Three-Dimensional In Vitro Skin and Skin Cancer Models Based on Human Fibroblast-Derived Matrix.

Three-dimensional in vitro skin and skin cancer models help to dissect epidermal-dermal and tumor-stroma interactions. In the model presented here, normal human dermal fibroblasts isolated from adult skin self-assembled into dermal equivalents with their specific fibroblast-derived matrix (fdmDE) over 4 weeks. The fdmDE represented a complex human extracellular matrix that was stabilized by its own heterogeneous collagen fiber meshwork, largely resembling a human dermal in vivo architecture. Complemented with normal human epidermal keratinocytes, the skin equivalent (fdmSE) thereof favored the establishment of a well-stratified and differentiated epidermis and importantly allowed epidermal regeneration in vitro for at least 24 weeks. Moreover, the fdmDE could be used to study the features of cutaneous skin cancer. Complementing fdmDE with HaCaT cells in different stages of malignancy or tumor-derived cutaneous squamous cell carcinoma cell lines, the resulting skin cancer equivalents (fdmSCEs) recapitulated the respective degree of tumorigenicity. In addition, the fdmSCE invasion phenotypes correlated with their individual degree of tissue organization, disturbance in basement membrane organization, and presence of matrix metalloproteinases. Together, fdmDE-based models are well suited for long-term regeneration of normal human epidermis and, as they recapitulate tumor-specific growth, differentiation, and invasion profiles of cutaneous skin cancer cells, also provide an excellent human in vitro skin cancer model.

Wnt-3a-activated human fibroblasts promote human keratinocyte proliferation and matrix destruction.

Aberrant Wnt regulation, detectable by nuclear translocation of beta-catenin, is a hallmark of many cancers including skin squamous cell carcinomas (SCCs). By analyzing primary human skin SCCs, we demonstrate that nuclear beta-catenin is not restricted to SCC cells but also detected in stromal fibroblasts, suggesting an important role for aberrant Wnt regulation also in the tumor microenvironment. When human keratinocytes and fibroblasts were treated with Wnt-3a, fibroblasts proved to be more responsive. Accordingly, Wnt-3a did not alter HaCaT cell functions in a cell-autonomous manner. However, when organotypic cultures (OTCs) were treated with Wnt-3a, HaCaT keratinocytes responded with increased proliferation. As nuclear beta-catenin was induced only in the fibroblasts, this argued for a Wnt-dependent, paracrine keratinocyte stimulation. Global gene expression analysis of Wnt-3a-stimulated fibroblasts identified genes encoding interleukin-8 (IL-8) and C-C motif chemokine 2 (CCL-2) as well as matrix metalloproteinase-1 (MMP-1) as Wnt-3a targets. In agreement, we show that IL-8 and CCL-2 were secreted in high amounts by Wnt-3a-stimulated fibroblasts also in OTCs. The functional role of IL-8 and CCL-2 as keratinocyte growth regulators was confirmed by directly stimulating HaCaT cell proliferation in conventional cultures. Most important, neutralizing antibodies against IL-8 and CCL-2 abolished the Wnt-dependent HaCaT cell hyperproliferation in OTCs. Additionally, MMP-1 was expressed in high amounts in Wnt-3a-stimulated OTCs and degraded the stromal matrix. Thus, our data show that Wnt-3a stimulates fibroblasts to secrete both keratinocyte proliferation-inducing cytokines and stroma-degrading metalloproteinases, thereby providing evidence for a novel Wnt deregulation in the tumor-stroma directly contributing to skin cancer progression.

Identification of a population of epidermal squamous cell carcinoma cells with enhanced potential for tumor formation.

Epidermal squamous cell carcinoma is among the most common cancers in humans. These tumors are comprised of phenotypically diverse populations of cells that display varying potential for proliferation and differentiation. An important goal is identifying cells from this population that drive tumor formation. To enrich for tumor-forming cells, cancer cells were grown as spheroids in non-attached conditions. We show that spheroid-selected cells form faster growing and larger tumors in immune-compromised mice as compared to non-selected cells. Moreover, spheroid-selected cells gave rise to tumors following injection of as few as one hundred cells, suggesting these cells have enhanced tumor-forming potential. Cells isolated from spheroid-selected tumors retain an enhanced ability to grow as spheroids when grown in non-attached culture conditions. Thus, these tumor-forming cells retain their phenotype following in vivo passage as tumors. Detailed analysis reveals that spheroid-selected cultures are highly enriched for expression of epidermal stem cell and embryonic stem cell markers, including aldehyde dehydrogenase 1, keratin 15, CD200, keratin 19, Oct4, Bmi-1, Ezh2 and trimethylated histone H3. These studies indicate that a subpopulation of cells that possess stem cell-like properties and express stem cell markers can be derived from human epidermal cancer cells and that these cells display enhanced ability to drive tumor formation.

Human skin keratinocytes can be reprogrammed to express neuronal genes and proteins after a single treatment with decitabine.

Patient-specific cell replacement therapy is fast becoming the future of medicine, requiring safe, effective methods for reprogramming a patient's own cells. Previously, we showed that a single transient transfection with a plasmid encoding Oct4 was sufficient to reprogram human skin keratinocytes (HSKs), and that this transfection resulted in a decrease in global DNA methylation. In more recent work we showed that decreasing global DNA methylation using the U.S. Food and Drug Administration-approved cancer treatment drug decitabine was sufficient to induce expression of endogenous Oct4. Here we report that a single treatment with decitabine, followed by 5 days in a defined neuronal transformation medium, then 7 days in a neuronal maintenance medium is sufficient to convert HSKs into cells that change their morphology substantially, gain expression of neuronal markers, and lose expression of keratinocyte markers. Within 1 week of treatment the cells express mRNA for ß3-tubulin and doublecortin, and at the end of 2 weeks express mRNA for NeuN, FOXP2, and NCAM1. Additionally, at the end of this protocol, neurofilament-1, nestin, synapsin, FOXP2, and GluR1 proteins are detectable by immunostaining. Thus, we demonstrate a simple method that begins the process for producing cells for cell replacement therapies without using exogenously introduced DNA.

Biochemistry of epidermal stem cells.

BACKGROUND: The epidermis is an important protective barrier that is essential for maintenance of life. Maintaining this barrier requires continuous cell proliferation and differentiation. Moreover, these processes must be balanced to produce a normal epidermis. The stem cells of the epidermis reside in specific locations in the basal epidermis, hair follicle and sebaceous glands and these cells are responsible for replenishment of this tissue. SCOPE OF REVIEW: A great deal of effort has gone into identifying protein epitopes that mark stem cells, in identifying stem cell niche locations, and in understanding how stem cell populations are related. We discuss these studies as they apply to understanding normal epidermal homeostasis and skin cancer. MAJOR CONCLUSIONS: An assortment of stem cell markers have been identified that permit assignment of stem cells to specific regions of the epidermis, and progress has been made in understanding the role of these cells in normal epidermal homeostasis and in conditions of tissue stress. A key finding is the multiple stem cell populations exist in epidermis that give rise to different structures, and that multiple stem cell types may contribute to repair in damaged epidermis. GENERAL SIGNIFICANCE: Understanding epidermal stem cell biology is likely to lead to important therapies for treating skin diseases and cancer, and will also contribute to our understanding of stem cells in other systems. This article is part of a Special Issue entitled Biochemistry of Stem Cells.

Treatment with the cancer drugs decitabine and doxorubicin induces human skin keratinocytes to express Oct4 and the OCT4 regulator mir-145.

Previously, we showed that transient transfection with OCT4 not only produced high expression of Oct4 in skin keratinocytes, but also caused a generalized demethylation of keratinocyte DNA. We hypothesized that DNA demethylation alone might allow expression of endogenous OCT4. Here, we report that treatment with the cancer drug decitabine results in generalized DNA demethylation in skin keratinocytes, and by 48 h after treatment, 96% of keratinocytes show expression of the endogenous Oct4 protein and the OCT4 repressor mir-145. This is true for keratinocytes only, as skin fibroblasts treated similarly show no OCT4 or mir-145 expression. Decitabine-treated keratinocytes also show increased mir-302c and proliferation similar to other Oct4(+) cells. Treatment with doxorubicin, another cancer drug, induces expression of mir-145 only in cells that already express OCT4, suggesting that Oct4 regulates its own repressor. Co-treatment with decitabine and doxorubicin results first in increased OCT4 and mir-145, then a decrease in both, suggesting that OCT4 and mir-145 regulate each other. The novel strategy presented here provides a regulatable system to produce Oct4(+) cells for transformation studies and provides a unique method to study the effects of endogenous Oct4 in cancer cells and the surrounding somatic cells.

Oxygen tension changes the rate of migration of human skin keratinocytes in an age-related manner.

Migration of keratinocytes to re-epithelialize wounds is a key step in dermal wound healing. In aged human skin, wound healing rates decrease and cellular damage by reactive oxygen species (ROS) accumulates. The relationship between age, ROS and human skin keratinocyte migration is not clearly understood. In this study, 4% and 21% oxygen tensions were used to modify levels of ROS produced by metabolism to model low and high oxidative stress conditions. When migration of keratinocytes from young and old primary skin was compared using an in vitro scratch assay, old keratinocytes migrated faster in high oxygen tension than did young keratinocytes, whereas young keratinocytes migrated faster in low oxygen tension. Although all young and old cells at the scratch margins showed intense increases in dihydroethidium oxidation immediately after scratching, the old keratinocytes grown at 21% oxygen demonstrated a greater decrease in the DHE oxidation following scratching and migrated the fastest. These results show that old and young keratinocytes respond to oxygen tension differently and support the hypothesis that keratinocyte migration is affected by the capacity to remove ROS.

Aging epidermis is maintained by changes in transit-amplifying cell kinetics, not stem cell kinetics.

It has been assumed that the slow rate of healing in aging epidermis is due to slowing of the epidermal stem cell proliferative rate. In this issue, Charruyer et al. report that this may not be the case. Using a long-term repopulating model, they demonstrate that epidermal stem cell kinetics are maintained. Instead, it is the compensatory action of the transit-amplifying (TA) cells that changes in aging skin and thus bears responsibility for slowed wound healing.

Are epidermal stem cells unique with respect to aging?

Epidermal stem cells are a population of somatic stem cells responsible for maintaining and repairing the epidermis of the skin. A malfunctioning epidermal stem cell compartment results in loss of the epidermis and death of the whole organism. Since the epidermis continually renews itself by sloughing a layer of cells every day, it is in a constant state of cellular turnover and requires continual cell replacement for life. Thus, maintaining a pristine epidermal stem cell population is of prime importance, even during aging. Unlike stem cells from internal tissues, epidermal stem cells show little response to aging. They do not appear to decrease in number or functionality with age, and do not show changes in gene expression, developmental responsiveness, or age-associated increases of reactive oxygen species. Thus, epidermal stem cells may be a unique somatic stem cell.

A stable niche supports long-term maintenance of human epidermal stem cells in organotypic cultures.

Stem cells in human interfollicular epidermis are still difficult to identify, mainly because of a lack of definitive markers and the inability to label human beings for label-retaining cells (LRCs). Here, we report that LRCs could be identified and localized in organotypic cultures (OTCs) made with human cells. Labeling cultures for 2 weeks with iododeoxyuridine (IdU) and then chasing for 6-10 weeks left <1% of basal cells retaining IdU label. Whole mounts demonstrated that LRCs were individually dispersed in the epidermal basal layer. Some LRCs, but not all, colocalized with cells expressing melanoma chondroitin sulfate proteoglycan, a putative stem cell marker. Although we found LRCs in both collagen- and scaffold-based OTCs, only the scaffold-OTCs supported long-term survival and regeneration. LRCs ' short survival in collagen-OTCs was not due to loss of appropriate growth factors from fibroblasts. Instead, it was due to expression of metalloproteinases, especially matrix metalloproteinase (MMP)-2 and MMP-14, which caused collagen fragmentation, matrix degradation, and dislocation of specific basement membrane components bound to epidermal integrins. Blocking MMP activation not only abrogated MMP-dependent matrix degradation but also increased longevity of the epidermis and the LRCs in these cultures. Such findings indicate that the stem cell niche, the microenvironment surrounding and influencing the stem cell, is essential for stem cell survival and function, including long-term tissue regeneration. Disclosure of potential conflicts of interest is found at the end of this article.

Cells isolated from the epidermis by Hoechst dye exclusion, small size, and negative selection for hematopoietic markers can generate B lymphocyte precursors.

Transdifferentiation has become a common claim for somatic stem cells, yet how such cells can be directed toward a specified cell lineage has not been well investigated. We previously demonstrated that when isolated epidermal stem cells were placed into an embryonic environment, their potential was extended beyond the keratinocyte lineage. Here, we present evidence that cells isolated using a modification of our published method for epidermal stem cells can be specifically directed to differentiate into B lymphocyte precursors. We found that these isolated cells co-cultured with S17 bone marrow stromal cells in cytokine-supplemented medium changed their cell surface marker profile and gene expression pattern to one characteristic of B lymphocyte precursors. Such cells also underwent variable, diversity, joining rearrangement at the immunoglobulin heavy-chain locus, a permanent genetic change unique to lymphocytes. This feature is limited to the cells isolated using the modified epidermal stem cell method, as cells isolated using the modified transit amplifying cell method could not be re-directed or reprogrammed. Such results demonstrate that cells from the epidermis can be directed to cross lineage boundaries to become mesodermally derived lymphocytes.

HSP70 and EndoG modulate cell death by heat in human skin keratinocytes in vitro.

We examined how young and old keratinocytes died from heat stress in vitro. We found that keratinocyte cell death was not due to oxidative stress as neither Mn-SOD nor Cu-Zn-SOD was produced in either young or old heated keratinocytes. Instead, analysis of the anti-apoptotic factors, Bcl2 and HSP70, and the pro-apoptotic factors, caspase 3, caspase 8, Apaf-1, cytochrome c, AIF, and EndoG, indicated that keratinocyte cell death occurred via the caspase-independent EndoG apoptotic pathway. We found that both young and old keratinocytes died via the same pathway, and that we could specifically reduce both young and old keratinocyte death by addition of the EndoG inhibitor NEM. Further analysis suggested that the difference between young and old keratinocyte death was due to the synthesis of HSP70 protein, with the increase in response to heat more pronounced in young keratinocytes than in old keratinocytes. When we inhibited HSP70 by adding quercetin, death was increased in both young and old keratinocytes, but more so in old keratinocytes. These data suggest that old keratinocytes may die more readily than young keratinocytes when heated because they synthesize HSP70 at a lower efficiency. Such findings suggest that HSP70 production may be age-dependent.

Epidermal stem cells are resistant to cellular aging.

The epidermis of the skin, acting as the primary physical barrier between self and environment, is a dynamic tissue whose maintenance is critical to the survival of an organism. Like most other tissues and organs, the epidermis is maintained and repaired by a population of resident somatic stem cells. The epidermal stem cells reside in the proliferative basal cell layer and are believed to persist for the lifetime of an individual. Acting through intermediaries known as transit amplifying cells, epidermal stem cells ensure that the enormous numbers of keratinocytes required for epidermal homeostasis to be maintained are generated. This continual demand for new cell production must be met over the entire lifetime of an individual. Breakdown of the epidermal barrier would have catastrophic consequences. This leads us to question whether or not epidermal stem cells represent a unique population of cells which, by necessity, might be resistant to cellular aging. We hypothesized that the full physiologic functional capacity of epidermal stem cells is maintained over an entire lifetime. Using murine skin epidermis as our model system, we compared several properties of young and old adult epidermal stem cells. We found that, over an average mouse's lifetime, there was no measurable loss in the physiologic functional capacity of epidermal stem cells, leading us to conclude that murine epidermal stem cells resist cellular aging.

De-differentiation of mouse interfollicular keratinocytes by the embryonic transcription factor Oct-4.

The embryonic transcription factor Oct-4 is often referred to as the master regulator of the undifferentiated state. Although its role in maintaining embryonic stem (ES) cell pluripotency is well established, its ability to directly reprogram committed somatic cells is not well defined. Using transient transfection, we tested its ability to revert mouse interfollicular epidermal basal keratinocytes to a more ES cell-like state. We found that the Oct-4-transfected keratinocytes expressed the Oct-4 target genes, Sox-2, Nanog, undifferentiated transcription factor 1 (Utf1), and Rex-1. We also noted an increase in developmental potential caused by Oct-4, with the transfected cells able to differentiate into neuronal cells when exposed to neuroectodermal differentiation medium. Control-transfected keratinocytes were unable to respond to the medium, and remained as keratinocytes. These findings suggest that Oct-4 may be the master regulator of the pluripotent state and demonstrate that differentiated somatic cells can be reverted into more developmentally potent cells through the use of a single factor. The latter finding has great implications for therapeutic cell-replacement applications using cells from easily accessible adult tissues, such as the skin.