentiation was considerably deregulated with cornified cells appearing in the granular layer, granular cells in the spinous layer and a sixfold increase in BrdU labeling over normal. In addition, hyperplastic HK1.fos/alpha epidermis exhibited aberrant loricrin, filaggrin and novel K13 expression associated with v-fos expression. Unlike adult HK1.TGF alpha controls, hyperplasia persisted in HK1.fos/alpha adults which also rapidly developed autonomous squamous cell papillomas. These results demonstrate that v-fos and TGF alpha over-expression can cooperate to reprogram keratinocyte differentiation and elicit the early stages of neoplasia. Moreover, TGF alpha over-expression appeared to play an early, initiating role in HK1.fos/alpha papilloma etiology, and a promotion role in the accelerated appearance of v-fos wound-associated preneoplastic phenotypes. However, the stable persistence of HK1.fos/alpha papillomas for up to 12 months, suggests that additional events are required for malignant conversion.