Noelia Andollo

In Vitro Effects of Three Blood Derivatives on Human Corneal Epithelial Cells

PURPOSE We compared the effects of three blood derivatives, autologous serum (AS), platelet-rich plasma (PRP), and serum derived from plasma rich in growth factors (PRGF), on a human corneal epithelial (HCE) cell line to evaluate their potential as an effective treatment for corneal epithelial disorders. METHODS The concentrations of epidermal growth factor (EGF), fibroblast growth factor (FGF), vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), platelet-derived growth factor (PDGF), and fibronectin were quantified by ELISA. The proliferation and viability of HCE cells were measured by an 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) colorimetric assay. Cell morphology was assessed by phase-contrast microscopy. The patterns of expression of several connexin, involucrin, and integrin α6 genes were analyzed by real-time RT-PCR. RESULTS We found significantly higher levels of EGF in PRGF compared to AS and PRP. However, AS and PRGF induced robust proliferation of HCE cells. In addition, PRGF cultured cells grew as heterogeneous colonies, exhibiting differentiated and non-differentiated cell phenotypes, whereas AS- and PRP-treated cultures exhibited quite homogeneous colonies. Finally, PRGF upregulated the expression of several genes associated with communication and cell differentiation, in comparison to AS or PRP. CONCLUSIONS PRGF promotes biological processes required for corneal epithelialization, such as proliferation and differentiation. Since PRGF effects are similar to those associated with routinely used blood derivatives, the present findings warrant further research on PRGF as a novel alternative treatment for ocular surface diseases.

Corneal Wound Healing Promoted by 3 Blood Derivatives: An In Vitro and In Vivo Comparative Study

Purpose: The aim of this study was to compare the effect on corneal wound healing of 3 differently manufactured blood derivatives [autologous serum (AS), platelet-rich plasma, and serum derived from plasma rich in growth factors (s-PRGF)]. Methods: Scratch wound-healing assays were performed on rabbit primary corneal epithelial cultures and human corneal epithelial cells. Additionally, mechanical debridement of rabbit corneal epithelium was performed. Wound-healing progression was assessed by measuring the denuded areas remaining over time after treatment with each of the 3 blood derivatives or a control treatment. Results: In vitro data show statistically significant differences in the healing process with all the derivatives compared with the control, but 2 of them (AS and s-PRGF) induced markedly faster wound healing. In contrast, although the mean time required to complete in vivo reepithelization was similar to that of AS and s-PRGF treatment, only wounds treated with s-PRGF were significantly smaller in size from 2.5 days onward with respect to the control treatment. Conclusions: All 3 blood derivatives studied are promoters of corneal reepithelization. However, the corneal wound-healing progresses differently with each derivative, being faster in vitro under AS and s-PRGF treatment and producing in vivo the greatest decrease in wound size under s-PRGF treatment. These findings highlight that the manufacturing process of the blood derivatives may modulate the efficacy of the final product.

Imprinting of mammalian male gametes is gene specific and does not occur at a single stage of differentiation

Epigenetic modifications such as DNA methylation and alterations to chromatin structure have been proposed as hallmarks of imprinting in somatic cells after fertilization. In the germ cell line, gene imprinting needs to be reset in order to transmit the correct sex-specific imprinting pattern to the next generation. The precise timing of imprint erasure and re-establishment for many genes remains to be determined and precise molecular mechanisms of genomic imprinting have not yet been fully characterized. Here, we have analysed the methylation state and DNase-I sensitivity of two genes with reciprocal genomic imprinting (U2af1-rs1 and H19 genes) in a male mouse primordial germ cell (PGC) derived cell line (EG-1), isolated post-natal spermatogonia and mature sperm cells. Our results show that establishment of imprinting of the U2af1-rs1 and H19 genes during male germ cell differentiation occurs at different stages of differentiation. Furthermore, the presence of DNase-I hypersensitive sites may constitute a molecular marker to identify alleles and subsequently acquire the appropriate methylation imprint. We propose that this molecular identifier may be present or absent for a specific gene according to the sex of the gamete.

Structural and functional preservation of specific sequences of DNA and mRNA in apoptotic bodies from ES cells.

Retinoic acid-induced apoptosis of embryonic stem (ES) cells is an experimental system which resembles the physiological programmed cell death that occurs during differentiation in embryonic development. Our aim was to analyze the involvement of epigenetic modifications such as DNA methylation and chromatin structure in the apoptotic process and to investigate the metabolic activity of apoptotic bodies. We found a relationship between DNA methylation and apoptosis, shown by a dose-dependent induction of apoptosis after treatment with the inhibitor of DNA methylation 5-aza-2′-deoxycytidine. Interestingly, we found a slight demthylation of specific sequences of the U2afl-rs1 imprinted gene in those RA treated cells which were specifically undergoing apoptosis. In addition, apoptotic bodies exhibited an unexpected open chromatin conformation accessible to the endonuclease DNase-I. Furthermore, we observed a structural and functional preservation of specific DNA sequences and mRNA. These results suggest that biological activities, such as transcription or protein synthesis, could be maintained even towards the end of the apoptotic process.

Epigenetic regulation of the imprinted U2af1-rs1 gene during retinoic acid-induced differentiation of embryonic stem cells

Epigenetic modifications such as DNA methylation and changes in chromatin structure are changes in the chemical composition or structure of DNA that work by regulating gene expression. Their mechanisms of action have been generally studied in imprinted genes. The present work analyzes the involvement of these mechanisms in the expression of the U2af1‐rs1 imprinted gene during the differentiation process of embryonic stem (ES) cells induced by retinoic acid. By DNA digestion with methylation‐dependent or independent restriction enzymes and consecutive Southern blot, we have found that methylation of the U2af1‐rs1 gene increases in differentiated ES cells and in embryoid bodies. However, northern blot and real‐time reverse transcription–polymerase chain reaction analysis showed a higher expression of the U2af1‐rs1 gene in differentiated ES cells and in embryoid bodies than in undifferentiated ones. On the other hand, the sensitivity to DNase‐I assay demonstrated an open chromatin conformation for differentiated cells with regard to undifferentiated ES cells. Our results suggest that the expression of the U2af1‐rs1 gene would be regulated by changes in chromatin structure rather than by DNA methylation during the RA‐induced process of differentiation of ES cells.

Serum from plasma rich in growth factors regenerates rabbit corneas by promoting cell proliferation, migration, differentiation, adhesion and limbal stemness

To evaluate the regenerating potential and the mechanisms through which the autologous serum derived from plasma rich in growth factors (s‐PRGF) favours corneal wound healing in vitro and in vivo.

Pluripotent Stem Cell Epigenetics During Development and Cancer

Studies on biological development and cancer have pointed out the importance of specific epigenetic environments to maintain the equilibrium between repressed and activated genes. It has been possible to establish that this kind of environment induces chromatin structure modification and heritable changes in gene functions without altering primary DNA sequencing. We show here recent results of our laboratory on the expression of two imprinted genes, U2af1-rs1 and H19, in normal and pluripotent male germinal cells and in embryonic stem cell after induction of differentiation and apoptosis by retinoic acid treatments. These experimental observations can shed new light for a better understanding of testis embryonal carcinoma biology.

Chromatin Structure and U2AF1-RS1 Gene Expression in Embryonic Stem Cells following RA-Induced Apoptosis

INTRODUCTION. Apoptosis is a genetically controlled, selective cellular death mechanism, which occurs during processes of differentiation and normal cellular development. AB1 embryonic stem cells were employed in our assays, based on previous studies where it was demonstrated that in vitro treatment with retinoic acid (RA) gave rise to apoptosis as well as cellular differentiation (1). In order to support the hypothesis that apoptosis is an active process even when apoptotic bodies appear, we decided to study the chromatin conformation of DNA (2) and the corresponding gene expression. To this end, we analyzed the U2af1-rs1 gene, which takes part in mRNA splicing. METHODS. AB1 cells after in vitro incubation with 1 µM RA for 48 and 72 hours, were stained with propidium iodine and analyzed by flow cytometry. Electron microscopy was employed to verify the ultrastructure of differentiated and apoptotic cells. In order to perform chromatin structure analysis, nuclei were isolated from non-differentiated and differentiated AB1 cells and from apoptotic bodies. After incubation at increasing concentrations of DNase-I, DNA was extracted from the nuclei, digested with Bgl II, electrophoresized and hybridized with a mouse U2af1-rs1 probe. Densitometric measurements on scanned x-ray films were performed with a Bio-Rad computer-assisted system. Total RNAs from non-differentiated and differentiated cells and from apoptotic bodies were hybridized with a U2af1-rs1 probe and with a mouse Gadph control probe. RESULTS. Following RA treatment, a decrease of the proliferative population and an increase of the non-proliferative population of AB1 cells was observed. In addition, apoptotical cell death was observed, especially after 72 hours RA-treatment. The ultrastructural phenotype of differentiated AB1 cells after RA-treatment consisted of an increment in the quantity and variety of organelles and the presence of eucromatic nuclei with evident nucleoli. The apoptotical fraction showed a characteristic morphology with cytoplasmic condensation and margination of the hypercondensed chromatin towards the nuclear periphery. Chromatin structure analysis of the U2af1-rs1 region revealed that differentiated cell DNA was more sensitive to DNase-I than undifferentiated cell DNA. Furthermore, we detected DNase-I sensitivity in apoptotic bodies. Northern blot analysis demonstrated that undifferentiated and differentiated cells and, even apoptotic bodies, expressed the U2af1-rs1 mRNA.