Molecular changes in endometrium origin stromal cells during initiation of cardiomyogenic differentiation induced with Decitabine, Angiotensin II and TGF- ß1.

Stem cells' differentiation toward cardiac lineage is a complex process dependent on various alterations in molecular basis and regulation pathways. The aim of the study is to show that endometrium-derived stromal cells - menstrual, endometrial and endometriotic, could be an attractive source for examination of the mechanisms underlying cardiomyogenesis. After treatment with Decitabine, Angiotensin II and TGF-ß1, cells demonstrated morphological dedifferentiation into early cardiomyocyte-like cells and expressed CD36, CD106, CD172a typically used to sort for human pluripotent stem cell-derived cardiomyocytes. RT-qPCR revealed changed cells' genetic profiles, as majority of cardiac lineage differentiation related genes and cardiac ion channels (calcium, sodium, potassium) coding genes were upregulated after 6 and 13 days of exposure. Additionally, analysis of expression of various signaling proteins (FOXO1, PDGFB, TGFBR1, mTOR, VEGFA, WNT4, Notch1) coding genes showed differences between cell cultures as they seem to employ distinct signaling pathways through differentiation initiation. Early stages of differentiation had biggest impact on cardiomyogenesis related proteins (Nkx-2.5, EZH2, FOXO3a, H3K9Ac) levels, as we noticed after conducting Western blot and as expected, early cardiac transcription factor Nkx-2.5 was highly expressed and localized in nucleus of differentiating cells. These findings led us to assess endometrium origin stromal cells' potential to differentiate towards cardiomyogenic lineage and better understand the regulation of complex differentiation processes in ex vivo model systems.

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.

The Effects of the Follicle-Stimulating Hormone on Human Follicular Fluid-Derived Stromal Cells.

The prevalence of infertility is getting higher over the years. The increasing age of first-time parents, although economically more desirable, can cause various biological problems from low natural conception rate to poor pregnancy outcomes. The growing demand for assisted reproductive technology procedures worldwide draws medical specialists' and scientists' attention to various elements which could lead to successful conception, such as follicular fluid (FF) and hormones. In this study, we analyzed the effects of exposure to follicle-stimulating hormone (FSH) on FF-derived stromal cells isolated from females admitted for treatment due to infertility, participating in assisted reproductive technologies procedures. We demonstrated that FF stromal cells are positive for mesenchymal stromal cell surface markers (CD90+, CD44+, CD166+) and showed that FSH has no impact on FF stromal cell morphology yet lowers proliferation rate. Using a real-time polymerase chain reaction method, we indicated that the expression of PTGS2 is significantly downregulated in FF sediment cells of patients who did not conceive; furthermore, we showed that FSH can affect the expression of ovarian follicle development and FSH response-related genes differentially depending on the length of exposure and that levels of ovulatory cascade genes differ in conceived and not-conceived patients' FF stromal cells. Using mass spectrometry analysis, we identified 97 proteins secreted by FF stromal cells. The identified proteins are related to stress response, positive regulation of apoptotic cell clearance and embryo implantation.

Characterization of Epigenetic and Molecular Factors in Endometrium of Females with Infertility.

Infertility is one of the most rapidly increasing global health concerns of the 21st century. Embryo quality and endometrial thickness and receptivity are the main factors for successful embryo implantation and pregnancy development. Nevertheless, until now, there has been a lack of understanding about the regulation of human endometrium function and its structure. This raises the demand for more research of the human endometrium in these fields. In our study, we analyzed the genetic and epigenetic changes of endometrial tissue's samples isolated from females admitted for treatment due to male infertility and females diagnosed with reproductive pathologies, who are preparing for assisted reproductive technologies procedures. Using real-time polymerase chain reaction method, we demonstrated that endometrium of females with reproductive pathology has significantly upregulated decidualization related genes HAND2, MUC1, CSF2, increased expression of angiogenesis related gene PDGFA, and increases of overall immune response and inflammation-related genes expression with significant changes of RELA and CXCL10 genes expression. Females with reproductive pathology have altered endometrium epigenetic regulation since expression of miRNAs-specifically, miRNA-34a, miRNA-223, and miRNA-125b-is lower in endometrium of females with reproductive pathology. Our findings suggest that the potential changes in genetic and epigenetic profile of endometrium from females with reproductive pathology could enrich the knowledge in the field of core biological knowledge and treatment of reproductive impairments.

Potential Prognostic Markers for Relapsed/Refractory vs. Responsive Acute Myeloid Leukemia.

Acute myeloid leukemia (AML) is a heterogeneous disease. A significant proportion of AML patients is refractory to clinical treatment or relapses. Our aim is to determine new potential AML clinical treatment prognosis markers. We investigated various cell fate and epigenetic regulation important gene level differences between refractory and responsive AML patient groups at diagnosis stage and after clinical treatment using RT-qPCR. We demonstrated that oncogenic MYC and WT1 and metabolic IDH1 gene expression was significantly higher and cell cycle inhibitor CDKN1A (p21) gene expression was significantly lower in refractory patients' bone marrow cells compared to treatment responsive patients both at diagnosis and after clinical treatment. Moreover, we determined that, compared to clinical treatment responsive patients, refractory patients possess a significantly higher gene expression of histone deacetylase 2 (HDAC2) and epigenetic DNA modulator TET1 and a significantly lower gene expression of lysine acetyltransferase 6A (KAT6A) and nucleosome remodeling and deacetylase (NuRD) complex component GATAD2A. We suggest that MYC, WT1, IDH1, CDKN1A, HDAC2, TET1, KAT6A and GATAD2A gene expression changes might characterize refractory AML. Thus, they might be useful for AML prognosis. Additionally, we suggest that epigenetic modulation might be beneficial in combination with standard treatment.

Prognostic Gene Predictors of Gestational Diabetes in Endometrium and Follicular Fluid of Women after Infertility.

Background and objectives. Gestational diabetes mellitus is an increasingly diagnosed metabolic disorder during pregnancy with unknown pathological pathways. Taking into account the growing numbers of women who are conceiving after assisted reproductive technologies, they comprise an engaging target group for gestational diabetes mellitus etiopathogenesis research. In terms of metabolism and genetics, as the evidence shows, both unexplained infertility and gestational diabetes mellitus pose challenges for their interpretation due to the complex bodily processes. Materials and Methods. Our study examined the expression of genes (IGF2, GRB10, CRTC2, HMGA2, ESR1, DLK1, SLC6A15, GPT2, PLAGL1) associated with glucose metabolism in unexplained infertility patients who conceived after in vitro fertilization procedure, were diagnosed with GDM and their findings were compared with control population. Results. There were no significant differences in gene expression of endometrium stromal cells between healthy pregnant women and women with gestational diabetes, although the significant downregulation of CRTC2 was observed in the follicular fluid of women with gestational diabetes mellitus. Moreover, expression of HMGA2 and ESR1 was significantly reduced in FF cells when compared to endometrial cells. Conclusions. These findings may indicate about the importance of follicular fluid as an indicator for gestational diabetes and should be explored more by further research.

Menstrual Blood-Derived Endometrial Stem Cells' Impact for the Treatment Perspective of Female Infertility.

When looking for the causes and treatments of infertility, much attention is paid to one of the reproductive tissues-the endometrium. Therefore, endometrial stem cells are an attractive target for infertility studies in women of unexplained origin. Menstrual blood stem cells (MenSCs) are morphologically and functionally similar to cells derived directly from the endometrium; with dual expression of mesenchymal and embryonic cell markers, they proliferate and regenerate better than bone marrow mesenchymal stem cells. In addition, menstrual blood stem cells are extracted in a non-invasive and painless manner. In our study, we analyzed the characteristics and the potential for decidualization of menstrual blood stem cells isolated from healthy volunteers and women diagnosed with infertility. We demonstrated that MenSCs express CD44, CD166, CD16, CD15, BMSC, CD56, CD13 and HLA-ABC surface markers, have proliferative properties, and after induction of menstrual stem cell differentiation into epithelial direction, expression of genes related to decidualization (PRL, ESR, IGFBP and FOXO1) and angiogenesis (HIF1, VEGFR2 and VEGFR3) increased. Additionally, the p53, p21, H3K27me3 and HyperAcH4 proteins' expression increased during MenSCs decidualization, they secrete proteins that are involved in the regulation of the actin cytoskeleton, estrogen and relaxin signaling pathways and the management of inflammatory processes. Our findings reveal the potential use of MenSCs for the treatment of reproductive disorders.

Pharmaceutical Drug Metformin and MCL1 Inhibitor S63845 Exhibit Anticancer Activity in Myeloid Leukemia Cells via Redox Remodeling.

Metabolic landscape and sensitivity to apoptosis induction play a crucial role in acute myeloid leukemia (AML) resistance. Therefore, we investigated the effect of metformin, a medication that also acts as an inhibitor of oxidative phosphorylation (OXPHOS), and MCL-1 inhibitor S63845 in AML cell lines NB4, KG1 and chemoresistant KG1A cells. The impact of compounds was evaluated using fluorescence-based metabolic flux analysis, assessment of mitochondrial Δψ and cellular ROS, trypan blue exclusion, Annexin V-PI and XTT tests for cell death and cytotoxicity estimations, also RT-qPCR and Western blot for gene and protein expression. Treatment with metformin resulted in significant downregulation of OXPHOS; however, increase in glycolysis was observed in NB4 and KG1A cells. In contrast, treatment with S63845 slightly increased the rate of OXPHOS in KG1 and KG1A cells, although it profoundly diminished the rate of glycolysis. Generally, combined treatment had stronger inhibitory effects on cellular metabolism and ATP levels. Furthermore, results revealed that treatment with metformin, S63845 and their combinations induced apoptosis in AML cells. In addition, level of apoptotic cell death correlated with cellular ROS induction, as well as with downregulation of tumor suppressor protein MYC. In summary, we show that modulation of redox-stress could have a potential anticancer activity in AML cells.