Heterogeneidad de las células mesenquimales en la membrana amniótica humana en partos a término

Resumo

Mesenchymal cells (MC) from the human amniotic membrane (hAM) exhibit immunological and regenerative characteristics that have attracted interest in reparative medicine. Several investigations have found a certain heterogeneity in these cells "in vitro", however, histological characterizations of this cells do not seem to exist in hAM at term "in vivo". Their knowledge may find differences related to their viability for culture or their differentiation potential and improve the understanding of the changes that occur in peripartum fetal membranes. Objectives of the present thesis: Experiment 1: 1) To characterize MC at the morphological level with light microscopy (LM) and transmission electron microscopy (TEM), looking for morphofunctional characteristics that show their heterogeneity and differences in synthetic activity, differentiation or senescence, 2) semi-quantitatively study of MC population to analyze the frequency of possible cell types in LM and TEM, 3) perform immunohistochemical techniques (growth factors, extracellular matrix proteins or cytoskeleton) on the CMa in order to establish a relationship between cell morphology and cell function. Experiment 2: 4) To review the histological structure in different zones of fetal membranes with LM, by means of conventional histological and histochemical techniques, specific for carbohydrates and collagen fibers, 5) to quantify in a semi-quantitatively manner the possible types of MC in the hAM zones. Subsequently perform statistical analysis to determine zonal differences in their frequency and total number, 6) immunohistochemically identification of fibronectin and vimentin in the fetal membrane zones, quantifying them by western blot and correlate them with the distribution of CMa types. In the first experiment, 4 periumbilical portions of different hAM at term, from cesarean deliveries, were processed for LM and TEM. On the samples processed for LM, different histochemical and immunohistochemical stains were performed, carrying out a qualitative analysis of the MC. In a second experiment, portions of the periumbilical, intermediate and rupture zone of 10 hAM, from eutocic births at term, were analyzed and processed for LM by performing different histochemical stains; and also protein quantification by western blot. The possible heterogeneity of the MC was grouped according to morphological criteria. The results showed that the MC presented morphological heterogeneity, finding three cell types (I-III) in LM, distinguishing, in addition, the Hofbauer cells in the semithin sections. Type III showed the most differentiated phenotype and its proportion in the rupture zone was higher than in the periumbilical zone. Type I, the most undifferentiated type, was more frequent in the latter (p<0.05). The three types were successively more intense to PAS, vimentin, fibronectin, concanavalin-A, VEGF, TGF-β2, PDGF-C and FGF-2 staining. With TEM, 4 types were identified and two important phenomena were observed, both more intense in type 3 and 4 cells: 1) vesiculation of the rough endoplasmic reticulum (RER) and exocytosis of its contents to extracellular matrix (EM), 2) dilatation of the RER by accumulation of a material, similar to EM, which was frequently released. The heterogeneity of the CMa in the different areas of the hAM was related to the amount of fibronectin and vimentin. In conclusion, the CMa population is heterogeneous and region specific along the hAM. The heterogeneity seems to be secondary to a fibroblastic differentiation process that ends in senescence, and which is related to an increase in the synthesis of growth factors. In addition, there is a stress of the RER characterized by dilatation and accumulation of fibronectin in its interior. These changes in the MC may be related to the biophysical phenomena associated with parturition and rupture of the fetal membranes, and may also be of interest for future work in regenerative medicine.