Control of growth and differentiation in vitro of human keratinocytes cultured in serum-free medium

Normal human proliferative keratinocytes can be serially cultured in serum-free medium. The medium and culture conditions that have been developed support the growth of these cells in the absence of a feeder layer and in the absence of contaminating fibroblasts. Using this system, the nutritional and hormonal factors that affect the growth and differentiation of these cells have been studied in the absence of undefined supplements. The data suggest that the control of proliferation and differentiation in keratinocytes may take place by unique mechanisms. The defined serum-free conditions that have been developed provide an excellent system for the study of nutritional disorders that affect human epidermal cells. In addition, the culture system can be used to propagate large numbers of cells for use in autografts for cases of severe burns.     

Glycosaminoglycan synthesis by human keratinocytes: cell growth and medium calcium effects

The influences of cell density, differentiation, and medium calcium levels on glycosaminoglycan biosynthesis were evaluated in cultured human epidermal keratinocytes. Following metabolic labeling with [35S]-sulfate and [3H]-glucosamine under steady state conditions in "high" medium calcium (greater than 1.0 mMol), the majority of sulfated glycosaminoglycans remained associated with the cell layers, whereas hyaluronic acid, which was present in smaller amounts than the sulfated products, was about equally distributed between the medium and the cell layers. Of the sulfated glycosaminoglycans, heparan sulfate and chondroitin 4/6-sulfate were the major species and were present in roughly comparable amounts, whereas dermatan sulfate was quantitatively the lesser of the products. The effects of "low" medium calcium (0.3 and 0.025 mM) were complex, although a consistent decrease in the incorporation of the [3H]-glucosamine precursor was found at high cell density, probably reflecting a decrease in its intracellular specific activity. In "high" calcium cultures, there was a strong inverse correlation (r = -0.92) between keratinocyte cell number and cellular production of sulfated glycosaminoglycans, whereas no such relationship was evident in cultures grown in "low" calcium medium at comparable cell density. Because keratinocyte differentiation is inhibited in the low calcium conditions, the results suggest that the decrease in production of sulfated glycosaminoglycans by confluent keratinocytes may actually correlate with differentiation rather than with cell number.     

Kinetics of withdrawal from the cell cycle in cultured human epidermal keratinocytes

In keratinizing epithelia one of the earliest changes in the process of terminal differentiation is cessation of replication or withdrawal from the cell cycle. In this report, we measured the loss of colony-forming ability, and confirmed that withdrawal from the cell cycle is a specific event that occurs during maturation of the keratinocyte in culture. In addition, the rate of withdrawal was assayed by labeling cultures for 24 h with [14C]dThd and then measuring the fraction of labeled cells that undergo repeated cycles of DNA synthesis. These additional cycles of replication were measured by feeding BrdUrd to the cultures and quantitating the distribution of 14C-labeled DNA in unsubstituted and BrdUrd-substituted DNA in CsCl density gradients. The results show that the fraction of 14C-labeled DNA undergoing replication decreases exponentially by 23% every 24 h. This cessation of replication could not be explained by a reduced level of replication in the entire culture since during each day of the experiment about 8% of the total DNA underwent replication. The exponential decrease in replication of 14C-labeled DNA represents withdrawal from the cell cycle. Since the doubling time for keratinocytes is approximately 24 h, these results suggest that following each cycle of replication, there is a probability of 0.23 that postreplicated cells will withdraw from the cell cycle.     

Supernatants from cultured human epidermal keratinocytes stimulate angiogenesis

Conditioned media from three different strains of human epidermal keratinocytes in culture were assayed on the chorioallantoic membrane of the chick embryo. Vascular responses were examined 4 days later stereo-microscopically and compared with controls—unconditioned medium and medium incubated with 3T3 cells. Specimens were also collected after 1,2,3 and 4 days for serial histological examination. Media from all three strains of keratinocytes stimulated statistically significant vascular growth relative to controls. Leukocytic infiltration was not demonstrated histologically at any time. In addition, quantitation of ectodermal epithelial hyperplasia in experimental groups did not reveal any significant difference when compared with controls. These results have demonstrated that epidermis may directly encourage vascular growth in the absence of significant inflammation.     

L-serine potentiates the mitogenic effects of growth factors on cultured human keratinocytes

L-Serine increased the growth of passaged human foreskin keratinocytes in terms of DNA and protein per dish of cells, and potentiated the mitogenic effects of serum and epidermal growth factor, and also insulin, keratinocyte growth factor, and a bovine pituitary extract. The effects of serine were apparent with as little as 0.05 mM. The stimulatory effects of these various growth factors were additive, without synergism. Exposure to L-[3H]serine resulted in labeling of the phospholipids phosphatidyl (Ptd) serine, Ptd ethanolamine, sphingomyelin, and, to a slight extent, Ptd choline. The time course of labeling suggested synthesis of Ptd serine was initiated; Ptd serine was converted to Ptd ethanolamine; little methylation of Ptd ethanolamine to form Ptd choline took place. Separation of nonradioactive phospholipids and phosphorus assay of individual lipids showed that serine-supplemented cells had only slightly increased content of Ptd serine and Ptd ethanolamine. The significance of these for activation of protein kinase C is discussed.     

Enzymatic properties of the 15-lipoxygenase of human cultured keratinocytes

The arachidonic acid 15-lipoxygenase or linoleic acid omega-6 lipoxygenase of human neonatal foreskin cultured keratinocytes converts arachidonic acid to 15-hydroxy-eicosatetraenoic acid and linoleic acid to 13-hydroxy-linoleic acid. A mean of 93% of the 15-lipoxygenase activity in sonicates of cultured keratinocytes was recovered in the 400,000 X g supernatant, attesting to the cytosolic localization of this enzyme. Optimal 15-lipoxygenase activity in the 400,000 X g supernatant was expressed at pH 6.7-7.3 and in the presence of calcium at a concentration of 2 mM or higher. Keratinocyte 15-lipoxygenase metabolized arachidonic acid (Km = 10.6 microM) and linoleic acid (Km = 9.5 microM) with similar efficiency. Nordihydroguaiaretic acid and 5,8,11,14-eicosatetraynoic acid both inhibited the conversion of arachidonic acid to 15-HETE with respective 50% inhibitory concentrations of 2.0 microM and 0.9 microM, while ATP, GTP, and cyclic AMP had no effect on activity at pH 6.8-7.2. The enzymatic properties of human keratinocyte 15-lipoxygenase thus resemble those of PMN leukocyte 15-lipoxygenase and the mediators generated may contribute to the regulation of cutaneous sensation and inflammation.     

Proliferating cell nuclear antigen/cyclin in cultured human keratinocytes

Expression of proliferating cell nuclear antigen (PCNA)/cyclin in cultured human keratinocytes was studied using an antibody from an SLE patient as the reagent. By indirect immunofluorescence staining, SV40-transformed human keratinocytes expressed PCNA/cyclin in 40-45% of the cells as a nulcear granular fluorescence. After synchronization of these cells, their nuclear distribution pattern during the S phase was sequential and showed a clear correlation with DNA synthesis. Primary cultured keratinocytes grown in high Ca+ medium expressed PCNA/cyclin in 10-15% of the cells with a similar staining pattern. These positively stained cells were confined to the basal and immediate suprabasal layers of the stratified culture sheet. The keratinocytes disaggregated by trypsin were separated according to cell size through a screen of Nitex monofilament cloth. The cells smaller than 15 microns in diameter synthesized abundant PCNA/cyclin, while the larger cells expressed very low levels. These results indicate that the expression of PCNA/cyclin correlates with DNA synthesis in cultured keratinocytes, but is not associated with their differentiation process.     

Insulin binding properties of normal and transformed human epidermal cultured keratinocytes

Insulin binding to its receptors was studied in cultured normal and transformed (A431 line) human epidermal keratinocytes. The specific binding was a temperature-dependent, saturable process. Normal keratinocytes possess a mean value of about 80,000 receptors per cell. Fifteen hours exposure of the cells to insulin (2 X 10(-7) M) lowered their receptor number (about 65% loss in available sites); these reappeared when the hormone was removed from the culture medium ("down-regulation" process). In the A431 epidermoid carcinoma cell line, there is a net decrease in insulin binding (84% of the initial bound/free hormone ratio in comparison with normal cells) essentially related to a loss in receptor affinity for insulin. Thus, cultured human keratinocytes which express insulin receptors may be a useful tool in understanding skin pathology related to insulin disorders.     

Reactivity of monoclonal anti-human skin basal cell antibody to cultured keratinocytes in serum-free medium

The reactivity of the monoclonal anti-human skin basal cell antibody (3B4-6) to cultured keratinocytes was examined by immunofluorescence techniques in order to verify the change in distribution of basal cell antigens during the in vitro differentiation of keratinocytes cultured in serum-free medium. The basal cells' antigen(s) determinants recognized by 3B4-6 were present in the cytoplasm of the cells at low concentration of Ca2+ (0.03 mM). High concentrations of Ca2+ (0.1 mM approximately 0.3 mM) induced the reactivity on the cell surface. This result was confirmed by flow cytometry analysis. The staining pattern was different from that of anti-involucrin antibody in 0.3 mM Ca2+ culture medium. This MoAb might be a useful tool for monitoring the degree of differentiation of cultured keratinocytes.     

Keratinocyte conditioned medium stimulates type IV collagenase synthesis in cultured human keratinocytes and fibroblasts

We have previously shown that conditioned medium from cultured human keratinocytes stimulates proliferation of a variety of cell types involved in wound healing. as well as re-epithelization of wounds in human skin in vitro. We now present evidence for an autocrine/paracrine control of the synthesis of type IV collagenases in human keratinocytes and fibroblasts. During wound healing, keratinocytes migrate over the wound bed, an activity coupled with lysis of basement membranes, and hence requiring the presence of collagenases. Collagenases are also needed for the production and remodelling of the granulation tissue. In order to study the autocrine/paracrine control of collagenase production in keratinocytes and fibroblasts, we stimulated these cells in culture with conditioned medium from cultured keratinocytes. Protease synthesis was determined by affinity labelling with ³H-diisopropylfluorophosphoridate (DFP) and by zymography. Keratinocyte-conditioned medium was found to increase the expression of 72 and 92 kDa type IV collagenase in human keratinocytes, and the 72 kDa collagenase in human fibroblasts. indicating that an autocrine/paracrine control mechanism is involved in collagenase production in these cell types during wound healing. This increased expression of collagenases could he partly responsible for the stimulated healing seen in wounds treated with sheets of cultured keratinocytes.     

Localized proliferative effect of preadipocytes on cultured human epidermal keratinocytes.

The effect of preadipocytes (ST 13) on cultured normal human epidermal keratinocytes (NHEK) was investigated. The growth of NHEK was accelerated with co-cultured ST 13 cells. This stimulative effect must have been localized around viable ST 13 cells, because neither the medium nor the surface conditioned by ST 13 or the ST 13 cell fragments showed any promotion of NHEK growth, and NHEK showed a compact, paving-like arrangement only when they were attached directly to ST 13 cells. It became clear that these compactly arranged keratinocytes have an active proliferative ability, since their nuclei showed a marked uptake of 5-bromodeoxyuridine (BrdU) and they were positively stained with anticytokeratin 37, a monoclonal antibody against the basal epidermis. Under electron microscopy, ST 13 preadipocytes were closely connected with NHEK. These results, together with those of previous reports, suggest that the localized proliferative effect of ST 13 cells on NHEK is due to cell-to-cell contact.     

体外培养人角质形成细胞周期与表皮生长因子受体表达检测

[摘 要] 目的 比较人角质形成细胞（KC）在角质形成细胞无血清培养基（K-SFM）和限制性角质形成细胞无血清培养基（DK-SFM）中的生长、增殖、传代和细胞性状变化的情况。方法 取健康男性包皮,将KC用K-SFM和DK-SFM培养,倒置显微镜下观察两组的细胞形态、数量、传代情况;用流式细胞仪检测细胞周期和细胞表皮生长因子受体（EGFR）的含量。结果 原代培养天12天,K-SFM培养基KC的细胞数量(304.70±83.08)明显多于DK-SFM(75.30±26.08),有统计学差异（P＜0.01）,K-SFM培养基KC细胞集落数(2.20±1.23)明显多于DK-SFM(0.00±0.00),有统计学差异（P＜0.01）。K-SFM组细胞可以传4代,DK-SFM组无法传代。K-SFM组G0-G1期细胞比例65.40%, DK-SFM组G0-G1期细胞比例84.10%;K-SFM组G2-M期细胞比例17.02%,DK-SFM组G2-M期细胞比例0.07%; K-SFM组S期细胞比例17.57%,D K-SFM组S期细胞比例组15.82%;K-SFM组细胞凋亡率1.04%,D-SFM组细胞凋亡率18.69%。K-SFM组细胞EGFR表达率为33.73%;DK-SFM组细胞EGFR表达率为3.19%。结论K-SFM培养基中KC细胞生长性状、增殖活性、细胞集落数、传代次数、EGFR表达量等指标均优于DK-SFM培养基。     

Interleukin-1 does not stimulate DNA synthesis of cultured human keratinocytes growth-arrested in growth-factor-depleted medium

Interleukin-1, a multifunctional cytokine, plays a central role in inflammatory processes. Several reports have appeared demonstrating that IL-1 stimulates growth of keratinocytes under certain experimental conditions, and we have shown previously that it can act as a strong stimulator of DNA synthesis in murine keratinocytes that have been growth-arrested by removal of growth factors (GF) from the medium for several days. Using the same assay system, we report here that, in contrast to cultured mouse keratinocytes, growth-arrested adult and newborn human keratinocytes do not respond to IL-1 with an increase in DNA synthesis. The experiments were performed with primary and secondary cells and cells propagated with and without feeder layer prior to the assay. A growth response to IL-1 in the absence of exogenous GF was only observed in non-growth-arrested human keratinocytes, i.e., when the cytokine was added for 24 to 48 h immediately after removal of other GF from the culture. Because keratinocytes continue to grow during this time anyway, although at a reduced rate, the additional growth observed with IL-1 could be explained as an enhancement of the growth-promoting effect by factors not previously completely removed from the culture. We would like to conclude from our results that, contrary to the findings in cultured mouse keratinocytes, IL-1 has no direct mitogenic effect on cultured human keratinocytes, but can still act as a growth promoter under certain conditions, apparently in concert with other GF.     

Dynamics of intracellular calcium in cultured human keratinocytes after localized cell damage

Recovery of cultured keratinocytes after scratch damage is considered to be a wound‐healing model. In this study, we observed changes in intracellular calcium concentration ([Ca²⁺]_i) in cultured human keratinocytes after scratch damage. Immediately after scratch damage, a wave of increased [Ca²⁺]_i radiated outward from the damaged area and then disappeared gradually. But, [Ca²⁺]_i remained elevated in a peripheral layer of cells around the damaged area for several minutes. This layer did not appear in calcium‐free medium. When the culture was switched to calcium‐free medium for 30 min immediately after scratch damage, then switched back to standard (Ca²⁺‐containing) medium, the recovery ratio after 24 h was approximately 25% lower than that of the culture in standard medium throughout. We speculate that delineation of damage sites by a layer of cells with increased [Ca²⁺]_i might be part of a signalling pathway that appropriately directs the wound‐healing process in epidermis.     

Fibroblast growth factor 10 induces proliferation and differentiation of human primary cultured keratinocytes

Fibroblast growth factor 10 is a novel member of the fibroblast growth factor family, which is involved in morphogenesis and epithelial proliferation. It is highly homologous to the keratinocyte growth factor (or fibroblast growth factor 7), a key mediator of keratinocyte growth and differentiation. Both fibroblast growth factor 10 and keratinocyte growth factor bind with high affinity to the tyrosine kinase keratinocyte growth factor receptor. Here we analyzed the effect of fibroblast growth factor 10 on primary cultures of human keratinocytes, grown in chemically defined medium, and we compared the proliferative and differentiative cell responses to fibroblast growth factor 10 with those induced by keratinocyte growth factor and epidermal growth factor. Cell counting, 5-bromo-2'-deoxyuridine incorporation, and western blot analysis showed that fibroblast growth factor 10, similarly to keratinocyte growth factor, not only is a potent mitogen for human keratinocytes, but also promotes the expression of both early differentiation markers K1 and K10 and late differentiation marker filaggrin in response to the Ca2+ signal, and seems to sustain the proliferative activity in suprabasal stratified cells. Immunoprecipitation/western blot analysis revealed that fibroblast growth factor 10, similarly to keratinocyte growth factor, is able to induce tyrosine phosphorylation of keratinocyte growth factor receptor and of cellular substrates such as PLCgamma.     

All-trans retinoic acid stimulates growth of adult human keratinocytes cultured in growth factor-deficient medium, inhibits production of thrombospondin and fibronectin, and reduces adhesion

Human epidermal keratinocytes were established in culture using a low-Ca2+ (0.15 mM), serum-free keratinocyte growth medium (KGM) as the culture medium. Early passage keratinocytes (i.e., between passages 3-8) were incubated for 1 or 2 d in KGM, in KGM supplemented with 1.4 mM Ca2+, or in growth factor-deprived keratinocyte basal medium (KBM). The cells were concomitantly treated with all-trans retinoic acid (0.1-2.5 micrograms/ml), and cell growth was quantitated at the end of the incubation period. The keratinocytes were simultaneously examined for adhesiveness and production of two extracellular matrix molecules, e.g., thrombospondin (TSP) and fibronectin (FN). Treatment with all-trans retinoic acid inhibited proliferation of keratinocytes that were rapidly growing in KGM. Proliferation was also inhibited in KGM supplemented with 1.4 mM Ca2+, but all-trans retinoic acid did not reverse the morphologic features associated with differentiation induced by high Ca2+. In contrast to these effects, all-trans retinoic acid treatment of keratinocytes in KBM, in which the cells were normally quiescent, stimulated growth. In the presence of optimal concentrations of all-trans retinoic acid (0.5 microgram/ml), the rate of keratinocyte proliferation in KBM was approximately 35% of the rate obtained in KGM (maximal proliferation rate). Keratinocyte adhesion (resistance to trypsin-mediated release from the substrate and attachment to the substrate) was inhibited by all-trans retinoic acid under all three conditions. In regard to extracellular matrix production, TSP production was inhibited by greater than 90% under all three conditions in the presence of all-trans retinoic acid. FN production was also inhibited but to a lesser degree. Concentrations of all-trans retinoic acid required to maximally inhibit keratinocyte adhesion and matrix production were higher (1.0-2.5 microgram/ml) than the concentration required to stimulate proliferation in KBM. These in vitro observations may have implications in the effects of retinoids on intact skin, including enhanced keratinocyte proliferation and thickening of the epidermis after topical application to photoaged skin and inhibition of proliferation and cell-cell cohesion after systemic administration in cases of psoriasis.     

他克莫司对人角质形成细胞增殖及干细胞因子分泌的影响

目的 探讨他克莫司对人角质形成细胞的增殖及干细胞因子（SCF）分泌的影响。方法 体外HaCaT细胞（人永生化角质形成细胞）培养,用MTT和ELISA分别测定他克莫司对HaCaT细胞增殖及SCF含量的影响。结果 103 nmol/L ~ 104 nmol/L的他克莫司对人HaCaT细胞增殖有抑制作用。10 nmol/L ~ 102 nmol/L的他克莫司可以促进HaCaT细胞SCF分泌量的增加;103 nmol/L的他克莫司对HaCaT细胞SCF的分泌无影响;高浓度104 nmol/L的他克莫司则可抑制HaCaT细胞SCF的分泌。结论 适当浓度的他克莫司能够促进角质形成细胞SCF的分泌。     

Nerve growth factor protects human keratinocytes from ultraviolet-B-induced apoptosis

Ultraviolet radiation is a potent inducer of apoptosis, whereas autocrine nerve growth factor protects human keratinocytes from programmed cell death. To evaluate the role of nerve growth factor in the mechanisms of ultraviolet B-induced apoptosis, cultured human keratinocytes were ultraviolet B irradiated following pretreatment with K252, a specific inhibitor of the tyrosine kinase high-affinity nerve growth factor receptor. Here we report that the addition of K252 significantly enhanced keratinocyte apoptosis. We then transfected normal human keratinocytes with pNUT-hNGF. Nerve growth factor overexpressing keratinocytes secreted the highest amounts of nerve growth factor in culture supernatants, were more viable, and had a higher rate of proliferation than mock-transfected cells. Whereas ultraviolet B radiation downregulated nerve growth factor mRNA and protein as well as the tyrosine kinase high-affinity nerve growth factor receptor in normal keratinocytes, it failed to do so in nerve growth factor-transfected cells. Moreover, nerve growth factor overexpressing keratinocytes were partially resistant to apoptosis induced by increasing doses of ultraviolet B at 24 and 48 h. These results indicate that downregulation of nerve growth factor function plays an important part in the mechanisms of ultraviolet B-induced apoptosis in human keratinocytes. In addition, ultraviolet B caused a decrease in BCL-2 and BCL-xL expression in mock-transfected keratinocytes, but not in nerve growth factor overexpressing cells. Finally, nerve growth factor prevented the cleavage of the enzyme poly(ADP-ribose) polymerase induced in human keratinocytes by ultraviolet B. These results are consistent with a model whereby the autocrine nerve growth factor protects human keratinocytes from ultraviolet B-induced apoptosis by maintaining constant levels of BCL-2 and BCL-xL, which in turn might block caspase activation.