Biología Celular

Although tight junctions (TJ) have been extensively studied in simple epithelial cells, it is still unknown whether their organization is coupled to cell differentiation in stratified epithelia. We studied the expression of TJ in RCE1(5T5) cells, an in vitro model which mimics the sequential steps of rabbit corneal epithelial differentiation. RCE1(5T5) cells expressed TJ components which were assembled once cells constituted differentiated epithelia, as suggested by the increase of transepithelial electrical resistance (TER) which followed a similar kinetic to the expression of the early differentiation marker Pax-6. TJ were functional as indicated by the establishment of an epithelial barrier nonpermeable to ruthenium red or a biotin tracer. In immunostaining experiments, TJ were located at the superficial cells from the suprabasal layers; Western blot and RT-PCR suggested that TJ were composed of claudins (cldn)-1,-2,-4, cingulin (cgn), occludin (ocln) and ZO-1. Semi-quantitative RT-PCR and TER measurements showed that TJ became organized when cells began to form a 3-5 layers stratified epithelium; TER increased once cells reached confluence, with a time course comparable to the raise in the expression of cgn, cldn-2 and-4. Nevertheless, cldn-1,-2, ZO-1 and ocln were present in the cells from the beginning of cultivation, suggesting that TER increases mainly depend on TJ assembly. While EGF increased epithelial barrier strength, retinoic acid disrupted it, increasing paracellular flux about 2-fold; this effect was concentration dependent and completely reversible. Our results suggest that TJ assembly is tightly linked to the expression of corneal epithelial terminal phenotype.

A method is described for assembling an evaporation manifold from a cell culture flask, which allows for efficient nitrogen evaporation of hexane from 24- and 96-well plates. The precursor parts are readily available in most research laboratories. The nitrogen evaporation manifold is inexpensive, could possibly be used with other organic solvents, and appears ideal for a small number of samples in a multi-well format.

Understanding of visual system function and the development of new therapies for corneal diseases and damages depend upon comprehension of the biological roles of the tissue. The in vitro cultivation of corneal epithelial cells and cell lines derived from them has become a powerful tool to analyze and understand such issues. Currently, researchers have developed well-defined and precisely described culture protocols and a collection of corneal epithelial cell lines. These cell lines have been obtained through different experimental approaches: (1) the ectopic expression of oncogenes, (2) the inactivation of p16 and p53 pathways and hTERT expression, and (3) the spontaneous establishment after serial cultivation of cells. The advantages or disadvantages for these approaches are discussed. In conclusion, the availability of several culture protocols and immortalized cell lines that express corneal epithelial phenotype will be useful for investigating issues such as gene regulation and tissue development, or for validating alternative methods in toxicology.

Aims: A major objective in the treatment of obese individuals is the development of agents that reduce body fat and improve metabolic homeostasis. Among a variety of biological effects, green tea exerts an anti-obesity effect; however, the mechanisms behind its activity on adipose tissue are uncertain. Tea contains high levels of (−)-epigallocatechin-3-gallate (EGCG), which is one of its main bioactive substances. Therefore, we studied the effects of EGCG on mouse embryonic fibroblast-adipose like cell line (3T3-L1) preadipocyte proliferation and differentiation. Main methods: 3T3-L1 cells were incubated with physiologically attainable (0.1-1 μM) and pharmacological (5 μM, 10 μM) concentrations of EGCG for various lengths of time. Cell proliferation was assessed by cell counting and cell cycle analysis. Adipose conversion was evaluated by lipid accumulation and expression of CCAAT/enhancer binding protein alpha (C/EBPα) and peroxisome proliferator-activated receptor gamma (PPARγ) transcription factors. Key findings: A dose dependent suppressive effect on preadipocyte proliferation was observed, with the highest reduction in cell number at 10 μM EGCG. On the other hand, adipose conversion was fully inhibited with 10 μM EGCG. Flow-cytometric analysis showed that 3T3-L1 cells treated with EGCG underwent an arrest of cell cycle at G2/M. The inhibition of the expression of C/EBPα and PPARγ was accompanied by the inhibitory effect of EGCG. Microscopic observation showed that 3T3-L1 cells treated with EGCG maintained the fibroblastic shape and failed to accumulate cytoplasmic fat droplets even after the induction of differentiation. Significance: Our results suggest that EGCG reduces adipogenesis through an arrest of cell cycle and a blockage of adipose phenotype expression. These results also suggest that the anti-obesity activity of green tea may be partially attributed to its suppressive effects in adipogenesis.

Noncritical segmental defect Interlocking nail fixation a b s t r a c t Background: The bone nonunion is an important complication of bone fracture repair. The existing models developed on small animal species prevent using osteosynthesis materials designed to be implanted in human bones. The goal of this study was to develop a nonunion process in a noncritical segmental tibial defect in sheep, a species analogous in size to humans. Materials and methods: The animals were divided into two groups of four animals each. In Group 1 (experimental), the defect was created by surgically stripping the periosteum from the edges of a distal tibial osteotomy, keeping the edges 5 mm apart, and placing an incomplete O-shaped silicone ring in the gap. Group 2 (control) was intervened with a simple fracture at the distal end of the tibia. In both groups an interlocking nail was used as a fixation system. Over 8 wk after surgery, radiographs and histologic and histomorphometric analyses were performed. Results: The control group showed a typical bone repair process. In contrast, the experimental group showed a fracture line with rounded edges and a scarce callus formation.

Established epithelial cell lines that retain their differentiation potential and growth regulatory characteristics can provide valuable tools for studying gene regulation, extracellular matrix synthesis or growth factor response. They are also useful for drug development and toxicity testing. Experiments were therefore carried out to optimize culture conditions for the long-term, serial transfer of corneal epithelial cells in the presence of 3T3 feeder layers; and to establish a permanent cell line. In such experiments, rabbit corneal epithelial cells were seeded at low inoculation densities, and transferred every 5 days. After 80 population doublings, an epithelial cell line, RCE1, emerged. The cell line is heteroploid, with an average population doubling time of 15.5 hours (vs 18 hours for primary cultures). When RCE1 cells reached confluence, they stratified to form a three- to five-layered epithelium and expressed the differentiation-related keratin pair K3/K12 as shown by immunoblot and immunostaining. Biosynthetic labeling of proliferating, confluent and stratified cultures further showed that RCE1 cells expressed keratin pairs K5/K14, K6/K16 and K3/K12, thus mimicking faithfully the stage-dependent differentiation of primary cultures of rabbit corneal keratinocytes. The results demonstrated that RCE1 cells provide a useful model for studying corneal cell growth and differentiation.

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.

Epithelial migration plays a central role in development, wound repair and tumor metastasis, but the role of intermediate filament in this important event is unknown. We showed recently that vimentin coexists in the same cell with keratin-KRT14 at the leading edge of the migrating epidermal cells, and knockdown of vimentin impaired colony growth. Here we demonstrate that vimentin co-localizes and co-immunoprecipitates with keratin-KRT14, and mutations in the-YRKLLEGEE-sequence of vimentin significantly reduced migration of the keratinocytes. Our data demonstrates that keratinocyte migration requires the interaction between vimentin and keratins at the-YRKLLEGEE-sequence at the helical 2B domain of vimentin. These findings have broad implications for understanding the roles of vimentin intermediate filaments in normal and neoplastic epithelial cells.

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.

Lactate dehydrogenase (LDH) and glucose-6-phosphate dehydrogenase (G-6-PDH) activities were studied during corneal epithelial growth and differentiation in cell culture. LDH and G-6-PDH activities increased up to 60 and 150-fold, respectively, when corneal epithelial cells constituted a differentiated four to ®ve layered epithelium; these increases showed a similar time-course to the expression of K3 keratin. Immunostaining experiments showed that in growing colonies, LDH staining is stronger in those cells that are K3 positive; in contrast, in con¯uent four to ®ve layered epithelia LDH and K3 were located in all cell layers, similar to the pattern found in frozen sections from rabbit central cornea. During growth and differentiation, the LDH isoenzyme set from corneal epithelial cells did not change; and it was different from those observed in cultured conjunctival, esophageal and epidermal cells. The augment in LDH activity was due to a 25-fold increase in the LDH-H mRNA and a 12-fold augment in LDH-M mRNA. A computer-assisted search led to identify AP2 and Sp1 binding sites in the LDH and G-6-PDH promoters, suggesting that their expression might share common regulatory mechanisms with the regulation of the differentiation-linked keratins. It is proposed that LDH may be an early marker of corneal epithelial differentiation, and its isozyme pattern could be distinctive from other epithelial cell lineages.

Sheets of cultured allogeneic human keratinocytes have been used for the treatment of burns and chronic leg ulcers but there has been no animal assay for the therapeutic action of these cultures. In order to analyze the effects of frozen cultures of human keratinocytes on wound healing, we have developed such an assay based on the rate of repair of full-thickness skin wounds in immunocompetent NMR1 mice. Reepithelialization of the control wounds, originating from the murine epithelium at the edge of the wound, occurred at a constant rate of advance of 150 µm/day. When frozen cultured human epidermal sheets were thawed at room temperature for 5-10 min and applied to the surface of the wound, the murine epithelium advanced at 267 µm/day. Most wounds treated with frozen cultures completely healed after 10 days, whereas most control wounds required 16 days. The accelerated reepithelialization did not depend on the presence of proliferative human keratinocytes in the frozen cultures. The cultures also promoted early formation of granulation tissue and laminin deposition over the surface of the wound bed. This simple assay should permit quantitative analysis of the effects on healing exerted not only by cultured cells, but also by proteins and small molecules.

Asymmetrical cell division in mammalian corneal epithelia is not widely accepted and it is not well characterized. Although some data led to propose that asymmetrical division occurs along the entire corneal epithelium, evidence from different laboratories implies that asymmetrical cell division in adult individuals could be confined to limbal epithelium, as suggested by the location of the corneal epithelial stem cells and the distribution of some molecular markers involved in regulation of stratification and cell differentiation. Nevertheless, most evidence sustaining the participation of asymmetric mitosis in corneal development and differentiation is merely an inference. Recent results based in cell culture experiments suggest that asymmetric division is part of the differentiation program; in such case, mitotic spindle orientation would be regulated by the structure, composition and active signaling pathways at the stem cell niche. Together, the results support the view that in adults, asymmetric division might be confined to limbus, and hence, the process takes place both in apico-basal oriented cells and in cells in which the mitotic spindle is horizontally oriented. In contrast, during development, asymmetrical divisions would be determined by intrinsic mechanisms involving cell polarity, predominantly occurring in apico-basal oriented cells. Future studies should be oriented to understand the regulation of the asymmetrical/symmetrical division, and the control of cell fate by the niche.

Pre-adipose 3T3-F442A cells exposed to fetal bovine serum or human growth hormone (adipogenic medium) become irreversibly committed to differentiation into adipocytes within 24-36 h. We show now that the action of the serine-threonine kinase inhibitor staurosporine is much more rapid since its addition in non-adipogenic medium resulted in commitment to adipocyte differentiation within 4-6 h. During this period, glycogen synthase kinase 3b was activated. Commitment depended on an increase in the intracellular calcium concentration that was modulated in part by a T-type calcium channel since mibefradil, amiloride, and NiCl 2 , which are selective blockers of the T-type channels, partially inhibited adipose differentiation. Studies of the inhibitory action of retinoic acid showed that a period of time after exposure to St was required in order to stabilize the commitment to adipose differentiation. It was concluded that the commitment of the cells consists of two stages. Commitment is promoted during the first one, and during the second there is a stabilization which still can be destabilized by the addition of retinoic acid or other drugs. The commitment becomes stable after 40 h of staurosporine treatment, and can no longer be prevented by retinoic acid. The identification of these two stages of commitment makes it possible to analyze in further detail early molecular events of the process and the nature of any other participating genes.

Pax-6 is a regulatory gene with a major role during visual system development, but its association with corneal epithelial differentiation is not clearly established. Using the RCE1-(5T5) cell line, which mimics corneal epithelial differentiation, we analyzed Pax-6 biological role. Immunostaining of proliferating colonies and confluent sheets showed that Pax-6-positive cells were also K3 keratin-positive, suggesting that Pax-6 is expressed in differentiating cells. Pax-6 mRNA was barely expressed in early cell cultures; but after confluence, its levels raised up to fivefold as demonstrated by Northern blot and RT-qPCR. The raise in Pax-6 expression preceded for 9 h the increase in LDH-H and LDH-M mRNAs, previously shown as early markers of corneal epithelial cell differentiation. The full-length mRNAs encoding for the two major Pax-6 isoforms were found at very low levels in proliferating cells, and abundantly expressed in the confluent stratified epithelia; Pax-6 mRNA was 2-to 2.5-fold more abundant than Pax-6(5a) mRNA. The ectopic expression of Pax-6 or Pax-6(5a) decreased proliferative ability leading to the formation of abortive, non-proliferative colonies. In contrast, culture conditions that delay or block corneal epithelial cell differentiation reduced or inhibited the expression of Pax-6. Collectively, results show that Pax-6 is the earlier differentiation marker expressed by corneal epithelial cells, and open the possibility for a major role of Pax-6 as the main driver of the differentiation of corneal epithelial cells.