HGH isoforms: cDNA expression, adipogenic activity and production in cell culture.

We have isolated, cloned and achieved functional expression of the cDNAs for both 22 kDa and 20 kDa human growth hormone (hGH) isoforms. A selective cDNA cloning strategy was used to preferentially and simultaneously obtain both hGH 22 kDa and hGH 20 kDa cDNAs. These were used to construct minigenes which were subcloned into two eukaryotic expression vectors and then introduced transiently in COS-7 cells and stably into CHO cells in culture. Transfection assays in COS-7 cells of both minigenes allowed the detection of the secreted hGH 22 kDa and hGH 20 kDa. These hGHs isoforms secreted into COS-7 medium were able to specifically promote differentiation of 3T3-F442A preadipocytes to adipose cells. Adipocyte differentiation was quantitated by Oil Red O triacylglycerol staining or glycerophosphate dehydrogenase activity. Furthermore, stable CHO cell lines have been derived that produce these hGH isoforms.

Cultivation of rabbit corneal epithelial cells in serum-free medium.

PURPOSE: To establish conditions for cultivation, serial growth, and normal differentiation of corneal epithelial cells in serum-free medium (SFM). METHODS: Rabbit corneal epithelial cells were co-cultured with lethally treated 3T3-cell feeder layers. Instead of serum, medium was supplemented with serum albumin, hormones, and other additives. Cell growth was quantitated spectrophotometrically with a new rhodamine-B staining protocol with a sensitivity range of 5 X 10(3) to 1 x 10(5) cells/cm2. Keratin expression was analyzed by immunostaining or sodium dodecyl sulfate-polyacrylamide gel electrophoresis of cell extracts. RESULTS: In SFM, without growth factors, cells grew no more than six to eight doublings, but when 10 ng/ml epidermal growth factor were added, serial transfer was possible, and epithelial cells grew to up to 18 to 20 doublings (three cell passages). Two cell colony types were seen: One type was composed of nonstratified proliferating cells, and the other of stratified cells expressing high levels of the differentiation-linked keratins K3 and K12. Confluent cultures formed a four- to five-layer stratified epithelium whose suprabasal cells were stained with anti-K12 antiserum. Acidic and basic fibroblast growth factors and epidermal growth factor reduced the expression of keratins K3 and K12. Transforming growth factor-alpha and epidermal growth factor led to the highest stimulation of cell proliferation. Limbal, peripheral, and central corneal epithelial cells showed similar clonal growth abilities, but colony size was larger for cells derived from limbal epithelium. CONCLUSIONS: These SFM conditions support the serial transfer, normal differentiation, and formation of typical corneal epithelium by cultured corneal epithelial cells and are useful in studying and assaying a variety of cytokines and compounds that modulate corneal epithelial cell proliferation and differentiation.

Intracytoplasmic triglyceride accumulation produced by dexamethasone in adult rat hepatocytes cultivated on 3T3 cells.

Glucocorticoids, such as hydrocortisone (HC) and dexamethasone (DEX), when administered to rats, induce lipid accumulation within hepatocytes (fatty liver). To investigate whether glucocorticoids can produce triglyceride (TG) accumulation as they do in vivo and the involved mechanisms, we have used primary cultures of rat hepatocytes which synthesized and secrete triglycerides into the culture medium. Hepatocytes cultivated on a feeder layer of lethally treated 3T3 cells were exposed for 2 weeks to micromolar concentrations of DEX. This glucocorticoid caused morphological alterations and cells accumulated lipid droplets in their cytoplasm; the TG content increased up to 6-fold in a concentration- and time-dependent manner. The removal of [14C]acetic or [14C]oleic acid from the culture medium was not altered in the cultures treated with 50 micrograms/ml DEX but the incorporation of [14C]acetic and [14C]oleic acid into TG in these cultures was about 13-fold and 60% higher than in non-treated cells, respectively. On the other hand, hepatocytes treated with 50 micrograms/ml DEX for 2 weeks showed a 16-fold decrease in TG release and 40% inhibition in protein export, whereas synthesis of total cellular proteins was not altered. Our results show that corticosteroids, such as DEX, caused lipid accumulation in liver cells through an increased synthesis and/or esterification of fatty acids, but mostly through a decrease in the secretion of TG.

Use of cultured human epidermal keratinocytes for allografting burns and conditions for temporary banking of the cultured allografts.

Five children who suffered burns clinically regarded as full skin thickness loss were grafted with cultured allogeneic skin from newborn prepuce. The wounds had remained open and infected without healing for about 20 days before the patients were received in the burn unit. To avoid losing surviving deep epidermal cells the wounds were débrided but not deeply excised and, a few days before allografting, they were washed with isodine solution and sterile water, and treated with silvadene cream application. All children received 76 cultured allografts of about 60 cm2 each. After allografting, the wounds were epithelized in 7-10 days and the allogeneic grafted skin began desquamation suggesting that the allograft did not 'take' permanently but was replaced by the newly formed skin. On the other hand, since allografting is an adequate therapy to provide early temporary coverage in extensively burned patients, we developed conditions for banking cultured skin to make it available for immediate use. The conditions described allow banking of the cultured grafts for 15-20 days with retention of clonal growth ability similar to that of unstored epithelia. The results show that cultured epidermal cells obtained from human newborn foreskin, when used as allografts for coverage of full skin or deep partial skin thickness burns, allow rapid epithelization of the burn wounds.

Combined use of allograft and autograft epidermal cultures in therapy of burns.

Cultivation of human epidermal keratinocytes made possible the use of cultured autografts as part of the therapy of extensively burned patients. On the basis of our early results using banked cultured allografts and autografts, we developed an integral and combined burn therapy comprising banked cultured allografts for rapid healing of skin donor sites and deep partial-thickness burns, conventional split-thickness skin autografting, and when needed, cultured autografts for full-thickness burns. We compared hospital stay in 32 burn patients treated with the combined therapy and in 39 who were not treated with cultured epidermis. Three groups of patients were defined: 15 to 29 percent (n = 12), 30 to 49 percent (n = 10), and more than 49 percent (n = 10) burned body surface area. We found a 20 to 29 percent decrease in hospital stay in patients with up to 49 percent burned body surface area and a 46 percent reduction in patients suffering more extensive burns. Survival rate of extensively burned patients also was increased. We took advantage of the availability of banked cultured allografts for ambulatory treatment, without hospitalization, of pediatric patients with 5 to 20 percent burned body surface area. We show for the first time the use and benefits of this combined therapy.

Controlled clinical study of skin donor sites and deep partial-thickness burns treated with cultured epidermal allografts.

Two clinical studies in donor sites and deep partial-thickness burns treated with banked cultured human epidermal allografts are described. Ten burn patients were subjected to donor split-thickness skin harvesting. The study was controlled, side-by-side comparative, blind, and randomized. Banked cultured epidermal allografts promoted a faster reepithelialization of the wounds; they epithelialized in an average of 6.9 days, whereas controls healed in an average of 11.1 days, giving a reduction of 37.8 percent in time to heal (p < 0.005). Allografted sites were less erythematous as compared with controls (p < 0.01), with more tendency to normopigmentation. In the deep partial-thickness burns study, 10 patients with 18 burned wounds were treated. Wounds treated with cultured allografts showed complete reepithelialization in about 3 to 6 days. The two clinical studies showed that banked cultured epidermal allograft promotes a significantly faster epithelialization of donor sites and deep partial-thickness wounds. These results support the idea that cultured allografts should be used routinely to improve treatment of burn patients and reduce their therapy time.

Controlled clinical study of deep partial-thickness burns treated with frozen cultured human allogeneic epidermal sheets.

Numerous studies, many uncontrolled, have suggested that the application of freshly prepared human allogeneic epidermal cultures promotes faster re-epithelialization of skin donor sites and deep partial-thickness burns. We describe the results of a study of deep partial-thickness burns treated with such cultures preserved in the frozen state. The study was controlled, side-by-side comparative, and randomized. Nine patients with deep partial-thickness burns and 2 patients with superficial partial-thickness burns were treated with the frozen cultures, with the use of adjacent wounds covered with petrolatum-coated gauze (Jelonet, Smith & Nephew Inc, Largo, Fla) as control wounds. The results showed that for the 2 superficial partial-thickness burns, the frozen cultures reduced healing time by 44%. For 5 of the patients with deep partial-thickness burns, the wounds treated with frozen cultures healed in a mean time of 5.6 days, whereas the control wounds healed in 12.2 days. More importantly, for the 4 other patients with deep partial-thickness burns, the wounds treated with the frozen cultures underwent complete re-epithelialization in a mean time of 4.2 days, but the control wounds were partially or mostly unhealed at up to 14 days. The results show that the frozen cultures not only accelerate the re-epithelialization of deep and superficial partial-thickness burns but also make it possible to heal such wounds that otherwise would not heal.

Differentiation of 3T3-F442A cells into adipocytes is inhibited by retinoic acid.

Retinoic acid at a concentration higher than 10(-6) M inhibited adipose conversion of 3T3-F442A cells in surface cultures. Since cells treated with 10(-5) M of the vitamin A analogue and the non-treated cells showed the same capacity to form colonies when seeded at low density, adipose conversion was not inhibited by a general toxic action of the retinoid. The inhibition produced was accompanied by changes in cell shape; adipose morphology was absent in retinoic acid treated cells, and they were more elongated and flattened without piling up. Adipose conversion of 3T3-F442A in Methocel suspended cultures was also inhibited by retinoic acid, suggesting the inhibition exerted by this compound was not related to the changes observed in cell shape. The inhibitory action of retinoic acid is manifested only when the cultures are treated before differentiation takes place; if the retinoid was added after some early events of adipose conversion, differentiation was not blocked. The inhibition was reversed when the drug was removed from the cultures and they were further incubated in adipogenic medium; incubation in medium without adipogenic factor did not permit reversion. On the other hand, vitamin A depleted serum showed a higher capacity to support adipose conversion than non-depleted serum. It is concluded that this vitamin A analogue blocks some early events before the cells undergo the commitment to differentiate into adipocytes.

Cultivation of adult rat hepatocytes on 3T3 cells: expression of various liver differentiated functions.

Adult rat hepatocytes were maintained in culture for at least 1 month without losing the expression of their differentiated functions; they were cultured on lethally treated 3T3 fibroblasts inoculated at 35,000 cells/cm2 with medium containing 10-25 micrograms/ml hydrocortisone. Hepatocytes showed their typical morphology; they formed bile canaliculi, microvilli, and intercellular junctions with desmosomes and nexus; some formed structures that may resemble the perisinusoidal space of Disse. In addition, they showed DNA synthesis and expressed some liver-specific functions. They synthesized albumin and other proteins, which were exported to the culture medium. Like parenchymal liver cells in vivo, de novo fatty acid synthesis and esterification took place, and more than 80% of the lipids synthesized by the hepatocytes were secreted into the medium as triglycerides; they also showed cytochrome-P450 activity that was inducible with phenobarbital, suggesting that the hepatocytes have the capacity to metabolize drugs. These culture conditions allow the study of various hepatocyte differentiated functions, and they may provide the means to analyze the effect on liver of hormones, viruses and hepatotoxic chemicals and drugs; they may also indicate conditions adequate for serial growth of hepatocytes.

Increasing activity of enzymes on pathway of triacylglycerol synthesis during adipose conversion of 3T3 cells.

When preadipose 3T3 cells pass from growing to resting state, they increase their rate of synthesis of triacylglycerol from precursors and convert to adipose cells. The behavior of two enzymes involved in the synthesis of triacylglycerol, glycerophosphate acyltransferase, and malic enzyme have been examined during the adipose conversion of 3T3-F442A in surface culture and in suspension culture stabilized with methylcellulose. Glycerophosphate acyltransferase activity rises sharply during the conversion and reaches a level of 80 times higher than that of another 3T3 subline in which practically no adipose conversion takes place (3T3-C2). The activity of malic enzyme also rises during the adipose conversion of 3T3-F442A and reaches a level of 15-fold higher than that of 3T3-C2. The activity of glycerophosphate acyltransferase responds more sharply than that of malic enzyme but the rise in activity is not sustained as long, so that the relative levels of the two enzymes change during the conversion. The adipose conversion appears to be the result of increases in the activity of the synthesizing enzymes, brought about by either of the two most physiological methods available for arresting the growth of cultured cells.

Adipose conversion of 3T3 cells depends on a serum factor.

The adipose conversion of 3T3-F442A cells depends on an adipogenic factor in serum. In the presence of this factor, cells grown to confluence become spherical, greatly increase the activity of their lipogenic enzymes, and accumulate triglyceride. In the absence of the adipogenic factor, the cells grow normally, but when they reach confluence they do not become spherical, do not accumulate triglyceride, and do not undergo any increase in activity of lipogenic enzymes. In cattle there is a great deal more of the adipogenic factor in the serum before birth than in the serum of grown animals. The nature of the adipogenic factor suggests that it may play an important role in the development of adipose tissue.

Suppression of the adipose conversion of 3T3 cells by acidified serum.

The adipose conversion of cultured 3T3-F442A cells in strongly inhibited if the fetal bovine serum of the culture medium is briefly acidified before it is used. The inhibitory factor is a polypeptide with an apparent molecular weight of 24,000, and is inactivated by pronase or trypsin. Cells grown to confluence in the presence of this factor do not become spherical or accumulate triglyceride; they also do not increase the activity of their triglyceride-synthesizing enzymes. The factor suppresses adipose conversion even in the presence of untreated serum. Once adipose conversion has begun in the absence of the inhibitory factor, subsequent addition of the factor does not arrest the conversion.

DNA synthesis and cell division related to adipose differentiation of 3T3 cells.

Adipose differentiation of 3T3-F442A cells in surface cultures depends on adipogenic factor present in the culture medium. We found that after stimulation with adipogenic serum, 3T3-F442A cell underwent a burst of DNA synthesis before adipose conversion was manifested by an augmented lipogenic enzyme activity. In differentiating cells, DNA synthesis, judged by a 100-fold higher rate of [3H]thymidine incorporation into TCA-insoluble material, was followed by a 100-fold increase in the activity of glycerophosphate acyltransferase. Cytosine arabinoside, added to the cultures at a concentration of 3 micrograms/ml, exerted 95% inhibition of [3H]thymidine incorporation and also inhibited adipose conversion. The burst of DNA synthesis correlated with a 2.5-fold increase in the amount of DNA and in the number of cells in the culture. The DNA content was the same in differentiated and nondifferentiated cells. We conclude that after the interaction with the adipogenic factor, the cells go through DNA synthesis and cell division essential for adipose conversion.

Thyroid hormone stimulates adipocyte differentiation of 3T3 cells.

Triiodothyronine added at 0.1 nM to 3T3-F442A cells cultured in adipogenic medium having endogenous hormone concentrations similar to those of hypothyroid serum stimulated adipose conversion; activities of both lipogenic enzymes, glycerophosphate dehydrogenase and malic enzyme, increased with hormone treatment. The number of adipocytes was also augmented by L-T3 addition but the number of fat cell clusters remained the same as compared to non-treated cultures, suggesting that thyroid hormone increased the number of adipocytes probably through stimulating selective multiplication of precursor adipose cells. Hormone addition to cells cultured with non-adipogenic medium did not promote conversion showing that L-T3 is not an adipogenic factor by itself. Triiodothyronine added at concentrations similar to those found in hyperthyroidism, from 10 nM up to 10 microM, also increased the proportion of adipocytes without changing the number of fat cell clusters, but they decreased the activity of both lipogenic enzymes and lipid accumulation in mature adipocytes. It can be concluded that during 3T3-F442A differentiation into adipocytes L-T3 increases the number of differentiated adipocytes and, at low concentrations, also enhances lipogenic enzyme activities, whereas at the hyperthyroid hormone levels these enzyme activities are significantly reduced, remaining at levels similar to those of cells cultured with hypothyroid medium. This cloned cell line seems to be a useful model to study thyroid hormone action at both molecular and cellular level.

Culture of proliferating and differentiating fat-storing cells in 3T3-conditioned medium.

There is growing evidence suggesting that hepatic fat-storing cells (FSC) or Ito cells have an important function in vitamin A storage and metabolism and in the synthesis of connective tissue components in normal liver and during fibrogenesis. The purified FSC acquire a fibroblastic morphology and their vitamin A content decreases in culture. We cultivated cells under in vitro conditions that allowed the expression of FSC morphological and functional characteristics for 3-4 weeks of primary culture. Cells were isolated from rat liver by the collagenase-perfusion method without further purification and cultured with 3T3-conditioned medium, which seemed to stimulate the selective proliferation of the FSC. After 8-10 days, round and stellate cells grew actively from a few precursor cells in the primary culture and were not subcultivated; the stellate cells had the ability to become round and vice versa and were highly motile. The cells had intracytoplasmic lipid droplets, a well developed rough endoplasmic reticulum, Golgi complex, numerous vesicles filled with electron-dense material, and extracellular matrix (ECM) components on their surface. Both stellate and round cells showed the presence of desmin by immunofluorescence and vitamin A autofluorescence, but lacked peroxidase activity. The culture conditions we describe allowed the selective proliferation of cells with morphological and functional characteristics of the FSC in the normal liver, raising the possibility of studying FSC proliferation and differentiation.

Extracellular matrix production by mouse 3T3-F442A cells during adipose differentiation in culture.

When cultured 3T3-F442A cells undergo adipose differentiation, they produce extracellular matrix (ECM) that is not present in undifferentiated cells. This ECM stains strongly with ruthenium red, tannic acid and with Alcian blue at both pH 1 and 2.5, showing histochemical characteristics similar to sulphated and non-sulphated glycosaminoglycans. Under the electron microscope, ECM was observed bound to the cell surface and in the intercellular space; it was composed of fibrils of several thicknesses with attached granules and fibrous long-spacing forms of collagen. In addition, adipocytes were observed as rounded cells interconnected with the ECM fibrils, thus giving rise to fat cell clusters similar to the adipocyte lobules found in adipose tissue. Since fat cell clusters in culture emerge by clonal expansion of one adipose precursor cell, we suggest that this ECM can keep daughter adipocytes interconnected during differentiation. ECM production by adipocytes might have some significance for the formation of fat cell lobules in vivo.

Frozen allogeneic human epidermal cultured sheets for the cure of complicated leg ulcers.

BACKGROUND: Skin ulcers due to venous stasis or diabetes are common among the elderly and are difficult to treat. Repeated applications of cell-based products have been reported to result in cure or improvement of leg ulcers of small size in a fraction of patients. OBJECTIVE: To examine the effects of frozen human allogeneic epidermal cultures for the treatment of acute and chronic ulcers. METHODS: We treated a series of 10 consecutive patients with leg ulcers of different etiology and duration with frozen human allogeneic epidermal cultures stored frozen and thawed for 5-10 minutes at room temperature before application. Three patients had ulcers with exposed Achilles or extensor tendon. The ulcers treated were as large as 160 cm2 in area and of up to 20-years' duration. After preliminary preparation of the wounds by debridement to remove necrotic tissue and application of silver sulfadiazine to control infection, thawed cultures were applied biweekly from 2 to 15 times depending on the size and complexity of the ulcer. RESULTS: All ulcers healed, including those with tendon exposure. After the first few applications, granulation tissue formed in the ulcer bed and on exposed tendons, and epidermal healing took place through proliferation and migration of cells from the margins of the wound. The time required for complete healing ranged from 1 to 31 weeks after the first application. CONCLUSION: The use of frozen human allogeneic epidermal cultures is a safe and effective treatment for venous or diabetic ulcers, even those with tendon exposure. It seems possible that any leg ulcer will be amenable to successful treatment by this method.