Comparative Proteomic Assessment of Normal vs. Polyhydramnios Amniotic Fluid Based on Computational Analysis.

Mass spectrometry-based proteomics have become a valued tool for conducting comprehensive analyses in amniotic fluid samples with pathologies. Our research interest is the finding and characterization of proteins related to normal vs. polyhydramnios (non-immune hydrops) pregnancy. Proteomic analysis was performed on proteins isolated from fresh amniotic fluid samples. Proteins were fractionated by 2DE using a different pI range (pI 3-11, pI 4-7) and analyzed with MALDI-TOF-MS. Furthermore, by using computational analysis, identified proteins in protein maps specific to normal vs. polyhydramnios pregnancy were compared and the quantities of expressed proteins were evaluated mathematically. Comparative analysis of proteome characteristic for the same polyhydramnios pregnancy fractionated by 2DE in different pI range (3-11 and 4-7) was performed and particular protein groups were evaluated for the quantification of changes within the same protein level. Proteins of normal and polyhydramnios pregnancies were fractionated by 2DE in pI range 3-11 and in pI range 4-7. Mass spectrometry analysis of proteins has revealed that the quantity changes of the main identified proteins in normal vs. polyhydramnios pregnancy could be assigned to immune response and inflammation proteins, cellular signaling and regulation proteins, metabolic proteins, etc. Specifically, we have identified and characterized proteins associated with heart function and circulatory system and proteins associated with abnormalities in prenatal medicine. The following are: serotransferrin, prothrombin, haptoglobin, transthyretin, alpha-1-antitrypsin, zinc-alpha-2-glycprotein, haptoglobin kininogen-1, hemopexin, clusterin, lumican, afamin, gelsolin. By using computational analysis, we demonstrated that some of these proteins increased a few times in pathological pregnancy. Computer assistance analysis of 2DE images suggested that, for the better isolation of the proteins' isoforms, those levels increased/decreased in normal vs. polyhydramnios pregnancy, and the fractionation of proteins in pI rage 3-11 and 4-7 could be substantial. We analyzed and identified by MS proteins specific for normal and polyhydramnios pregnancies. Identified protein levels increased and/or modification changed in case of non-immune hydrops fetus and in cases of cardiovascular, anemia, growth restriction, and metabolic disorders. Computational analysis for proteomic characterization empower to estimate the quantitative changes of proteins specific for normal vs. polyhydramnios pregnancies.

Epigenetic regulation of amniotic fluid mesenchymal stem cell differentiation to the mesodermal lineages at normal and fetus-diseased gestation.

Human mesenchymal stem cells isolated from amniotic fluid (AF-MSCs) demonstrate the potency for self-renewal and multidifferentiation, and can, therefore, be a potential alternative source of stem cells adapted for therapeutic purposes. The object of this study is to evaluate the efficacy of MSCs from AF when the pregnancy is normal or when the fetus is affected during pregnancy to differentiate into mesodermal lineage tissues and to elucidate epigenetic states responsible for terminal adipogenic and osteogenic differentiation. The morphology of AF-MSCs from two cell sources and the expression of the cell surface-specific (CD44, CD90, and CD105) markers and pluripotency (Oct4, Nanog, Sox2, and Rex1) genes were quite similar and underwent mesodermal lineage differentiation because this is shown by the typical cell morphology and of genes' expression specific for adipogenic (peroxisome proliferator-activated receptor-É£, adiponectin) and osteoblastic (alkaline phosphatase, osteopontin, and osteocalcin) differentiation. Terminal lineage-specific differentiation was related to differential expression of miR-17, miR-21, miR-34a, and miR-146a, decreased levels of acetylated H4 and H3K9, trimethylated H3K4 and H3K9, and the retention of H3K27me3 along with a reduction in the levels of HDAC1, DNMT1, and PRC1/2 proteins (BMI1/SUZ12). No significant distinction could be identified in the levels of expression of all epigenetic or pluripotency markers between undifferentiated MSCs isolated from AF of normal gestation and pregnancy where the fetus was damaged and between those differentiated toward adipocytes or osteoblasts. The expressional changes of those marks and microRNAs that occurred during terminal differentiation to mesodermal tissues indicate subtle epigenetic regulation in AF-MSCs when the condition of the fetus is healthy normal or diseased. More detailed studies of epigenetic mechanisms may offer a better understanding of AF-MSCs differentiation in fetus-diseased conditions and their usage in an autologous therapeutic application and prenatal disease research.

Epigenetic alterations in amniotic fluid mesenchymal stem cells derived from normal and fetus-affected gestations: A focus on myogenic and neural differentiations.

Amniotic fluid-derived mesenchymal stem cells (AF-MSCs) are autologous to the fetus and represent a potential alternative source for the regenerative medicine and treatment of perinatal disorders. To date, AF-MSCs differentiation capacity to non-mesodermal lineages and epigenetic regulation are still poorly characterized. The present study investigated the differentiation potential of AF-MSCs toward neural-like cells in comparison to the mesodermal myogenic lineage and assessed epigenetic factors involved in tissue-specific differentiation. Myogenic and neural differentiation assays were performed by the incubation with specific induction media. Typical MSCs markers were determined by flow cytometry, the expression of lineage-specific genes, microRNAs and chromatin modifying proteins were examined by RT-qPCR and Western blot, respectively. AF-MSCs of normal and fetus-affected gestations had similar stem cells characteristics and two-lineage potential, as characterized by cell morphology and the expression of myogenic and neural markers. Two-lineage differentiation process was associated with the down-regulation of miR-17 and miR-21, the up-regulation of miR-34a, miR-146a and DNMT3a/DNMT3b along with the gradual decrease in the levels of DNMT1, HDAC1, active marks of chromatin (H4hyperAc, H3K9ac, H3K4me3) and the repressive H3K9me3 mark. Differentiation was accompanied by the down-regulation of PRC1/2 proteins (BMI1/SUZ12, EZH2) and the retention of the repressive H3K27me3 mark. We report that both AF-MSCs of normal and fetus-affected gestations possess differentiation capacity toward myogenic and neural lineages through rather similar epigenetic mechanisms that may provide potential applications for further investigation of the molecular basis of prenatal diseases and for the future autologous therapy.

DNA methyltransferases inhibitors effectively induce gene expression changes suggestive of cardiomyogenic differentiation of human amniotic fluid-derived mesenchymal stem cells via chromatin remodeling.

Human amniotic fluid-derived mesenchymal stem cells (AF-MSCs) are a new potential stem cell source for cell therapy and regenerative medicine. These are fetal mesenchymal stem cells with multilineage differentiation potential found in amniotic fluid. The aim of the present study was to evaluate in vitro differentiation initiation of AF-MSCs into cardiac progenitors upon application of inhibitors of DNA methyltransferases (DNMT), such as Decitabine (DEC; 5-aza-2'-deoxycytidine) and Zebularine (ZEB). We assessed epigenetic changes and explored patterns of genes, enriched in association with hyperacetylated H4 after induced differentiation. Upregulation of cardiomyogenesis-related genes (TNNT2, MYH6, ACTN2, and DES) and cardiac ion channels genes, downregulation of pluripotency genes markers as well as increase in Connexin43 expression indicated cardiomyogenic commitment. Evaluation of global epigenetic changes showed that levels of chromatin modifying enzymes, such as Polycomb repressive complex 2 proteins (EZH2, SUZ12), DNMT1, histone deacetylases 1 and 2 were reduced to the similar extent by both differentiation agents. Levels of specific histone marks keeping active state of chromatin (H3K4me3, H3K9Ac, and H4hyperAc) increased and marks of repressed chromatin state (H3K27me3 and H3K9me3) decreased after DEC or ZEB treatment. Chip-Seq analysis after chromatin immunoprecipitation with H4hyperAc demonstrated enrichment of around 100 functionally annotated genes, related to chromatin reorganization and cardiomyogenesis and confirmed relation between H4 hyperacetylation and gene expression. Our results demonstrate that both DEC and ZEB can be potentially used as cardiomyogenic differentiation inducers in AF-MSCs, and they cause various genetic and epigenetic changes resulting in global chromatin remodeling.

Histone Modifications Pattern Associated With a State of Mesenchymal Stem Cell Cultures Derived From Amniotic Fluid of Normal and Fetus-Affected Gestations.

Human amniotic fluid (AF)-derived mesenchymal stem cells (MSCs) sharing embryonic and adult stem cells characteristics are interesting by their multipotency and the usage for regenerative medicine. However, the usefulness of these cells for revealing the fetal diseases still needs to be assessed. Here, we have analyzed the epigenetic environment in terms of histone modifications in cultures of MSCs derived from AF of normal pregnancies and those with fetal abnormalities. The comparison of MSCs samples from AF of normal pregnancies (N) and fetus-affected (P) revealed two distinct cultures by their proliferation potential (P I and P II). Cell populations from N and P I samples had similar growth characteristics and exhibited quite similar cell surface (CD44, CD90, CD105) and stemness markers (Oct4, Nanog, Sox2, Rex1) profile that was distinct in slower growing and faster senescent P II cultures. Those differences were associated with changes in 5-Cyt DNA methylation and alterations in the expression levels of chromatin modifiers (DNMT1, HDAC1/2), activating (H4ac, H3K4me3), and repressive (H3K9me2/me3, H3K27me3) histone marks. MSCs isolated from AF with the genetic or multifactorial fetal diseases (P II samples) were enriched with repressive histone marks and H4K16ac, H3K9ac, H3K14ac modifications. This study indicates that differential epigenetic environment reflects a state of AF-MSCs dependently on their growth, phenotype, and stemness characteristics suggesting a way for better understanding of epigenetic regulatory mechanisms in AF-MSCs cultures in normal and diseased gestation conditions. J. Cell. Biochem. 118: 3744-3755, 2017. © 2017 Wiley Periodicals, Inc.

Senescence-Associated Molecular and Epigenetic Alterations in Mesenchymal Stem Cell Cultures from Amniotic Fluid of Normal and Fetus-Affected Pregnancy.

Human amniotic-fluid-derived mesenchymal stem cells (AF-MSCs) are interesting for their multilineage differentiation potential and wide range of therapeutic applications due to the ease of culture expansion. However, MSCs undergo replicative senescence. So far, the molecular mechanisms that underlie fetal diseases and cell senescence are still poorly understood. Here, we analyzed senescence-associated morphologic, molecular, and epigenetic characteristics during propagation of MSCs derived from AF of normal and fetus-affected pregnancy. AF-MSCs cultures from both cell sources displayed quite similar morphology and expression of specific cell surface (CD44, CD90, and CD105) and stemness (Oct4, Nanog, Sox2, and Rex1) markers but had interindividual variability in proliferation capability and time to reach senescence. Within passages 4 and 8, senescent cultures exhibited typical morphological features, senescence-associated ß-galactosidase activity, increased levels of p16, and decreased levels of miR-17 and miR-21 but showed differential expression of p21, p53, and ATM dependently on the onset of cell senescence. These differences correlated with changes in the level of chromatin modifiers (DNMT1 and HDAC1) and polycomb group proteins (EZH2, SUZ12, and BMI1) paralleling with changes in the expression of repressive histone marks (H3K9me3 and H3K27me3) and stemness markers (Oct4, Nanog, Sox2, and Rex1). Therefore epigenetic factors are important for AF-MSCs senescence process that may be related with individuality of donor or a fetus malignancy status.

Human Amniotic Fluid Mesenchymal Stem Cells from Second- and Third-Trimester Amniocentesis: Differentiation Potential, Molecular Signature, and Proteome Analysis.

Human amniotic fluid stem cells have become an attractive stem cell source for potential applications in regenerative medicine and tissue engineering. The aim of this study was to characterize amniotic fluid-derived mesenchymal stem cells (AF-MSCs) from second- and third-trimester of gestation. Using two-stage protocol, MSCs were successfully cultured and exhibited typical stem cell morphological, specific cell surface, and pluripotency markers characteristics. AF-MSCs differentiated into adipocytes, osteocytes, chondrocytes, myocytes, and neuronal cells, as determined by morphological changes, cell staining, and RT-qPCR showing the tissue-specific gene presence for differentiated cell lineages. Using SYNAPT G2 High Definition Mass Spectrometry technique approach, we performed for the first time the comparative proteomic analysis between undifferentiated AF-MSCs from late trimester of gestation and differentiated into myogenic, adipogenic, osteogenic, and neurogenic lineages. The analysis of the functional and expression patterns of 250 high abundance proteins selected from more than 1400 demonstrated the similar proteome of cultured and differentiated AF-MSCs but the unique changes in their expression profile during cell differentiation that may help the identification of key markers in differentiated cells. Our results provide evidence that human amniotic fluid of second- and third-trimester contains stem cells with multilineage potential and may be attractive source for clinical applications.