Elutriation of human keratinocytes and melanocytes fromin vitro cultured epithelium

Summary Human epidermal keratinocytes grown in culture and at different stages of differentiation are shown to be viably separated by elutriation. A specific fraction enriched in melanocytes was obtained. Elutriation of cells obtained fromin vitro cultured epithlium could prove useful in studies concerning the biochemistry and molecular markers of cells isolated from normal epithelium and from different pathologies.     

Crosstalk in skin: melanocytes,keratinocytes, stem cells,and melanoma

In the vertebrate embryo, melanocytes arise from the neural crest, migrate to and colonize the basal layer within the skin and skin appendages. Post-migratory melanocytes are securely attached to the basement membrane, and their morphology, growth, adhesion, and migration are under control of neighboring keratinocytes. Melanoma is a malignant tumor originated from melanocytes or their progenitor cells. During melanocyte transformation and melanoma progression, melanocytes lose their interactions with keratinocytes, resulting in uncontrolled proliferation and invasion of the malignant cells. Melanoma cells at the advanced stages often lack melanocytic features and resemble multipotent progenitors, which are a potential melanocyte reservoir in human skin. In this mini-review, we will summarize findings on cell-cell interactions that are responsible for normal melanocyte homeostasis, stem cell self-renewal, and differentiation. Our ultimate goal is to define molecules and pathways, which are essential for normal cell-cell interactions but deregulated in melanoma formation and progression.     

Coculture of human keratinocytes and melanocytes: differentiated melanocytes are physiologically organized in the basal layer of the cultured epithelium

Human epidermal keratinocytes differentiate in vitro into a stratified epithelium suitable for grafting on burned patients. In this paper, we show that differentiated melanocytes are present in the cultured epithelium. In particular, we have found that i) melanocytes proliferate in the same culture conditions that allow keratinocyte growth, ii) during the culture the ratio between keratinocytes and melanocytes tends to remain constant, iii) melanocytes organize into the basal layer of the cultured epithelium independently of the presence of dermis, develop dendritic arborizations with melanosome-containing processes and transfer melanosomes into keratinocyte cytoplasm.     

Diversity of pigmentation in cultured human melanocytes is due to differences in the type as well as quantity of melanin

Cultured human melanocytes differ tremendously in visual pigmentation, and recapitulate the pigmentary phenotype of the donor's skin. This diversity arises from variation in type as well as quantity of melanin produced. Here, we measured contents of eumelanin (EM) and pheomelanin (PM) in 60 primary human melanocyte cultures (51 neonatal and nine adults), and correlated some of these values with the respective activity and protein levels of tyrosinase, and the melanocortin-1 receptor (MC1R) genotype. Melanocytes were classified into four phenotypes (L, L+, D, D+) as depicted by visual pigmentation using light microscopy, and by the pigmentary phenotype of the donor's skin. There were large differences in total melanin (TM) and EM, which increased progressively for L, L+, D and D+ melanocytes. TM content, the sum of EM and PM, showed a good correlation with TM measured spectrophotometrically, and with the activity and protein levels of tyrosinase. Log EM/PM ratio did not correlate with MC1R genotype. We conclude that: (i) EM consistently correlates with the visual phenotype; (ii) lighter melanocytes tend to be more pheomelanic in composition than darker melanocytes; (iii) in adult melanocyte cultures, EM correlates with the ethnic background of the donors (African-American > Indian > Caucasian); and (iv) MC1R loss-of-function mutations do not necessarily alter the phenotype of cultured melanocytes.     

Structural and functional evaluation of modifications in the composite skin graft: cryopreserved dermis and cultured keratinocytes.

Structural and functional aspects of modifications in the composite skin graft consisting of cultured keratinocytes and cryopreserved dermis were determined. Cryopreserved human cadaveric dermis separated from skin by short and mild trypsinization was compared with dermis obtained by prolonged incubation in medium and with fresh dermis obtained by the same methods. All types of dermis were shown to retain normal ultrastructure and topographic organization, as detected by scanning and transmission electron microscope and immunofluorescence analysis. However, in fresh skin, the layers were more firmly attached, mechanical separation was more difficult, and residual epidermis often remained attached to the dermis. Keratinocytes attached better, began replication earlier, and generally reached higher cell numbers when cultured on trypsinized dermis than on medium-treated dermis. The performance of several modifications in the reconstitution and grafting procedures of the composite skin graft after transplantation to athymic mice was examined. Cultured epidermis combined onto trypsinized or medium-treated whole and meshed dermis, dermis pregrafted and allowed to take before transplanting epidermis on top, and keratinocytes grown into multiple epithelia on top of trypsinized meshed or whole dermis prior to grafting. The best grafting results were obtained with an "instant" reconstituted skin model: multiple epithelia grown in vitro combined immediately before grafting onto meshed trypsinized dermis. The transplantation results of this modification were significantly better than those of all the other modifications, including initial growth of keratinocytes into multiple epithelia on top of trypsinized dermis prior to grafting.     

Autophagy induction can regulate skin pigmentation by causing melanosome degradation in keratinocytes and melanocytes

Autophagy regulates cellular turnover by disassembling unnecessary or dysfunctional constituents. Recent studies demonstrated that autophagy and its regulators play a wide variety of roles in melanocyte biology. Activation of autophagy is known to induce melanogenesis and regulate melanosome biogenesis in melanocytes. Also, autophagy induction was reported to regulate physiologic skin color via melanosome degradation, although the downstream effectors are not yet clarified. To determine the role of autophagy as a melanosome degradation machinery, we administered several autophagy inducers in human keratinocytes and melanocytes. Our results showed that the synthetic autophagy inducer PTPD‐12 stimulated autophagic flux in human melanocytes and in keratinocytes containing transferred melanosomes. Increased autophagic flux led to melanosome degradation without affecting the expression of MITF. Furthermore, the color of cell pellets of both melanocytes and keratinocytes was visibly lightened. Inhibition of autophagic flux by chloroquine resulted in marked attenuation of PTPD‐12‐induced melanosome degradation, whereas the expression of melanogenesis pathway genes and proteins remained unaffected. Taken together, our results suggest that the modulation of autophagy can contribute to the regulation of melanocyte biology and skin pigmentation.     

Patterns of junctional communication in skin: Studies on cultured keratinocytes

Junctional communication has long been suggested to play a role in coordinating the development of multicellular tissues. A better understanding of the patterns of communication between cells in such tissues is important for the identification of areas where this process may have a role. We have investigated the patterns of communication in cultures of human epidermal keratinocytes by iontophoretic injection of Lucifer Yellow CH, using involucrin expression as a marker of cells undergoing terminal differentiation. Cells that lack involucrin (i.e., the basal, proliferating cells) transfer dye preferentially to other involucrin-negative cells, whereas involucrin-positive cells either are not coupled or transfer dye with similar frequency to involucrin-positive and involucrin-negative neighbors. This decrease in communication associated with terminal differentiation was observed in both the presence and the absence of assembled desmosomes. Our observations lead us to speculate that loss of junctional communication may influence the commitment of basal keratinocytes to terminal differentiation.     

Expression of P450 enzymes in rat whole skin and cultured epidermal keratinocytes

The complement and level of expression of P450 enzymes in male Fischer F344 rat whole skin and cultured keratinocytes were investigated using a panel of mono-specific antibodies. In whole skin microsomal fraction, immunoreactive bands corresponding to CYP2B12, CYP2C13, CYP2D1, CYP2D4, CYP2E1, CYP3A1, and CYP3A2 were detected whereas CYP1A1, CYP1A2, and CYP2C12 were absent. Skin levels were all between 0.1% and 4.7% of those found in liver, except for CYP2D4, which was not detected in liver. Keratinocytes were isolated from rat skin, cultured for up to 42 days, and changes in the levels of CYP3A1, CYP3A2, and CYP2E1 determined. Levels were low in isolated keratinocytes, but they increased markedly in culture, reaching a maximum at 10-14 days, where they were similar to those found in fresh skin. This suggests that primary keratinocytes grown in culture for 10-14 days may provide a useful experimental model to study P450-catalysed metabolism of xenobiotics in skin.     

In ovo gene manipulation of melanocytes and their adjacent keratinocytes during skin pigmentation of chicken embryos

During skin pigmentation in avians and mammalians, melanin is synthesized in the melanocytes, and subsequently transferred to adjacently located keratinocytes, leading to a wide coverage of the body surface by melanin‐containing cells. The behavior of melanocytes is influenced by keratinocytes shown mostly by in vitro studies. However, it has poorly been investigated how such intercellular cross‐talk is regulated in vivo because of a lack of suitable experimental models. Using chicken embryos, we developed a method that enables in vivo gene manipulations of melanocytes and keratinocytes, where these cells are separately labeled by different genes. Two types of gene transfer techniques were combined: one was a retrovirus‐mediated gene infection into the skin/keratinocytes, and the other was the in ovo DNA electroporation into neural crest cells, the origin of melanocytes. Since the Replication‐Competent Avian sarcoma‐leukosis virus long terminal repeat with Splice acceptor (RCAS) infection was available only for the White leghorn strain showing little pigmentation, melanocytes prepared from the Hypeco nera (pigmented) were back‐transplanted into embryos of White leghorn. Prior to the transplantation, enhanced green fluorescent protein (EGFP)⁺Neo^r+‐electroporated melanocytes from Hypeco nera were selectively grown in G418‐supplemented medium. In the skin of recipient White leghorn embryos infected with RCAS‐mOrange, mOrange⁺ keratinocytes and transplanted EGFP⁺ melanocytes were frequently juxtaposed each other. High‐resolution confocal microscopy also revealed that transplanted melanocytes exhibited normal behaviors regarding distribution patterns of melanocytes, dendrite morphology, and melanosome transfer. The method described in this study will serve as a useful tool to understand the mechanisms underlying intercellular regulations during skin pigmentation in vivo.     

Functional characterization of cultured keratinocytes after acute cutaneous burn injury

Background

In addition to forming the epithelial barrier against the outside environment keratinocytes are immunologically active cells. In the treatment of severely burned skin, cryoconserved keratinocyte allografts gain in importance. It has been proposed that these allografts accelerate wound healing also due to the expression of a favourable - keratinocyte-derived - cytokine and growth factor milieu.

Methods

In this study the morphology and cytokine expression profile of keratinocytes from skin after acute burn injury was compared to non-burned skin. Skin samples were obtained from patients after severe burn injury and healthy controls. Cells were cultured and secretion of selected inflammatory mediators was quantified using Bioplex Immunoassays. Immunohistochemistry was performed to analyse further functional and morphologic parameters.

Results

Histology revealed increased terminal differentiation of keratinocytes (CK10, CK11) in allografts from non-burned skin compared to a higher portion of proliferative cells (CK5, vimentin) in acute burn injury. Increased levels of IL-1α, IL-2, IL-4, IL-10, IFN-γ and TNFα could be detected in culture media of burn injury skin cultures. Both culture groups contained large amounts of IL-1RA. IL-6 and GM-CSF were increased during the first 15 days of culture of burned skin compared to control skin. Levels of VEGF, FGF-basic, TGF-ß und G-CSF were high in both but not significantly different. Cryoconservation led to a diminished mediator synthesis except for higher levels of intracellular IL-1α and IL-1ß.

Conclusion

Skin allografts from non-burned skin show a different secretion pattern of keratinocyte-derived cytokines and inflammatory mediators compared to keratinocytes after burn injury. As these secreted molecules exert auto- and paracrine effects and subsequently contribute to healing and barrier restoration after acute burn injury therapies affecting this specific cytokine/growth factor micromilieu could be beneficial in burned patients.     

Cell kinetic characterization of cultured human keratinocytes from normal and psoriatic individuals

Psoriasis is a chronic skin disease characterized by epidermal hyperproliferation, disturbed differentiation, and inflammation. It is still a matter of debate whether the pathogenesis of psoriasis is based on immunological mechanisms, on defective growth control mechanisms, or possibly on a combination of both. Several in vivo cell biological differences between psoriatic lesional epidermis and normal epidermis have been reported. However, it is not clear whether these changes are causal or consequential. In case that keratinocytes from psoriatic patients have genetically determined deficiencies or polymorphisms with respect to autocrine growth regulation and the response to inflammatory cytokines, we hypothesize that these differences should be maintained in culture. Here we have started a systematic comparison of first passage keratinocytes cultured from normal skin and uninvolved psoriatic skin to address the question whether there are intrinsic differences in basic cell cycle parameters. In an established, defined culture system using keratinocyte growth medium (KGM) we have determined: (i) cell cycle parameters of exponentially growing keratinocytes, (ii) induction of quiescence by transforming growth factor β₁ (TGF-β₁), and (iii) restimulation from the G₀-phase of the cell cycle. Bivariate analysis of Iodo-deoxyuridine incorporation and relative DNA content was performed by flow cytometry. Within the limitations of this model no gross differences were found between normal and psoriatic keratinocytes with respect to S-phase duration (T_s), total cell cycle duration (T_c), responsiveness to TGF-β₁ and the kinetics for recruitment from G₀. In psoriatic keratinocytes we found a lower amount of cells in S-phase and a shorter duration of G₁, compared to normal keratinocytes. The methodology developed here provides us with a model for further studies on differences between normal and psoriatic keratinocytes in their response to immunological and inflammatory mediators. © 1996 Wiley-Liss, Inc.     

Cell-cycle withdrawal in cultured keratinocytes

Cell-cycle withdrawal is the irreversible arrest of replication that occurs in keratinocytes early in terminal differentiation. According to the epidermal proliferation unit (EPU) model of renewal, withdrawal takes place in a subset of cells that have completed a final cycle of amplification replication. Using a recently developed double-labelling assay, we followed cell-cycle withdrawal in growing cultures of epidermal keratinocytes and correlated these results with population-growth kinetics. The levels of withdrawal measured were much too high to be consistent with the population-growth kinetics. These unexpectedly high levels could be explained by postulating that withdrawal takes place in a specific subset of cells, as described by the EPU model. Other possible explanations were entertained but, for various reasons, were considered unlikely. To learn whether withdrawal occurred in basal or suprabasal cells, confluent cultures were pulse labeled with 3H-thymidine, and the position of the labeled cells was monitored by autoradiography during the period of cold chase. The results indicated that, in cultured keratinocytes, withdrawal takes place while the cells are still in the basal compartment.