The outcome of tissue cryopreservation on the cellular, molecular and epigenetic characteristics of endometrial tissue and stromal cells.

RESEARCH QUESTION: What impact does the cryopreservation of endometrial tissue have on cell characteristics and molecular and epigenetic profile changes in endometrial tissue and stromal cells? DESIGN: Cellular properties, such as proliferation efficiency, surface marker expression and the differentiation potency of endometrial stromal cells (ESC) isolated from fresh (Native) and cryopreserved (Cryo) tissue were compared. Moreover, changes in the expression of genes associated with pluripotency, endometrial function and epigenetic regulation and microRNA (miRNA, miR) were assessed, as were levels of DNA methylation and histone modifications. RESULTS: Native and Cryo cells exhibit very similar profiles including cell surface marker expression, differentiation potency and histone modifications, except for a decrease in proliferative potency and cell surface marker SUSD2 expression in Cryo cells. It was demonstrated that endometrial tissue cryopreservation led to an up-regulated expression of genes associated with pluripotency (NANOG, OCT4 [also known as POU5F1]). This confirms that despite being recovered from cryopreserved differentiated tissue, cells retained their stemness properties. In addition, alterations in DNA methyltransferase (DNMT1, DNMT3A, DNMT3B) gene regulation were observed, along with a down-regulation of hsa-miR145-5p in Cryo ESC. CONCLUSIONS: These findings contribute to a deeper understanding of the complex effects of endometrial tissue cryopreservation, providing insights for both medical and basic research applications. Since different tissues possess unique characteristics, it is essential to select the most suitable cryopreservation method for each tissue individually. Furthermore, the study findings indicate the potential utility of slow-cooling cryopreservation for both normal and pathological endometrial tissue samples, with the purpose of isolating stromal cell cultures.

Anti-neuroinflammatory microRNA-146a-5p as a potential biomarker for neuronavigation-guided rTMS therapy success in medication resistant depression disorder.

Treatment-resistant depression (TRD) is a challenging issue to address. Repetitive transcranial magnetic stimulation (rTMS) is commonly used but shows varying efficacy, necessitating a deeper understanding of depression physiology and rTMS mechanisms. Notably, an increasing amount of recent data has displayed the connection of TRD and its clinical outcome with chronic inflammatory processes. The current study included 19 TRD patients undergoing rTMS and 11 depressed patients responding to medication as a comparison group. We assessed therapeutic efficacy using MADRS, HAM-D-17, GAD-7, and PHQ-9 tests. Inflammatory markers, neurotrophins, and associated miRNAs were measured in patients blood serum before and during treatment. A control group of 18 healthy individuals provided baseline data. The results of our study showed significantly higher levels of pro-inflammatory interleukins-6 and - 8 in TRD patients compared to drug-responders, which also related to more severe symptoms before treatment. In addition, TRD patients, both before and during treatment, exhibited higher average blood serum concentrations of pro-inflammatory interleukin-18 and lower levels of anti-neuroinflammatory miR-146a-5p compared to healthy controls. We also observed that the expression of miR-16-5p, miR-93-5p, and especially miR-146a-5p correlated with clinical changes following rTMS. Our study confirmed that TRD patients possess a higher inflammatory status, while the anti-neuroinflammatory miR-146a-5p was demonstrated to have a considerable potential for predicting their rTMS treatment success.

Effects of human placenta cryopreservation on molecular characteristics of placental mesenchymal stromal cells.

Cryopreservation of placenta tissue for long-term storage provides the opportunity in the future to isolate mesenchymal stromal cells that could be used for cell therapy and regenerative medicine. Despite being widely used, the established cryopreservation protocols for freezing and thawing still raise concerns about their impact on molecular characteristics, such as epigenetic regulation. In our study, we compared the characteristics of human placental mesenchymal stromal cells (hPMSCs) isolated from fresh (native) and cryopreserved (cryo) placenta tissue. We assessed and compared the characteristics of native and cryo hPMSCs such as morphology, metabolic and differentiation potential, expression of cell surface markers, and transcriptome. No significant changes in immunophenotype and differentiation capacity between native and cryo cells were observed. Furthermore, we investigated the epigenetic changes and demonstrated that both native and cryo hPMSCs express only slight variations in the epigenetic profile, including miRNA levels, DNA methylation, and histone modifications. Nevertheless, transcriptome analysis defined the upregulation of early-senescence state-associated genes in hPMSCs after cryopreservation. We also evaluated the ability of hPMSCs to improve pregnancy outcomes in mouse models. Improved pregnancy outcomes in a mouse model confirmed that isolated placental cells both from native and cryo tissue have a positive effect on the restoration of the reproductive system. Still, the native hPMSCs possess better capacity (up to 66%) in comparison with cryo hPMSCs (up to 33%) to restore fertility in mice with premature ovarian failure. Our study demonstrates that placental tissue can be cryopreserved for long-term storage with the possibility to isolate mesenchymal stromal cells that retain characteristics suitable for therapeutic use.

Effect of 3D Spheroid Culturing on NF-κB Signaling Pathway and Neurogenic Potential in Human Amniotic Fluid Stem Cells.

Human amniotic fluid stem cells (hAFSCs) are known for their advantageous properties when compared to somatic stem cells from other sources. Recently hAFSCs have gained attention for their neurogenic potential and secretory profile. However, hAFSCs in three-dimensional (3D) cultures remain poorly investigated. Therefore, we aimed to evaluate cellular properties, neural differentiation, and gene and protein expression in 3D spheroid cultures of hAFSCs in comparison to traditional two-dimensional (2D) monolayer cultures. For this purpose, hAFSCs were obtained from amniotic fluid of healthy pregnancies and cultivated in vitro, either in 2D, or 3D under untreated or neuro-differentiated conditions. We observed upregulated expression of pluripotency genes OCT4, NANOG, and MSI1 as well as augmentation in gene expression of NF-κB-TNFα pathway genes (NFKB2, RELA and TNFR2), associated miRNAs (miR103a-5p, miR199a-3p and miR223-3p), and NF-κB p65 protein levels in untreated hAFSC 3D cultures. Additionally, MS analysis of the 3D hAFSCs secretome revealed protein upregulation of IGFs signaling the cascade and downregulation of extracellular matrix proteins, whereas neural differentiation of hAFSC spheroids increased the expression of SOX2, miR223-3p, and MSI1. Summarizing, our study provides novel insights into how 3D culture affects neurogenic potential and signaling pathways of hAFSCs, especially NF-κB, although further studies are needed to elucidate the benefits of 3D cultures more thoroughly.

Metabolic Profile and Neurogenic Potential of Human Amniotic Fluid Stem Cells From Normal vs. Fetus-Affected Gestations.

Human amniotic fluid stem cells (hAFSCs) possess some characteristics with mesenchymal stem cells (MSCs) and embryonic stem cells and have a broader differentiation potential compared to MSCs derived from other sources. Although hAFSCs are widely researched, their analysis mainly involves stem cells (SCs) obtained from normal, fetus-unaffected gestations. However, in clinical settings, knowledge about hAFSCs from normal gestations could be poorly translational, as hAFSCs from healthy and fetus-diseased gestations may differ in their differentiation and metabolic potential. Therefore, a more thorough investigation of hAFSCs derived from pathological gestations would provide researchers with the knowledge about the general characteristics of these cells that could be valuable for further scientific investigations and possible future clinical applicability. The goal of this study was to look into the neurogenic and metabolic potential of hAFSCs derived from diseased fetuses, when gestations were concomitant with polyhydramnios and compare them to hAFSCs derived from normal fetuses. Results demonstrated that these cells are similar in gene expression levels of stemness markers (SOX2, NANOG, LIN28A, etc.). However, they differ in expression of CD13, CD73, CD90, and CD105, as flow cytometry analysis revealed higher expression in hAFSCs from unaffected gestations. Furthermore, hAFSCs from "Normal" and "Pathology" groups were different in oxidative phosphorylation rate, as well as level of ATP and reactive oxygen species production. Although the secretion of neurotrophic factors BDNF and VEGF was of comparable degree, as evaluated with enzyme-linked immunosorbent assay (ELISA) test, hAFSCs from normal gestations were found to be more prone to neurogenic differentiation, compared to hAFSCs from polyhydramnios. Furthermore, hAFSCs from polyhydramnios were distinguished by higher secretion of pro-inflammatory cytokine TNFα, which was significantly downregulated in differentiated cells. Overall, these observations show that hAFSCs from pathological gestations with polyhydramnios differ in metabolic and inflammatory status and also possess lower neurogenic potential compared to hAFSCs from normal gestations. Therefore, further in vitro and in vivo studies are necessary to dissect the potential of hAFSCs from polyhydramnios in stem cell-based therapies. Future studies should also search for strategies that could improve the characteristics of hAFSCs derived from diseased fetuses in order for those cells to be successfully applied for regenerative medicine purposes.

Small Molecule Treatments Improve Differentiation Potential of Human Amniotic Fluid Stem Cells.

Human amniotic fluid stem cells (AFSC) are an exciting and very promising source of stem cells for therapeutic applications. In this study we investigated the effects of short-term treatments of small molecules to improve stem cell properties and differentiation capability. For this purpose, we used epigenetically active compounds, such as histone deacetylase inhibitors Trichostatin A (TSA) and sodium butyrate (NaBut), as well as multifunctional molecules of natural origin, such as retinoic acid (RA) and vitamin C (vitC). We observed that combinations of these compounds triggered upregulation of genes involved in pluripotency (KLF4, OCT4, NOTCH1, SOX2, NANOG, LIN28a, CMYC), but expression changes of these proteins were mild with only significant downregulation of Notch1. Also, some alterations in cell surface marker expression was established by flow cytometry with the most explicit changes in the expression of CD105 and CD117. Analysis of cellular energetics performed using Seahorse analyzer and assessment of gene expression related to cell metabolism and respiration (NRF1, HIF1α, PPARGC1A, ERRα, PKM, PDK1, LDHA, NFKB1, NFKB2, RELA, RELB, REL) revealed that small molecule treatments stimulate AFSCs toward a more energetically active phenotype. To induce cells to differentiate toward neurogenic lineage several different protocols including commercial supplements N2 and B27 together with RA were used and compared to the same differentiation protocols with the addition of a pre-induction step consisting of a combination of small molecules (vitC, TSA and RA). During differentiation the expression of several neural marker genes was analyzed (Nestin, MAP2, TUBB3, ALDH1L1, GFAP, CACNA1D, KCNJ12, KCNJ2, KCNH2) and the beneficial effect of small molecule treatment on differentiation potential was observed with upregulated gene expression. Differentiation was also confirmed by staining TUBB3, NCAM1, and Vimentin and assessed by secretion of BDNF. The results of this study provide valuable insights for the potential use of short-term small molecule treatments to improve stem cell characteristics and boost differentiation potential of AFSCs.

Pleiotropic effects of statins via interaction with the lipid bilayer: A combined approach.

Statins are effective inhibitors of cholesterol biosynthesis, largely used for prevention of cardiovascular diseases induced by hypercholesterolemia. However, their use in different clinical trials clearly indicate that the general benefits observed with statins are also related to effects beyond the cholesterol lowering. Increasing evidences suggest that some of these cholesterol-independent or "pleiotropic" effects of statins involve the interaction and modification of the membrane bilayers. In this manuscript, using a combined approach based on biophysical (electrochemical impedance spectroscopy on tethered bilayer lipid membranes) and biological methods (hemolysis on erythrocytes and immunocytochemistry on cancer cells), we demonstrate that lipophilic, but not hydrophilic statins are capable of reducing the damage caused by cholesterol-dependent cytolysins. This protection correlates with statins lipophilicity and capacity to interact with the lipid bilayer. Our data suggests lipophilic statins associate with membranes and interfere with the ability of cholesterol dependent cytolysins, to bind to membrane cholesterol. Evaluation of the capacity of statins to modulate cell membrane properties is essential for developing a correct therapeutic approach for cardiovascular diseases as well as for understanding the potential of this class of drugs as adjuvants for drug delivery.

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.

Amyloid ß oligomers inhibit growth of human cancer cells.

Effects of amyloid beta (Aß) oligomers on viability and function of cell lines such as NB4 (human acute promyelocytic leukemia), A549 (human lung cancer (adenocarcinomic alveolar basal epithelial tumor)) and MCF-7 (human breast cancer (invasive breast ductal carcinoma)) were investigated. Two types of Aß oligomers were used in the study. The first type was produced in the presence of oligomerization inhibitor, hexafluoroisopropanol (HFIP). The second type of amyloids was assembled in the absence of the inhibitor. The first type preparation was predominantly populated with dimers and trimers, while the second type contained mostly pentadecamers. These amyloid species exhibited different secondary protein structure with considerable amount of antiparallel ß sheet structural elements in HFIP oligomerized Aß mixtures. The effect of the cell growth inhibition, which was stronger in the case of HFIP Aß oligomers, was observed for all cell lines. Tests aiming at elucidating the effects of the amyloid species on cell cycles showed little differences between amyloid preparations. This prompts us to conclude that the effect on the cancer cell proliferation rate is less specific to the biological processes developing inside the cells during the proliferation. Therefore, cell growth inhibition may involve interactions with the peripheral parts of the cancer cells, such as a phospholipid membrane, and only in case of the NB4 cells, where accumulation of amyloid species inside the cells was detected, one may imply the opposite. In general, cancer cells were much less susceptible to the damaging effects of amyloid oligomers compared to earlier observations in mixed neuronal cell cultures.

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.

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.

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.