3T3 Feeder Cells Research Articles

The in vitro and in vivo proliferative capacity of serum-free cultivated human conjunctival epithelial cells

Purpose. To investigate the in vitro and in vivo proliferative capacity of human conjunctival epithelial cells cultured in serum-free media, and to compare this with current methods that utilize serum-containing media and 3T3 feeder layers. Methods. Human conjunctival epithelial cells were cultivated in serum-free media alone, serum-free media with a 3T3 feeder layer, and serum-containing media with a 3T3 feeder layer. The areas of outgrowth, colony-forming efficiencies and number of population doublings were compared. The in vivo proliferative potential was assessed by analyzing the number of cells generated by the implantation of cultured cells into athymic mice. Cultured cells were evaluated for the expression of cytokeratins K3, K4, K12, K19, as well as the gel-forming goblet cell mucin, MUC5AC. Results. Cells cultivated in serum-free media, serum-free media and feeder cells, and serum-containing media and feeder cells achieved colony-forming efficiencies of 14.5 ± 4.1%, 10.1 ± 3.1%, and 20.4 ± 6.7%, respectively, and number of population doublings of 24.8 ± 4.3, 14.8 ± 3.6, and 30.0 ± 5.0, respectively. Nine-day old athymic mice conjunctival cysts derived from serum-free cultures comprised 1.29 × 10 6 ± 0.46 × 10 6 cells, while cysts derived from serumcontaining cultures comprised 1.30 × 10 6 ± 0.53 × 10 6 cells. The degree of epithelial stratification was similar in both conditions. Serum-free cultivated conjunctival cells retained their in vivo characteristics and expressed K4, K19 and MUC5AC. The presence of MUC5AC mRNA in these cells was confirmed by RT-PCR. Conclusions. Conjunctival epithelial cells propagated in serum-free media demonstrated a similar in vivo proliferative capability, as compared to serum-containing media with 3T3 feeder cells. This has important clinical implications, as the serum-free ex vivo expansion of cells for clinical transplantation overcomes the problems associated with the use of animal serum and cells.

Senescence-associated genes in normal human oral keratinocytes

The current study was undertaken to identify senescence-associated (SA) genes in cultured normal human oral keratinocytes (NHOK). Primary NHOK were serially subcultured in vitro as dispersed cells in low (0.15 mM) Ca 2+ medium until senescence. The SA genes of NHOK were identified by comparing the expression levels of 3195 human genes between exponentially replicating and senescing cultures. Approximately 5% of the screened genes were upregulated in senescing NHOK by a factor greater than 3 compared with rapidly dividing NHOK culture. Among them, we identified discrete gene groups, i.e., cyclin-dependent kinase inhibitors, G-protein-coupled receptors, apolipoproteins, matrix metalloproteinases, and mitochondrial proteins. To validate the microarray results, we confirmed the enhanced expression of a few selected SA genes, i.e., gpr1, apo-D, apo-E, apo-L, mmp-1, mmp-3, cyb561, cyp1b1, and cyp4b1, by reverse transcription-PCR. These SA genes were upregulated in three independent cultures of NHOK at high population doubling (PD) levels compared with those of low PDs. The enhanced expression of these SA genes was also found in senescing NHOK maintained in 3T3 feeder cell system, as well as in the chemically defined medium containing low Ca 2+. These results indicate that the onset of senescence in NHOK is associated with altered expression of the SA genes, which represent discrete gene groups, independently of the donor variation or culture conditions.

Temporal in vitro expansion of the luminal lineage of human mammary epithelial cells achieved with the 3T3 feeder layer technique.

Human mammary epithelial cells from reduction mammoplasties were serially propagated in vitro from single cells and/or cell clusters using the NIH 3T3 cell feeder layer technique. In seven passages 46 cell population doublings, corrected for plating efficiency were achieved. The plating efficiency of epithelial cells in the primary culture was 0.2%. During subsequent passages it rose to 10-12% and decreased sharply towards the end of the culture life. In the third and fourth passages temporal prevalence of luminal cells was observed. The critical conditions for prevalence of the luminal phenotype were found to be the initial dissociation and optimum seeding density during subculturing. In primary cultures, after optimum dissociation of 0.15 cm3 mammary tissue with 0.05% collagenase A (Boehringher-Mannheim) in Eagle's MEM for 16 h at 37 degrees C, the yield on day 13 was 20 large colonies of 8-10 mm diameter. About 30% of the epithelial cells, which stained positively for the luminal cell marker cytokeratin 19, occupied colony centres. The remaining 70% were actin positive myoepithelial cells at the periphery. In subsequent passages, when using the optimum seeding density of 2 x 10(5) cells per 60 mm culture dish, the proportion of luminal cells gradually increased to 90% on day 35 in the fourth passage. A sudden rise in the proportion of rapidly growing myoepithelial cells to 65% was observed in the fifth passage. In the sixth and seventh passage small colonies were formed, most of which contained at least one keratin-19-positive (luminal) cell. Cells of human breast carcinomas are considered to be of luminal origin. Therefore, the described approach can be useful in studies of cell and molecular biology of mammary carcinomas.

A new system for the cultivation of keratinocytes on acellular human dermis with the use of fibrin glue and 3T3 feeder cells.

The growth of human keratinocytes on human acellular dermis in 4 different culture systems was compared. Epidermis was completely separated and removed from dermis after skin samples had been soaked in 0.1% trypsin at 4 degrees C for 1 week. Forty pieces of saline-washed dermis, 1 cm2 each, were randomized into 4 groups: in group A, human keratinocytes that had undergone 2 to 3 cell passages were seeded (30 x 10(4) cell/cm2) onto the dermis and sprayed with a thin layer of fibrin glue and proliferative 3T3 feeder cells that had been growing separately on the culture dish; in group B, the dermis was only sprayed with fibrin glue; in group C, the dermis was treated with 3T3 cells only; and in group D, the dermis was not sprayed with anything. The dermis samples in all groups were raised on a grid to provide an air-liquid culture system. Histology results of the composite grafts at 2 weeks were assessed as having either scanty colonies of keratinocytes (SCK), continuous stratified epithelium (CSE), or no observable keratinocyte growth. Eight out of the ten dermis samples (80%) in group A demonstrated CSE, and 30% of the samples in group B showed SCK. There were 10% SCK and 20% CSE in group C, and in group D, 30% SCK and 10% CSE were found. The good results in group A indicated that the fibrin glue facilitated the seeding efficiency of the keratinocytes on the dermis and that the vital factors released from the 3T3 feeder cells enhanced the growth and differentiation of the keratinocytes. This model provides an optimal system for the cultivation of keratinocytes on acellular dermis.

Conjunctival epithelial cells cultured on human amniotic membrane fail to transdifferentiate into corneal epithelial-type cells.

Previous studies on the use of human amniotic membrane (HAM) in rabbit stem cell deficiency models have found the new epithelium growing over the HAM to express cornea-specific keratins (K3 and K12) in 40% of the cases, suggesting that HAM may have induced conjunctival epithelial cells to transdifferentiate into cornea-type epithelial cells. The current study was performed to determine whether HAM could induce transdifferentiation of conjunctival epithelia] cells when cultured in vitro. Conjunctival grafts taken from the fornices of New Zealand white rabbits (6-12 weeks old) were placed over HAMs and lifted to an air-media interface using polypropylene double rings. These cultures were maintained in supplemented hormonal epithelial medium with and without 3T3 feeder cells. Rabbit corneal epithelial cells were cultured similarly using strips of keratolimbal grafts placed over HAM. The cultures were terminated at various times between the 8th and 15th day. The cultured epithelial cells were examined histologically and immunohistochemically using monoclonal antibodies AK-2 (to K12 keratin), AM-3 (to goblet cell mucin), and AE-5 (to K3 keratin). Both conjunctival and corneal epithelial cells cultured on HAMs showed multilayered, differentiated epithelial structures. On immunohistochemical examination, both epithelial cells stained positive for AE-5. None of the cultured conjunctival epithelial cells stained positively for AK-2, while the corneal epithelial cells showed positive staining with AK-2. There were no AM-3-positive goblet cells in either epithelial cell culture. There was no difference in the immunohistochemical patterns between cultures with or without 3T3 feeder cells. However, culture without feeder cells seemed to manifest a more degenerative appearance than those with feeders. HAM does not induce transdifferentiation of conjunctival epithelial cells into corneal-type epithelial cells under the in vitro culture conditions used in this study.

AUTOLOGOUS TRANSPLANTATION OF UROTHELIUM INTO DEMUCOSALIZED GASTROINTESTINAL SEGMENTS: EVIDENCE FOR EPITHELIALIZATION AND DIFFERENTIATION OF IN VITRO EXPANDED AND TRANSPLANTED UROTHELIAL CELLS

Purpose Our study established a technique for in vitro expansion and subsequent transplantation of autologous urothelial cells into vascularized seromuscular segments from stomach and colon in sheep. The proof of proliferation and differentiation of the transplanted urothelium in the absence of resident urothelium is considered to be a prerequisite for use of this technique in bladder augmentation. Materials and Methods Autologous sheep urothelial cells were expanded in vitro and grown on collagen membranes for sheet grafting. Using a vital stain, viability and confluency status of the urothelial graft were determined before transplantation into demucosalized segments isolated from the sheep stomach and colon gastrointestinal pouches. The gastrointestinal segments were sewn up and remained in the abdomen as small pouches stiched to the abdominal wall. Take and differentiation of transplanted cells within the pouch were assessed two and three weeks later using histological and immunohistological means. Results Urothelial cells grew well on collagen membranes. A confluency status > 40% and co-culturing with 3T3 feeder cells favored successful transplantation. Two weeks after transplantation a multilayered urothelial-like epithelium was found to line the lumen of the pouch. The epithelium was characterized by a distinct urothelium-typical distribution of basal and luminal keratins and the expression of the umbrella cell-specific marker uroplakin III. Moreover, the epithelium had an underlying basal lamina which focally contained collagen type IV. Conclusions The data indicate that in vitro expanded urothelial cells are capable of epithelializing demucosalized gastrointestinal segments forming a genuine, differentiated “neo” urothelium.

Establishment of a Tissue Culture System for Hamster Sebaceous Gland Cells

Background: A human sebaceous gland culture system is very useful in studying the functions of the sebaceous glands at the cellular level. On the other hand, the success rates for culture systems and the experimental results from these human culture systems exhibit significant fluctuations, depending on the condition of the donor skin. Objective: The purpose of this study was to establish a stable culture system in order to investigate the functions of the sebaceous glands under uniform conditions. Methods: Sebaceous glands from the auricles of 5-week-old hamsters were isolated and seeded onto a 3T3 cell feeder layer. The proliferation, lipid production and response to androgens of these sebaceous-gland-derived cells (SGDC) were then examined. Results: The SGDC showed outgrowth, formed colonies and became confluent in a 35-mm dish culture after 14 days in primary culture. The amount of intracellular lipids significantly increased following the peak of the cell proliferation. The composition of the intracellular lipids from the hamster SGDC was identical to human SGDC, except that the hamster SGDC did not contain any squalene. Testosterone and 5α-dihydrotestosterone markedly stimulated the proliferation of these SGDC in a dose-dependent manner. Conclusion: This tissue culture system will be a useful tool for the study of sebaceous gland function at the cellular level.

Regulation of K3 keratin gene transcription by Sp1 and AP-2 in differentiating rabbit corneal epithelial cells.

Rabbit corneal epithelial cells cultured in the presence of 3T3 feeder cells undergo biochemical differentiation, as evidenced by their initial expression of K5 and K14 keratins characteristic of basal keratinocytes, followed by the subsequent expression of K3 and K12 keratin markers of corneal epithelial differentiation. Previous data established that mutations of an Sp1 site in a DNA element, E, that contains overlapping Sp1 and AP-2 motifs reduce K3 gene promoter activity by 70% in transfection assays. We show here that Sp1 activates while AP-2 represses the K3 promoter. Although undifferentiated corneal epithelial basal cells express equal amounts of Sp1 and AP-2 DNA-binding activities, the differentiated cells down-regulate their Sp1 activity slightly but their AP-2 activity drastically, thus resulting in a six- to sevenfold increase in the Sp1/AP-2 ratio. This change coincides with the activation and suppression of the differentiation-related K3 gene and the basal cell-related K14 keratin gene, respectively. In addition, we show that polyamines, which are present in a high concentration in proliferating basal keratinocytes, can inhibit the binding of Sp1 to its cognate binding motif but not that of AP-2. These results suggest that the relatively low Sp1/AP-2 ratio as well as the polyamine-mediated inhibition of Sp1 binding to the E motif may account, in part, for the suppression of the K3 gene in corneal epithelial basal cells, while the elevated Sp1/AP-2 ratio may be involved in activating the K3 gene in differentiated corneal epithelial cells. Coupled with the previous demonstration that AP-2 activates the K14 gene in basal cells, the switch of the Sp1/AP-2 ratio during corneal epithelial differentiation may play a role in the reciprocal expression of the K3 and K14 genes in the basal and suprabasal cell layers.

Immortalization and characterization of proximal tubule cells derived from kidneys of spontaneously hypertensive and normotensive rats

Epithelial cell lines from the proximal tubule of SHR and WKY rats were generated by microdissection, cell growth on 3T3 cell feeder layers, and transduction of the SV40 large T-antigen gene. The cell lines that formed confluent, electrically-resistive monolayers (basal conductance 1 to 20 mS/cm2) were selected for further study. Of these, cell lines generated from one rat did not show evidence of T-antigen expression or integration, and apparently immortalized spontaneously. Cell lines from three other rats expressed high levels of T-antigen, and showed evidence of integration of one or more copies of T-antigen. All cell lines formed polarized monolayers with apical microvilli, tight junctional complexes, and convolutions of the basolateral plasma membrane. Most cell lines grew in the absence of extracellular glucose indicating a capacity for gluconeogenesis. Sodium succinate cotransport and P2-purinergic receptor mediated signaling were demonstrated in all lines tested. The cell lines also showed that Na/H exchanger activity is regulated by angiotensin II. The results indicate that these cell lines express a proximal tubular phenotype, and are morphologically and functionally similar to primary cultures. These rat cell lines represent a new, potentially useful cell model for elucidating the cellular and molecular mechanisms of genetic differences in proximal tubule Na+ reabsorption.

Formation of asymmetric unit membrane during urothelial differentiation.

Mammalian urothelium undergoes unique membrane specialization during terminal differentiation making numerous rigid-looking membrane plaques (0.3-0.5 micron diameter) that cover the apical cell surface. The outer leaflet of these membrane plaques is almost twice as thick as the inner leaflet hence the name asymmetric unit membrane (AUM). Ultrastructural studies established that the outer leaflet of AUM is composed of 16 nm particles forming two dimensional crystals, and that each particle forms a 'twisted ribbon' structure. We showed recently that highly purified bovine AUMs contain four major integral membrane proteins: uroplakins Ia (27 kD), Ib (28 kD), II (15 kD) and III (47 kD). Studies of the protease sensitivity of the different subdomains of uroplakins and other considerations suggest that UPIa and UPIb have 4 transmembrane domains, while UPII and UPIII have only one transmembrane domain. Chemical crosslinking studies showed that UPIa and UPIb, which share 39% amino acid sequence, are topologically adjacent to UPII and UPIII, respectively, thus raising the possibility that there exist two biochemically distinct AUM particles, i.e., those containing UPIa/UPII vs. UPIb/UPIII. Bovine urothelial cells grown in the presence of 3T3 feeder cells undergo clonal growth forming stratified colonies capable of synthesizing and processing all known uroplakins. Transgenic mouse studies showed that a 3.6 kb 5'-flanking sequence of mouse uroplakin II gene can drive the expression of bacterial LacZ gene to express in the urothelium. Further studies on the biosynthesis, assembly and targeting of uroplakins will offer unique opportunities for better understanding the structure and function of AUM as well as the biology of mammalian urothelium.

Epidermis as a Secretory Tissue: An In Vitro Tissue Model to Study Keratinocyte Secretion

In addition to protective functions, keratinocytes secrete a variety of effector molecules that may have local or distant effects. To explore the secretory activity of keratinocytes we have developed a two-chamber culture model in which a fully differentiated stratified epithelium is present in the upper chamber and secreted protein is collected in a lower chamber. A collagen matrix is not used and during collection of secreted protein, 3T3 feeder cells are absent. Keratinocytes secrete protein at the rate of 0.67 microgram/h/10(6) cells, encompassing a molecular weight range from several kD to over 180 kD. Secretion is an unexplored area of keratinocyte biology and this model will allow investigation of this activity.

Upper Human Hair Follicle Contains a Subpopulation of Keratinocytes with Superior In Vitro Proliferative Potential

We and others have shown previously that corneal keratinocyte stem cells can proliferate in vitro better than their progeny cells. In this paper, we applied this approach to the identification of hair follicular stem cells. When human scalp hair follicles were placed in explant culture, the bulge area yielded best outgrowths. In another experiment, we isolated different subpopulations of human follicular keratinocytes by micro-dissection, dispersed them by trypsin/EDTA into single cells, and grew them in the presence of 3T3 feeder cells. The keratinocytes were then subcultured under identical conditions to compare their in vitro life span. Our results indicate that the life span of keratinocytes of the upper follicle (containing mainly the isthmus area) > sebaceous gland > lower follicle (between the bulge and bulb) > bulb (containing the matrix cells). The cultured upper follicular keratinocytes tend to be small and relatively uniform in size. The poor in vitro growth of matrix cells may reflect their non-stem cell nature and/or special growth requirement(s) satisfied in vivo by the neighboring dermal papilla cells. Unexpectedly, we found that the upper follicular keratinocytes grow even better than epidermal keratinocytes. The existence of a subpopulation of keratinocytes with an in vitro growth potential superior than other known keratinocytes of the skin supports the hypothesis that follicular stem cells reside in the upper follicle. Our data also raise the possibility that putative follicular stem cells are involved not only in forming the follicle, but also in the long-term maintenance of the epidermis. Finally, we discuss the possibility that keratinocyte stem cells, as defined by their in vivo slow-cycling nature, are absent in culture.

Culture of hair matrix and follicular keratinocytes

In recent years, a variety of in vitro models for the cultivation of hair follicles and their constituents have been developed. Outer root sheath (ORS) keratinocytes (KC) have been mainly studied in explant cultures, planted on bovine eye lens capsules, collagen substrata, 3T3 cell feeder layers, or dermal equivalents, yielding outgrowth of a multilayered stratified epithelium with some biochemical and ultrastructural characteristics of keratinocytic differentiation. More recently, ORS KC cultures have also been initiated from single cell suspensions, and organotypic cultures have been obtained by recombination with dermal cells, inducing a higher degree of epidermal differentiation. Presumptive human hair matrix cells have been isolated from plucked anagen hair follicles and have been successfully propagated on 3T3 cell or normal human fibroblast feeder layers, giving rise to multilayered stratified KC cultures. In contrast, only preliminary data exist concerning the cultivation of bulge cells that have been suggested to represent follicular stem cells. In conclusion we dispose of several in vitro models today to cultivate ORS KC and hair matrix cells that have increased our knowledge on the regulation of the human hair cycle by soluble factors and dermal-epidermal interactions. Further comparative studies on ORS KC, bulge cells and matrix cells have to be carried out to confirm the distinct character of these hair KC subsets.

Culture of Hair Matrix and Follicular Keratinocytes.

In recent years, a variety of in vitro models for the cultivation of hair follicles and their constituents have been developed. Outer root sheath (ORS) keratinocytes (KC) have been mainly studied in explant cultures, planted on bovine eye lens capsules, collagen substrata, 3T3 cell feeder layers, or dermal equivalents, yielding outgrowth of a multilayered stratified epithelium with some biochemical and ultrastructural characteristics of keratinocytic differentiation. More recently, ORS KC cultures have also been initiated from single cell suspensions, and organotypic cultures have been obtained by recombination with dermal cells, inducing a higher degree of epidermal differentiation. Presumptive human hair matrix cells have been isolated from plucked anagen hair follicles and have been successfully propagated on 3T3 cell or normal human fibroblast feeder layers, giving rise to multilayered stratified KC cultures. In contrast, only preliminary data exist concerning the cultivation of bulge cells that have been suggested to represent follicular stem cells. In conclusion we dispose of several in vitro models today to cultivate ORS KC and hair matrix cells that have increased our knowledge on the regulation of the human hair cycle by soluble factors and dermal-epidermal interactions. Further comparative studies on ORS KC, bulge cells and matrix cells have to be carried out to confirm the distinct character of these hair KC subsets.

DOK, a cell line established from human dysplastic oral mucosa, shows a partially transformed non-malignant phenotype.

There are many reports of cell lines being established from human oral squamous-cell carcinomas but apparently none of cell lines from dysplastic or "pre-malignant" oral mucosa. We describe here the isolation and characterization of a cell line, DOK (dysplastic oral keratinocyte), from a piece of dorsal tongue showing epithelial dysplasia. The tissue was obtained from a 57-year-old man who was a heavy smoker prior to the appearance of a white patch on his tongue. Eleven years later a squamous-cell carcinoma developed at the site and was excised. Subsequently the remaining dysplasia was removed, and it was from a piece of this that the primary cell cultures which eventually gave rise to DOK were initiated. The DOK line has been single-cell cloned and is apparently immortal. It grows in the absence of 3T3 feeder cells, is anchorage-dependent for growth and is non-tumorigenic in nude mice. The keratin profile of the cells shows a striking similarity to that of the original tongue dysplasia. The karyotype of DOK is aneuploid and complex. By PCR and oligonucleotide hybridization on dot blots, codons 12, 13 and 61 of Ha-ras, Ki-ras and N-ras in DNA extracted from DOK cells were shown to be normal. Immunohistochemistry showed no abnormal, i.e., elevated expression of the onco-suppressor protein p53. Because of its origin and partially transformed phenotype, DOK presents an opportunity to study whether specific carcinogens associated with tobacco and areca nut can cause malignant transformation of oral keratinocytes in vitro.

Radiosensitivity measurement of keratinocytes and fibroblasts from radiotherapy patients

Genetic diversity is believed to influence cellular radiosensitivity and individual variability in normal tissue reactions to radiotherapy. To measure normal cell radiosensitivity in vitro, we investigated a culture technique that yields keratinocyte and fibroblast cell cultures from small skin biopsy samples (average weight 32 mg). This technique uses 3T3 NIH cells as feeder cells, culture medium containing dialyzed fetal calf serum, low calcium, and various growth factors for keratinocyte growth. A calcium concentration of 4 × 10 −3 M and the use of lethally irradiated NIH 3T3 feeder cells were critical to the success of this method. Primary keratinocyte cultures were successfully obtained from nine biopsy specimens, and radiosensitivity measurements were obtained in six of the resulting strains. Keratinocytes were, in general, more radioresistant than fibroblasts derived from the same specimen. We conclude that radiosensitivity assessment of keratinocyte and fibroblast cultures derived from small punch biopsy specimens is feasible. Further studies can now be carried out to determine the degree of variability between individuals and the relationship between in vitro keratinocyte and fibroblast radiosensitivity and their value in predicting normal tissue responses to radiotherapy.

Assessing the differentiation state of cultured bovine urothelial cells: elevated synthesis of stratification-related K5 and K6 keratins and persistent expression of uroplakin I

Although significant progress has recently been made in culturing mammalian urothelial cells, relatively little is known about their biochemical differentiation. In this paper, we assessed the differentiation state of cultured bovine urothelial cells by analyzing their keratins and a cell surface marker, uroplakin I. Urothelial cells were serially cultured either in a serum-free medium, or in a serum-containing medium in the presence of 3T3 feeder cells, with similar results. Despite their stratified appearance, both normal urothelium and cultured urothelial cells synthesize mainly K8, K18 and K19, keratins that are typically seen in simple epithelia. However, cultured urothelial cells synthesize a greatly increased amount of K5 and K6 keratins, which are usually expressed by stratified epithelia but present only in trace amounts in normal urothelium. These data indicate that, as far as keratin synthesis is concerned, cultured urothelial cells undergo an altered pattern of differentiation towards a more 'stratified phenotype'; this unusual finding has interesting implications for urothelial evolution. In the meantime, many superficial cells in cultured urothelial colonies make uroplakin I, a 27 x 10(3) Mr protein subunit of the asymmetrical unit membrane (AUM) characteristic of urothelial (superficial) umbrella cells. These results indicate that cultured urothelial cells undergo, at least in part, AUM biogenesis. Cultured urothelial cells thus provide a useful experimental model system for studying certain early steps of AUM formation.

Initial hyperproliferation and incomplete terminal differentiation of cultured human keratinocytes from lesional and uninvolved psoriatic skin

Human epidermal keratinocytes (KCs) were isolated from lesional and from uninvolved skin of 8 patients with chronic plaque-like psoriasis and from the normal skin of 8 healthy volunteers. Primary KC cultures, grown on 3T3 cell feeder layers, were examined over a period of 4 weeks and their plating efficiency, colony growth area, DNA synthesis and ultrastructural cell differentiation were evaluated. Psoriatic KCs formed colonies one day earlier than non-psoriatic controls and proliferated faster during the first 2 weeks, as assessed by the mean colony growth area and 3H-thymidine incorporation. After 4 weeks, however, no significant differences were observed between the in vitro proliferation parameters of normal and psoriatic KCs. At the ultrastructural level, cultures of lesional psoriatic KCs consisted of more cell layers with adherent transitional cells and incomplete formation of cornified envelopes, even after 4 weeks, while KCs from uninvolved psoriatic skin were characterized by a transient delay of in vitro maturation. These results indicate that the characteristic hyperproliferation of psoriatic KCs may only be maintained over a short period of primary culture, whereas defective terminal differentiation of lesional psoriatic KCs was maintained throughout the culture period.

Macrophage Lineage Switching of Murine Early Pre-B Lymphoid Cells Expressing Transduced fms Genes

fms genes encoding either wild-type or constitutively activated colony-stimulating factor 1 receptors (CSF-1R) were introduced by retroviral infection into long-term mouse lymphoid cultures. Four early pre-B-cell lines transformed by the feline v-fms oncogene underwent spontaneous and irreversible differentiation to macrophages when transferred from RPMI 1640 to Iscove modified Dulbecco medium. Expression of wild-type human CSF-1R in early pre-B cells conferred no proliferative advantage unless human CSF-1 was added to the culture medium. A clonal, factor-dependent early pre-B-cell line (D1F9), selected for continuous growth on NIH 3T3 cell feeder layers producing human CSF-1, could be maintained in RPMI 1640 medium containing interleukin-7 (IL-7) but also differentiated to macrophages when grown in Iscove modified Dulbecco medium containing human CSF-1. The macrophages retained parental immunoglobulin gene rearrangements and proviral insertions, lost B-cell antigens, expressed butyrate esterase and MAC-1, were actively phagocytic, and no longer survived in IL-7. Unlike factor-independent v-fms transformants, the irreversible commitment of D1F9 cells to differentiate in the macrophage lineage could be suppressed by IL-7, depended on human (but not mouse) CSF-1, and was inhibited by an antibody to human CSF-1R. Signals mediated by transduced CSF-1R can therefore play a deterministic role in cell differentiation.

Macrophage lineage switching of murine early pre-B lymphoid cells expressing transduced fms genes.

fms genes encoding either wild-type or constitutively activated colony-stimulating factor 1 receptors (CSF-1R) were introduced by retroviral infection into long-term mouse lymphoid cultures. Four early pre-B-cell lines transformed by the feline v-fms oncogene underwent spontaneous and irreversible differentiation to macrophages when transferred from RPMI 1640 to Iscove modified Dulbecco medium. Expression of wild-type human CSF-1R in early pre-B cells conferred no proliferative advantage unless human CSF-1 was added to the culture medium. A clonal, factor-dependent early pre-B-cell line (D1F9), selected for continuous growth on NIH 3T3 cell feeder layers producing human CSF-1, could be maintained in RPMI 1640 medium containing interleukin-7 (IL-7) but also differentiated to macrophages when grown in Iscove modified Dulbecco medium containing human CSF-1. The macrophages retained parental immunoglobulin gene rearrangements and proviral insertions, lost B-cell antigens, expressed butyrate esterase and MAC-1, were actively phagocytic, and no longer survived in IL-7. Unlike factor-independent v-fms transformants, the irreversible commitment of D1F9 cells to differentiate in the macrophage lineage could be suppressed by IL-7, depended on human (but not mouse) CSF-1, and was inhibited by an antibody to human CSF-1R. Signals mediated by transduced CSF-1R can therefore play a deterministic role in cell differentiation.