Growth of melanocytes in human epidermal cell cultures.

Epidermal cell cultures were grown in keratinocyte-conditioned medium for use as burn wound grafts; the melanocyte composition of the grafts was studied under a variety of conditions. Melanocytes were identified by immunohistochemistry based on a monoclonal antibody (MEL-5) that has previously been shown to react specifically with melanocytes. During the first 7 days of growth in primary culture, the total number of melanocytes in the epidermal cultures decreased to 10% of the number present in normal skin. Beginning on day 2 of culture, bipolar melanocytes were present at a mean cell density of 116 +/- 2/mm2; the keratinocyte to melanocyte ratio was preserved during further primary culture and through three subpassages. Moreover, exposure of cultures to mild UVB irradiation stimulated the melanocytes to proliferate, suggesting that the melanocytes growing in culture maintained their responsiveness to external stimuli. When the sheets of cultured cells were enzymatically detached from the plastic culture flasks before grafting, melanocytes remained in the basal layer of cells as part of the graft applied to the patient.

Interactions of arterial cells: III. Stathmokinetic analyses of smooth muscle cells cocultured with endothelial cells.

Arterial endothelial cells (EC) or their conditioned medium (ECCM) can alter the proliferation of cocultured arterial smooth muscle cells (SMC). Previously, we have shown, as have others, that EC regulate the growth of cocultured SMC depending on the density of both cell types. To ascertain the rate of cell-cycle traverse in preconfluent arterial SMC cocultured with arterial EC or ECCM (derived from preconfluent EC), we have conducted a series of stathmokinetic experiments using flow cytometry to determine where specific changes may occur in the cell cycle. Results of our experiments indicate for the first time that ECCM stimulates the proliferation of preconfluent SMC by significantly shortening the residence times in the G1 and S phases of the cell cycle. The predominant relative effect occurs within the early G1 (G1A) compartment where pretreatment with ECCM shortens the residence time by approximately 55%. Furthermore, we have observed that preincubation of serum-free ECCM with antiplatelet-derived growth factor (PDGF) antibody abolishes any mitogenic effect on SMC. This suggests that EC secrete PDGF-like molecules which enhance the proliferation rate of preconfluent, cocultured SMC. These findings support the hypothesis that arterial EC may secrete mitogens which stimulate arterial SMC proliferation in the vascular wall.

Grafting of cultured allogeneic epidermis on second- and third-degree burn wounds on 26 patients.

Twenty-six individuals with second- and third-degree burn wounds have been grafted with cultured allogeneic epidermal cells. These epidermal cell grafts were grown in culture from cadaver skin according to a technique which we have developed. After being grafted with cultured allogeneic epidermal cells, superficial wounds, e.g., donor sites, healed within 7 days, compared to 14 days for mirror image control sites. Deep second-degree burn wounds which were excised before grafting with cultured cells healed in a mean time of 10 days. Deep second-degree burn wounds which were not excised before grafting healed in a mean time of 14 days. The cultured cells produced rapid healing in 11 of the 12 patients with deep second-degree burn wounds. The deep second-degree wounds grafted with cultured allogeneic epidermal cells healed with results which were comparable to the deep second-degree wounds which were autografted. Grafts of cultured allogeneic epidermal cells placed on full-thickness, or third-degree burn, wounds did not grow well.

Effect of age on the capacity of the bone marrow and the spleen cells to generate B lymphocytes.

The effect of age on the regeneration of the B cell population was studied by cell transfer methods, using the allotype-congenic mouse strains BALB/c (Igha) and C.B-17 (Ighb) as donors of old and young bone marrow (BM) and spleen cells, and C.AL-20 (Igho) as recipients. This design allowed us to identify the origin of the sIgD+ B cells present in the recipients. It was found that in a simple cell transfer, BM cells or spleen cells of aged donors could reconstitute the peripheral B cell population of irradiated, thymectomized recipients essentially as effectively as could BM or spleen cells from young donors. However, when BM cells from aged donors and from young donors were mixed and were used to reconstitute a single recipient, the cells from the aged donor were less efficient than were the cells from the young donor. We found that sIgD+ B cells of young donor origin predominated in the peripheral B cell population of the recipient at 3 to 6 wk after cell transfer. In the BM of the recipients, however, there was no difference in the incidence of sIgD+ B cells derived from the young and the old donors. When recipients were reconstituted with a mixture of spleen cells from old and young mice, the sIgD+ cells of young donor allotype showed a tendency to predominate in the peripheral B cell population, although this predominance was not statistically significant. Under such competitive conditions, the spleen cells of aged donors were less efficient than the BM of aged donors in reconstituting the sIgD+ B cell population of the recipient's BM, but were more efficient in reconstituting the splenic sIgD+ cells. Thus, a subtle defect in the B cell precursor population of the BM and the spleen of aged mice has been demonstrated. The role of T cells in the generation of sIgD+ cells was also analyzed.

Grafting of skin ulcers with cultured autologous epidermal cells.

We treated five adult individuals with six full-thickness chronic ulcerations in the skin caused by venous insufficiency, sickle cell anemia, or surgical wounds. Each patient received applications to the ulcerations of sheets of autologous epidermal cells grown in culture. All patients experienced relief of pain after grafting. Four of the six ulcers healed completely in 21 to 35 days, and three of the four remained healed for up to 2 years. One ulceration recurred within 2 months. Our experience suggests that cultured autologous epidermal grafts can provide continuous covering, relief from pain, and rapid healing of chronic debilitating ulcerations of the skin.

Human keratinocyte culture. Identification and staging of epidermal cell subpopulations.

Stratification of human epidermal cells into multilayered sheets composed of basal and suprabasal layers (resembling the stratum germinativum and stratum spinosum of the epidermis) was studied in a dermal component-free culture system. Although no stratum corneum developed in vitro, this culture system provided a method to study early events in human keratinocyte differentiation. Multiparameter flow cytometric analysis of acridine orange-stained epidermal cells from these cultures revealed three distinct subpopulations differing in cell size, RNA content, and cell cycle kinetics. The first subpopulation was composed of small basal keratinocytes with low RNA content and a long generation time. The second subpopulation consisted of larger keratinocytes, having higher RNA content and a significantly shorter generation time. Finally, the third subpopulation contained the largest cells, which did not divide, and represent the more terminally differentiated keratinocytes. This in vitro approach provides discriminating cytochemical parameters by which the maturity of the epidermal cell sheets can be assessed prior to grafting onto human burn patients.

Interaction of arterial cells. I. Endothelial cells alter cholesterol metabolism in co-cultured smooth muscle cells.

Results of previous in vivo experiments indicated that the presence of arterial endothelium modifies cholesteryl ester (CE) metabolism and the retention of low density lipoproteins (LDL) in injured arteries. We describe herein the effects of bovine arterial endothelial cells (ENDO) on the CE cycle, fluid phase endocytosis, and cell proliferation in co-cultured bovine arterial smooth muscle cells (SMC). Following several days of cultivation on confluent SMC, ENDO were removed from SMC by treatment of the co-cultures with 1.0% collagenase (type II). Removal of only ENDO from the co-culture dishes was confirmed by immunofluorescent staining for Factor VIII antigen, hemotoxylin-eosin staining, and biochemical analyses. We observed that ENDO grown to 75% confluency on confluent SMC induced: 1) a reduction of CE hydrolysis as a result of decreased lysosomal CE hydrolytic activity in SMC as compared to SMC cultured alone; and 2) an increase in the rate of incorporation of labeled oleate into CE as a result of increased acyl CoA:cholesterol O-acyltransferase activity in SMC as compared to SMC cultured alone. Neither endothelial cell-derived culture media (ECDM) nor fibroblasts modulated CE metabolism in co-cultured SMC. Additional experiments showed that the presence of endothelial cells or ECDM decreased the proliferation of co-cultured SMC by 50%, but enhanced the endocytotic rate of labeled sucrose into SMC threefold. Results of experiments described herein demonstrate that, in addition to providing a thrombo-resistant surface and regulating permeability, endothelial cells may also serve to modulate cholesteryl ester metabolism in smooth muscle cells derived from the arterial wall.

Tobacco constituents are mitogenic for arterial smooth-muscle cells.

Tobacco glycoprotein (TGP) purified from flue-cured tobacco leaves, tar-derived material (TAR), the water soluble, nondialyzable, delipidized extract of cigarette smoke condensate, rutin-bovine serum albumin conjugates, quercetin, and chlorogenic acid are mitogenic for bovine aortic smooth-muscle cells, but not adventitial fibroblasts. The mitogenicity appears to depend on polyphenol epitopes on carrier molecules. Ellagic acid, another plant polyphenol, inhibited arterial smooth-muscle proliferation. These results suggest that a number of ubiquitous, plant-derived substances may influence smooth-muscle cell proliferation in the arterial wall.

Loss of HLA-DR expression by human epidermal cells after growth in culture.

These studies present evidence that when human epidermal cells are grown in culture they lose both the ability to stimulate the proliferation of allogeneic T lymphocytes in vitro and their expression of HLA-DR antigens. Our results also show that epidermal cells incubated with anti-HLA-DR serum lose their ability to stimulate the proliferation to allogeneic T lymphocytes in a mixed skin cell-lymphocyte reaction.

Grafting of burn patients with allografts of cultured epidermal cells.

Epidermal cells from cadaver skin grown in culture into confluent sheets of stratified cells were grafted on to partial thickness burn wounds in three patients. The burn areas covered with these allogeneic cultured epidermal allografts were tangentially excised deep second-degree burns which routinely would have been covered with split-thickness autografts. The burn wounds grafted with cultured allografts healed within three days and remained healthy for the 9 months of observation. Since epidermal cell cultures may be grown continuously, cultured allografts may serve as alternative biological dressings, or grafts, for deep second-degree burn wounds. They produce accelerated healing and an excellent cosmetic result, and they reduce the need for split-thickness autografts.

Increased sensitivity of lymphocytes from people over 65 to cell cycle arrest and chromosomal damage.

Flow cytometry revealed that, in the presence of tritiated thymidine, a greater percentage of phytohemagglutinin-stimulated lymphocytes from old human donors were arrested in the G2 or M phase than were cells from young donors. Furthermore, lymphocytes from old donors showed significantly more chromosomal damage than did lymphocytes from young donors. Lymphocyte cultures from old or young donors not exposed to tritiated thymidine had the same percentage of cycling lymphocytes in G2 or M, although the number of lymphocytes stimulated by phytohemagglutinin to enter the cell cycle was significantly lower in cultures from old donors. Thus, the impaired incorporation of tritiated thymidine by phytohemagglutinin-exposed lymphocytes from old humans reflects both an impaired proliferative response to phytohemagglutinin and an increased sensitivity to the radiobiological effects of tritiated thymidine.

Prostacyclin modulates cholesteryl ester hydrolytic activity by its effect on cyclic adenosine monophosphate in rabbit aortic smooth muscle cells.

We tested the hypothesis that prostacyclin (PGI2), 6-keto-prostaglandinF1 alpha(6-keto-PGF1 alpha), and several E series prostaglandins (PG) may affect the activity of cholesteryl ester (CE) hydrolase since our previous experiments indicated that smooth muscle cells (SMC) in neointima of injured rabbit aorta (a) acquire the capacity to produce PGI2 and (b) have increased lysosomal CE hydrolytic (acid cholesteryl ester hydrolase [ACEH])activity. Using cultured SMC from rabbit thoracic aorta, we demonstrated that PGI2, 6-keto-PGF1 alpha, and 6-keto-PGE1 enhanced ACEH activity fourfold. No significant effects on ACEH activity were observed with PGE1 or PGE2. Preincubation of SMC with an inhibitor of adenylate cyclase activity (dideoxyadenosine) abolished the effect of these PG on CE hydrolytic activity. Addition of dibutyryl cAMP to these SMC significantly increased ACEH activity. Although concentrations of PGI2 used significantly increased cAMP levels, proliferation of these SMC was not observed. In related experiments, we determined if the addition of PGI2, 6-keto-PGF1 alpha, or 6-keto-PGE1 to cultured aortic SMC would enhance the egress of unesterified cholesterol and CE from these SMC. A significant loss of total cholesterol from PG-treated SMC was observed at the end of 14 d. Results suggest that increased synthesis of PGI2 by neointimal SMC in the injured aortic wall may, at least in part, explain the changes in CE catabolism and accumulation following injury. These PG may also be important in CE metabolism and accumulation in human arteries.

Immunologic studies of aging. V. Impaired proliferation of PHA responsive human lymphocytes in culture.

The impaired response to mitogens of lymphocytes from old persons has been shown to result from the presence of fewer responsive cells and the failure of these cells to divide normally after stimulation. We present additional evidence that the successive divisions of mitogen-responsive lymphocytes from old people are impaired. We labeled sister chromatids with BrdU and identified cells that were dividing for the first, second, or third time in culture. This analysis showed that lymphocyte cultures from old people contained only one-half the number of lymphocytes dividing for a second time and less than one-quarter the number of cells dividing for a third time compared with lymphocyte cultures from young people. It was shown that these changes reflected an absolute decrease in the number of second and third generation cells in cultures from old persons. No alterations in the cytokinetics of proliferation by lymphocytes from old people were found. These results provide additional evidence that mitogen-responsive lymphocytes from healthy elderly people do not undergo as many divisions in cultures with PHA as do lymphocytes from young persons.

Human epidermal cell cultures: growth and differentiation in the absence of differentiation in the absence of dermal components or medium supplements.

Human epidermal cells grew and differentiated in vitro, provided that the pH of the culture medium was at 5.6-5.8, the seeding density was optimal (approximately 2.5 x 10(5) cells per cm2), and the incubation temperature was maintained at 35-37 degrees C. Under these conditions, epidermal cells from many different skin locations grew to confluency within 15-20 days and formed multi-layered sheets whose differentiated structure resembled that of the full depth of skin epidermis. Cell proliferation and differentiation did not require a feeder layer, a collagen substrate, a high concentration of fetal bovine serum, or added hormones. The sheets of differentiated epidermal cells could be dissociated from the plastic surfaces of the tissue culture flasks. The use of such cultured cells for wound dressing is proposed.

A quantitative micro-complement fixation method in studies of human wart viruses.

This report offers a description of a quantitative micro-complement fixation method (Cikes, 1975) for detecting human wart virus antigens and their specific antibodies, and proof of the specificity of the reactions being detected. The increased sensitivity demonstrated by chromium-release measurement is compared to the results of visual interpretation of complement fixation.