Mature miR-99a Upregulation in the Amniotic Fluid Samples from Female Fetus Down Syndrome Pregnancies: A Pilot Study.

Background and Objective: Although Down syndrome is the most frequent aneuploidy, its pathogenic molecular mechanisms are not yet fully understood. The aim of our study is to quantify-by qRT-PCR-the expression levels of both the mature forms and the pri-miRNAs of the microRNAs resident on chromosome 21 (miR(21)) in the amniotic fluid samples from Down syndrome singleton pregnancies and to estimate the impact of the differentially expressed microRNAs on Down syndrome fetal heart and amniocytes transcriptomes. Materials and methods: We collected amniotic fluid samples harvested by trained obstetricians as part of the second trimester screening/diagnostic procedure for aneuploidies to assess the trisomy 21 status by QF-PCR and karyotyping. Next, we evaluated-by Taqman qRT-PCR-the expression levels of both the mature forms and the pri-miRNA precursors of the microRNAs resident on chromosome 21 in amniotic fluid samples from singleton Down syndrome and euploid pregnancies. Further, we combined miRWalk 3.0 microRNA target prediction with GEO DataSets analysis to estimate the impact of hsa-miR-99a abnormal expression on Down syndrome heart and amniocytes transcriptome. Results: We found a statistically significant up-regulation of the mature form of miR-99a, but not pri-miR-99a, in the amniotic fluid samples from Down syndrome pregnancies with female fetuses. GATHER functional enrichment analysis of miRWalk3.0-predicted targets from Down syndrome amniocytes and fetal hearts transcriptome GEODataSets outlined both focal adhesion and cytokine-cytokine receptor interaction signaling as novel signaling pathways impacted by miR-99a and associated with cardiac defects in female Down syndrome patients. Conclusions: The significant overexpression of miR-99a, but not pri-miR-99a, points towards an alteration of the post-transcriptional mechanisms of hsa-miR-99a maturation and/or stability in the female trisomic milieu, with a potential impact on signaling pathways important for proper development of the heart.

Explants-isolated human placenta and umbilical cord cells share characteristics of both epithelial and mesenchymal stem cells.

In recent years, identification of new sources of adult stem cells developed rapidly, pursuing to find easily available tissues, which will give rise to homogenous stem cells populations. Up to present, bone marrow-derived mesenchymal stem cells (BM-MSCs) are unanimously considered to fulfill the criteria for being used in clinical settings, but adipose stem cells, placental and umbilical cord stem cells, and other tissue-derived stem cells are making their way to being used at least in autologous transplantation. We isolated cellular populations from placental tissue and umbilical cord using the explants method. The placental (PL) and umbilical cord (UC)-derived cells were cultured and expanded in appropriate conditions for generation of stem cells. We assessed the stemness characteristics of the tissue-isolated cells and compared them to an established MSCs line. For this purpose, we determined the immunophenotype, morphological and ultrastructural characteristics, as well as functional abilities of PL- and UC-derived cells. Flow cytometric evaluation of cells revealed presence of CD90, CD73, and CD105 stem cells markers, while the cells were negative for CD34, CD45 and HLA-DR. Immunocytochemical staining showed that 100% of PL- and UC-derived cells are positive for vimentin and CD105 expression, while cytokeratin was revealed in less than 10% in both tissue-isolated cells. Morphological and ultrastructural characteristics of cells exposed analogous cellular size and intracellular organization, similar to MSCs, but detailed view of UC-derived cells by transmission electron microscopy (TEM) demonstrated presence of intercellular junctions-desmosomes, similar to epithelial cells. Both PL- and UC-derived cells confirmed their trilineage potential, being able to differentiate into adipocytes, osteoblasts, and chondrocytes in different proportions. Flow chamber in vitro assay was used to determine to what extent PL- and UC-derived cells are able to adhere to substrates (VCAM and ICAM) and we showed progressively decreased adhesion of both cellular types, inversely proportional to the generated shear stress. We may conclude that explants-isolated placental and umbilical cord cells are endowed with characteristics of both epithelial and mesenchymal stem cells, and purification procedures are additionally required for safe use of these cells in diverse clinical applications.