ESC Cell Research Articles

Two‐Way Regulation of MgFe‐LDH on ESC Cell Fate via Temporal Selective Activation of Different Cell Membrane Receptors

AbstractStem cell fate regulation by biomaterials has considerable therapeutic potential; however, the mechanisms whereby biomaterials instruct stem cells for accurate fate regulation still need to be elucidated. Layered double hydroxide (LDH) nanoparticles are widely used for tissue regeneration because of their excellent physicochemical properties and biocompatibility. This study reveals that MgFe‐LDH has bidirectional regulatory effects on the differentiation of mouse embryonic stem cells into neural progenitor cells, essentially suppressing cell differentiation on the 1st day and promoting cell differentiation on the 3rd day. Single‐cell transcriptome sequencing and simulated computation of structural analysis demonstrate that MgFe‐LDH interacts with the membrane receptors leukemia inhibitory factor receptor (LIFR, 15 nm above the cell membrane) and pached1 (PTCH1, 6 nm above the cell membrane) on the 1st and 3rd day of differentiation, respectively. LIFR has more contact with MgFe‐LDH than PTCH1 and relatively weaker electrostatic interaction energies (−11010.0 and −9829.3 kJ mol−1 for PTCH1 and LIFR, respectively). These factors determine receptor binding and activation priority, followed by cell fate regulation. The results indicate that MgFe‐LDH regulate the activity and fate of stem cells dually, thus creating possibilities for personalized therapy in neurodevelopment and regenerative medicine through an in‐depth study of cell membrane receptors.

TIM-3 regulates the proliferation by BDNF-mediated PI3K/AKT axis in the process of endometriosis

BackgroundT cell immunoglobulin and mucin domain-containing molecule-3 (TIM-3) initially discovered on the surface of Th1 cells, negatively regulates immune responses and mediates apoptosis of Th1 cells. An increasing number of studies have since shown that TIM-3 is crucial in the genesis and development of immune diseases, cancers, and chronic infectious illnesses. However, the effect of TIM-3 on endometriosis is still unknown.MethodsQuantitative real-time polymerase chain reaction, western blotting, and immunohistochemistry were used to measure TIM-3 levels in endometriosis. Cell Counting Kit-8, 5-ethynyl-2’-deoxyuridine, colony-forming, Transwell® migration, Matrigel® invasion, and flow cytometry assays were used to explore the function of TIM-3 in vitro, and xenograft experiments in nude mice were used to assess its role in vivo. According to the RNA seq, brain-derived neurotrophic factor (BDNF) was screened. The involvement of specific proliferation-related signaling molecules was determined by transfecting a plasmid and adding an inhibitor in vivo and in vitro.ResultsTIM-3 mRNA and protein expression levels were significantly higher in eutopic and ectopic endometrial tissues than in normal endometrial tissues. By examining the effects of TIM-3 overexpression and knockdown on cell proliferation, migration, and invasion in vitro, and lesions formation in vivo, we found that the expression of TIM-3 was positively correlated with cell proliferation and clone formation in vitro, as well as lesions growth in nude mice. By adding the phosphatidylinositol 3 kinase/protein kinase B(PI3K/AKT) pathway inhibitor LY294002 and knocking down PI3K, we further verified that TIM-3 promotes proliferation in vivo and in vitro via the PI3K pathway. By transfecting the plasmid into ESC cells and gave inhibitors to endometriotic rats models, we tested that TIM-3 regulates the proliferation by BDNF-mediated PI3K/AKT axis.ConclusionTIM-3 can promote the proliferation of endometriosis by BDNF-mediated PI3K/AKT axis in vivo and in vitro, which may provide a new therapeutic target for the treatment of endometriosis.

Rab27b promotes endometriosis by enhancing invasiveness of ESCs and promoting angiogenesis.

Endometriosis (EMS) is an estrogen-dependent disease which is characterized with estrogen-dependent growth of ectopic endometrium and increased local estrogen production. EMS performs tumor-like biological functions such as invasiveness and angiogenesis. Rab27b is a member of the Rab family of GTPases, which is strongly associated with the growth, invasion and metastasis of a variety of tumors. However, little is known about the function of Rab27b in EMS. In this study, we intended to investigate the impact of Rab27b and its downstream molecule in the development of EMS. Normal endometrium and endometriotic lesions were collected to investigate the expression levels of Rab27b. Then, ESCs were transfected with Rab27b siRNA. We analyzed the influence of Rab27b on the proliferation and invasive activity of ESCs. Conditioned media harvested from Rab27b siRNA-treated ESCs were used to treat HUVECs. HUVEC Tube formation and ELISA were performed to explored the interactions between ESCs and HUVEC. In addition, ESCs were treated with different concentrations of estrogen. Based on biological database predictions, we explored possible mechanisms through which estrogen regulates the expression of Rab27b. The expressions of Rab27b were significantly higher in endometriotic lesions than that in normal endometrium. Rab27b can promote the cell proliferation, migration and invasion of ESCs. The elevated expression of Rab27b, on the one hand, promotes the secretion of MMP9 and increases the invasiveness of ESCs. On the other hand, Rab27b may play a key role in the communication between ESC and endothelial cells, by simulating VEGF secretion and neovascularization. Besides, estrogen upregulated phosphorylated FOXO1 levels in ectopic ESCs, resulting in the promotion of Rab27b expression levels. Rab27b plays a key role in the development of EMS, which may provide new insights into the pathogenesis of EMS. Our findings may also contribute to the development of therapeutic interventions for EMS.

Abstract A013: Induced pluripotent stem cells derived from normal human esophageal epithelial cells identify transcription factor networks contributing to esophageal carcinogenesis

Abstract Esophageal cancer (EsC) is the sixth leading cause of cancer-related mortality worldwide. Esophageal squamous cell cancers (ESCC) typically arise in the upper and mid-esophagus, whereas esophageal adenocarcinomas (EAC) arise in the distal esophagus and gastroesophageal junction. Presently, epigenetic mechanisms contributing to initiation, progression, and dissemination of EsC have not been fully elucidated. The present study was undertaken to determine if induced pluripotent stem cells (iPSCs) derived from normal esophageal epithelial cells (Eso-iPSCs), EsC cell lines cultured in stemness enriched conditions, and cancer stem-like cells isolated from primary EsC specimens could be used to identify unique primitive stem-like transcription factor networks associated with epigenetic plasticity, chemoresistance, and metastatic potential of EsC cells. RNA-seq analysis demonstrated that genes differentially expressed in Eso-iPSC overlapped with transcriptome signatures in lung-iPSC (Lu-iPSC) which we previously generated to identify novel therapeutic targets in lung cancers; similar to Lu-iPSC, Eso-iPSC transcriptome signatures overlapped considerably more with small cell lung cancers (SCLC) compared to non-small cell lung cancers (NSCLC), possibly reflecting greater stemness in SCLC. We next compared transcriptome signatures of Eso-iPSC with stem-like-EAC enrichment models (cell lines and PDX). 6% and 8% of differentially expressed genes in Eso-iPSC uniquely overlapped with EAC or ESCC lines, respectively, whereas 24% were common to Eso-iPSC, EAC cells, and ESCC cells. Culturing of EAC cells on matrigel or propagation of EAC as primary patient derived xenografts increased commonalities of Eso-iPSC and EAC transcriptome signatures. Lastly, we compared transcriptomes of Eso-iPSC with Barrett’s esophagus (BE; a premalignant condition in which the squamous epithelium of the distal esophagus is replaced by metaplastic columnar epithelial cells) and EAC samples. By combining and stringently filtering transcriptome, ATAC-seq, DNA methylome, and ChIP-seq datasets for tissue- and disease-specific signatures, we identified several potentially druggable, master TFs mediating stemness/plasticity, chemoresistance, and metastatic potential which are shared between EsC lines and primary tumor specimens. Enrichment techniques promoted acquisition of more stem-like states in cultured EAC cells as reflected in multi-omic signatures of these cells relative to Eso-iPSC and primary EAC specimens. Collectively, these findings establish proof of principal for our experimental approach which is now being used to further characterize epigenetic mechanisms contributing to esophageal carcinogenesis and to identify novel targets for EsC therapy. Citation Format: Haitao Wang, Ruihong Wang, Katherine P. Prothro, Sichuan Xi, Weilong Hou, Xinwei Wu, Tuana Tolunay, Vivek Shukla, Mary R. Zhang, Stephanie J. Shiffka, Sudheer Gara, David S. Schrump. Induced pluripotent stem cells derived from normal human esophageal epithelial cells identify transcription factor networks contributing to esophageal carcinogenesis. [abstract]. In: Proceedings of the AACR Special Conference: Cancer Epigenomics; 2022 Oct 6-8; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2022;82(23 Suppl_2):Abstract nr A013.

Cytotoxic, antioxidant, and antimicrobial activities of Celery (Apium graveolens L.).

Celery (Apium graveolens Linn, Family: Apiaceae) is a common edible herb used as a spice in the traditional medicine of several nations since time immemorial. The whole plant is extensively used in cooking as soups and salads. A. graveolens has various pharmacological properties such as anticancer, anti-obesity, anti-hepatotoxic, and antihypertensive agents. Hence, it is of interest to document the in vitro cytotoxic, antioxidant, and antimicrobial activity of A. graveolens. The plants were collected in the local market, shade dried, and different parts of the plants were extracted with 70% ethanol using a cold maceration process. Antioxidant tests were performed based on the various radical scavenging methods. Antimicrobial activity and MIC were completed using the respective cup-plate and two-fold serial dilution method. In vitro cytotoxic studies were achieved by the MTT; Sulphorhodamine B assayed total cell protein content. DLA and ESC cells determined the short-term toxicity. The leaf extract exhibited significant antioxidant properties against NO, DPPH, ABTS, LPO, and HPO methods. Thus, potential inhibition against Gram-positive, Gram-negative, and fungal strains within the MIC ranges of 250-500 µg/ml was observed. All the extracts of the plant presented in the study revealed greater cytotoxicity effects against five respective cancer cell lines, L6, Vero, BRL 3A, A-549, L929, and L-929 with the ranging of 443-168.5 µg/ml. Thus, we show that A. graveolens possess a potential cytotoxic, antioxidant, and antimicrobial activity.

Development of immortalized human hepatocyte-like hybrid cells by fusion of multi-lineage progenitor cells with primary hepatocytes.

Human primary hepatocytes (PHs) are critical to studying liver functions, drug metabolism and toxicity. PHs isolated from livers that are unacceptable for transplantation have limited expansion and culture viability in vitro, in addition to rapidly deteriorating enzymatic functions. The unsuitability of immortalized hepato-carcinoma cell lines for this function has prompted studies to develop hepatocyte-like cells from alternative sources like ESC, iPS, and other stem cell types using differentiation protocols. This study describes a novel technique to produce expandable and functional hepatocyte-like cells from the fusion of an immortalized human umbilical cord blood derived cell line (E12 MLPC) to normal human primary hepatocytes. Multi-lineage progenitor cells (MLPC) comprise a small subset of mesenchymal-like cells isolated from human umbilical cord blood. MLPC are distinguishable from other mesenchymal-like cells by their extended expansion capacity (up to 80 cell doublings before senescence) and the ability to be differentiated into cells representative of endo-, meso- and ectodermal origins. Transfection of MLPC with the gene for telomerase reverse transcriptase (TERT) resulted in clonal cell lines that were capable of differentiation to different cellular outcomes while maintaining their functional immortality. A methodology for the development of immortalized hepatocyte-like hybrid cells by the in vitro fusion of human MLPC with normal human primary hepatocytes is reported. The resultant hybrid cells exhibited homology with hepatocytes by morphology, immunohistochemistry, urea and albumin production and gene expression. A medium that allows stable long-term expansion of hepatocyte-like fusion cells is described.

In vitro Differentiation of Human TERT-Transfected Multi-Lineage Progenitor Cells (MLPC) into Immortalized Hepatocyte-Like Cells.

BackgroundResearch directed towards drug development, metabolism, and liver functions often utilize primary hepatocytes (PH) for preliminary in vitro studies. Variability in the in vitro functionality of PH and the unsuitability of hepatocarcinoma cells for these studies have driven researchers to look to ESC, iPS, and other stem cell types using differentiation protocols to provide more reliable and available cells. This study describes the development of hepatocyte-like cells through the in vitro differentiation of human TERT-immortalized cord blood-derived multi-lineage progenitor cells (MLPC). The E12 clonal cell line derived from polyclonal TERT-transfected cells was used throughout the study.MethodsE12 MLPC were subjected to a three-step differentiation protocol using alternating combinations of growth factors, cytokines, and maturational factors. Cells at various stages of differentiation were analyzed for consistency with PH by morphology, immunohistochemistry, urea production, and gene expression.ResultsE12 MLPC were shown to significantly change morphology with each stage of differentiation. Coincidental with the morphological changes in the cells, immunohistochemistry data documented the differentiation to committed endoderm by the expression of SOX-17 and GATA-4; the progression to committed hepatocyte-like cells by the expression of a large number of markers including α-fetoprotein and albumin; and the final differentiation by the expression of nuclear and cytoplasmic HNF4. Fully differentiated cells demonstrated gene expression, urea production, and immunohistochemistry consistent with PH. A methodology and medium formulation to continuously expand the E12-derived hepatocyte-like cells is described.ConclusionThe availability of immortalized hepatocyte-like cell lines could provide a consistent tool for the study of hepatic diseases, drug discovery, and the development of cellular therapies for liver disorders. Utilization of these techniques could provide a basis for the development of bridge therapies for liver failure patients awaiting transplant.

Generation of Dip2a homozygous knockout murine ES cell line IBMSe001-A-1 via CRISPR/Cas9 technology.

DIP2A mutation is associated with abnormal brain development and diseases including dyslexia, autism and Alzheimer's disease. However, the role and the involved mechanisms remain unknown. To study the biological function of DIP2A during mESCs neural differentiation in early neural development, we generated a Dip2a homozygous knockout 46C ESC cell line using CRISPR/Cas9 genome editing technology. The eighth exon of Dip2a gene was replaced with PGK-Puro-P2A-mCherry. This 46C-Dip2a KO cell line offers a useful resource to investigate the molecular mechanisms of DIP2A in the process of cell fate determination, as well as a potential source of building disease mouse model.

Inhibition of MAGEA2 regulates pluripotency, proliferation, apoptosis, and differentiation in mouse embryonic stem cells.

Mouse embryonic stem cells (mESCs)exhibit self-renewal and pluripotency, can differentiate into all three germ layers, and serve as an essential model in stem cell research and for potential clinical application in regenerative medicine. Melanoma-associated antigen A2 (MAGEA2) is not expressed in normal somatic cells but rather in different types of cancer, especially in undifferentiated cells, such as in the testis, differentiating cells, and ESCs. However, the role of MAGEA2 in mESCs remains to be clarified. Accordingly, in this study, we examined the expression and functions of MAGEA2 in mESCs. MAGEA2 messenger RNA (mRNA) expression was decreased during mESCs differentiation. MAGEA2 function was then evaluated in knockdown mESC. MAGEA2 knockdown resulted in decreased pluripotency marker gene expression in mESCs consequent to increased Erk1/2 phosphorylation. Decreased MAGEA2 expression inhibited mESC proliferation via S phase cell cycle arrest with a subsequent decrease in cell cycle-associated genes Cdk1, Cdk2, Cyclin A1, Cyclin D1, and Cdc25a. Apoptotic mESCs markedly increased along with cleaved forms of caspases 3, 6, and 7 and PARP expression, confirming caspase-dependent apoptosis. MAGEA2 knockdown significantly decreased embryoid body size in vitro when cells were differentiated naturally and teratoma size in vivo, concomitant with decreased ectoderm marker gene expression. These findings suggested that MAGEA2 regulates ESC pluripotency, proliferation, cell cycle, apoptosis, and differentiation. The enhanced understanding of the regulatory mechanisms underlying diverse mESC characteristics will facilitate the clinical application of mESCs.

Investigation into the expression levels of MAGEA6 in esophageal squamous cell carcinoma and esophageal adenocarcinoma tissues.

Esophageal carcinoma (EC) is the sixth most deadly of all cancers. It is among the most malignant cancers due to its highly aggressive nature and low survival rate. The incidence of EC is high in Asia, particularly in Southern areas including China, Iran and Japan. There is a large body of evidence to suggest an association between the melanoma antigen gene (MAGE) family and the initiation of cancer; however, there is no clear evidence to suggest an association between EC and MAGE. Discovery of the chemical and physiological processes relevant to the occurrence of EC is vital for clinicians to diagnose and treat this highly aggressive cancer. The present study focused on the association of EC with the expression of MAGE family member A6 (MAGEA6) at the mRNA and protein levels using gene chip, reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and immunohistochemistry. The expression of MAGEA6 in human esophageal squamous cell carcinoma (ESCC) and esophageal adenocarcinoma (EAC) tissue samples were compared with those in paracancerous tissue. The result of the gene chip assay revealed that as the generation grew, there was a significant increase in MAGEA6 transcription in the esophageal epithelial cell line, SHEE Different ESC cell lines also exhibited a significantly higher transcription of MAGEA6 compared with the HaCaT cell line, as determined via reverse transcription-quantitative PCR. An higher positive rate of MAGEA6 expression in ESCC and EAC tissues was also revealed when compared with paracancerous tissues, as determined via immunohistochemistry. The results indicated that MAGEA6 is highly transcribed and expressed in the development of EC and may therefore serve as a novel biomarker for the diagnosis or treatment of EC.

X-ray fluorescence nano-imaging of long-term operated solid oxide electrolysis cells

In this work, we investigate using nanoscale X-ray fluorescence spectroscopy the degradation processes of a cathode and an electrolyte supported cell, produced by two different suppliers, that have been long-term operated in electrolysis mode. High-resolution compositional analysis in combination with high lateral resolution are presented in the format of a 2D map with 50 nm resolution for Ni, Y, Ce, Gd, La, Sr, Co, Fe. We report on the different elements that are diffusing already during the sintering process as well as their evolution after long-term operation. In both type of cell, Co is the element that is highly segregating from the LSCF electrode during the electrochemical reaction. The CGO barrier does not prevent the diffusion of Sr in the electrolyte of the CSC cell while it does in the ESC cell. In addition, the contact layer between the hydrogen electrode and the electrolyte in the ESC cell seems to prevent the Ni depletion. A high concentration of gadolinium from the CGO layer is visible at the CGO/electrolyte interface on the oxygen electrode side as well as on the hydrogen electrode side in the ESC cell. The gadolinium concentration significantly increases during the cell operation to form a layer of a few micrometers.

Human Induced Pluripotent Stem Cells in the Curative Treatment of Diabetes and Potential Impediments Ahead.

The successful landmark discovery of mouse and human inducible pluripotential stem cells (iPSC's) by Takahashi and Yamanaka in 2006 and 2007 has triggered a revolution in the potential generation of self-compatible cells for regenerative medicine, and further opened up a new avenue for "disease in dish" drug screening of self-target cells (Neofytou et al. 2015). The introduction of four 'Yamanaka' transcription factors through viral or other transfection of mature cells can induce pluripotency and acquired plasticity. These factors include transduction with octamer-binding transcription factor-4 (Oct-4), nanog homeobox (Nanog), sex-determining region Y-box-2 (Sox-2) and MYC protooncogene (cMyc). Such cells become iPSC's (Takahashi and Yamanaka 2006). These reprogrammed cells exhibit increased telomerase activity and have a hypomethylated gene promotor region similar to embryonic stem cells (ESC's). These milestone discoveries have generated immense hope that diseases such as diabetes could be treated and effectively cured by transplantation of self-compatible, personalized autologous stem cell transplantation of β-cells that release physiological insulin under glycemic control (Maehr et al. 2009; Park et al. 2008) (Fig. 1). Diabetes is a profligate disease of disordered glucose metabolism resulting from an absolute or relative deficiency of insulin, the consequences of which lead to immense socio-economic societal burden. While there are many different types of diabetes, the two major types (type 1 diabetes (T1DM) and type 2 diabetes (T2DM) are caused respectively by immune-mediated destruction (T1DM) or malfunctioning (T2DM) insulin-producing β-cells within the endocrine pancreas, the islets of Langerhans (Atkinson et al. 2011; Holman et al. 2015; You and Henneberg 2016). Almost 425million people are affected by the global burden of diabetes, and this is predicted to increase by 48% (629million) by 2045 (International Diabetes Federation Atlas 8th Ed 2018). Whole pancreas or islet cell transplantation offer an effective alternative to injected insulin, but both require lifelong potent immunosuppression to control both allo-and autoimmunity. Whole pancreas transplantation involves invasive complex surgery and is associated with greater morbidity and occasional mortality, while islet transplantation involves a minimally invasive intraportal hepatic infusion. Generally, whole pancreas transplantation provides greater metabolic reserve, but this may be matched by cumulative multiple islet infusions to achieve insulin independence. An additional challenge of islet transplantation is progressive loss of complete insulin independence over time, which may be multifactorial, the dominant factor however being ineffective control of autoimmunity. Both whole pancreas and islet transplantation are restricted to patients at risk of severe hypoglycemia that cannot be stabilized by alternate means, or in recipients that are already immunosuppressed in order to sustain a kidney or other solid organ transplant. The risks of chronic immunosuppression and the scarcity of human organ donors mean that both of these transplantation therapies cannot presently be extended to the broader diabetic population (Shapiro 2011; Shapiro et al. 2006). Recent progress in xenotransplantation of multiple knock-out 'humanized' pig islets could offer one potential solution, perhaps aided by clustered regularly interspaced short palindromic repeats/CRISPR associated-9 (CRISPR/Cas-9) gene editing approaches, but this remains to be proven in practice. Human stem cell derived new β-cell products could effectively address the global supply challenge for broad application across all forms of diabetes, but recurrent autoimmunity may still remain an insurmountable challenge. Considerable progress in the generation of human stem cell derived SC-β cells from ESC, iPS and other adult cell sources such as mesenchymal stem cells (MSCs) offer huge hope that a personalized, 'syngeneic' cell could be transplanted without risk of alloimmunity, thereby securing sufficient supply to meet future global demand (Cito et al. 2018).

Direct conversion of fibroblasts traces the way back to our first organ-the placenta.

Cell fate determination has always fascinated scientists predominantly embryologists and challenged them to decipher master genes or networks of transcriptional factors which together with cell signaling cascades act as key regulators for the cell lineage allocation. With the rediscovery of the pluripotent precursor cells in an adult body and the possibility to induce pluripotency in somatic cells (iPSCs), as sources for alternative therapeutic options in translational medicine, this field has attracted high interest in medical research and application (1). iPSC have several advantages over embryonic stem cells such as avoiding tumor formation and to circumvent ethical concerns. Meanwhile the understanding of pluripotency has broadened thanks to abundant studies on converting ESC and somatic cells into another cell fate using predominantly transfection of a bunch of lineage specific transcription factors. While many studies focused on the regulation of ESC conversion less is known about the hierarchically organized network of transcriptional factors which determine the trophoblast lineage for placenta development.

Enhanced cyclooxygenase-2 expression levels and metalloproteinase 2 and 9 activation by Hexachlorobenzene in human endometrial stromal cells

Hexachlorobenzene (HCB) is an organochlorine pesticide that induces toxic reproductive effects in laboratory animals. It is a dioxin-like compound and a weak ligand of the aryl hydrocarbon receptor (AhR). Endometriosis is characterized by the presence of functional endometrial tissues outside the uterine cavity. Experimental studies indicate that exposure to organochlorines can interfere with both hormonal regulation and immune function to promote endometriosis. Altered expression of metalloproteinases (MMPs) in patients with endometriosis, suggests that MMPs may play a critical role. In the endometriotic lesions, prostaglandin E2 (PGE2) produced by cyclooxygenase-2 (COX-2), binds to its EP4 receptor (EP4), and via c-Src kinase induces MMPs activation, promoting endometriosis. We examined the HCB action on MMP-2 and MMP-9 activities and expression, COX-2 levels, PGE2 signaling, and the AhR involvement in HCB-induced effects. We have used different in vitro models: (1) human endometrial stromal cell line T-HESC, (2) primary cultures of Human Uterine Fibroblast (HUF), and (3) primary cultures of endometrial stromal cells from eutopic endometrium of control (CESC) and subjects with endometriosis (EESC). Our results show that HCB enhances MMP-2 and MMP-9 activities in T-HESC, HUF and ESC cells. The MMP-9 levels were elevated in all models, while the MMP-2 expression only increased in ESC cells. HCB enhanced COX-2 and EP4 expression, PGE2 secretion and the c-Src kinase activation in T-HESC. Besides, we observed that AhR is implicated in these HCB-induced effects. In conclusion, our results show that HCB exposure could contribute to endometriosis development, affecting inflammation and invasion parameters of human endometrial cells.

Modeling the Dynamic AMD-Associated Chronic Oxidative Stress Changes in Human ESC and iPSC-Derived RPE Cells.

Here we use human embryonic stem cells (hESCs) and human-induced pluripotent stem cell (hiPSC)-derived retinal pigment epithelium (RPE) cells to model chronic oxidative stress in vitro. This model allows us to understand the evolution of chronic stress response in RPE in vivo, as well as to monitor microRNAs changes. Finally, we use this in vitro model to identify a partial agonist of NRF2 that is protective against reactive oxygen species (ROS)-induced cytotoxicity. The hESCs and hiPSCs were differentiated toward an RPE fate. Upon maturation, RPE cells were subjected to chronic oxidative stress using Paraquat (PQ). The cells were then analyzed using immunocytochemistry and quantitative RT-PCR to look for changes in gene expression and microRNA changes. Small molecules targeting NRF2 pathways were utilized to look for protection against oxidative stress-induced apoptosis. We show that 160 μM PQ can be used to generate a model of chronic oxidative stress in RPE cells derived from hESCs and hiPSCs. Using this model, we characterize the NRF2 pathway effectors during the early and late stages of chronic oxidative stress and identify microRNAs changes during oxidative stress. We find that hsa-miR144 modulates NRF2 activity during ROS stress. Lastly, we found a small molecule modulator of NRF2 that plays a protective role against oxidative stress-induced RPE apoptosis. In summary, pluripotent stem cell-derived retinal cells can be used to model retinal diseases in a dish. This can provide an unprecedented opportunity to understand the evolution of disease processes and allow us to identify novel therapeutics.

Tip60 complex binds to active Pol II promoters and a subset of enhancers and co-regulates the c-Myc network in mouse embryonic stem cells.

BackgroundTip60 (KAT5) is the histone acetyltransferase (HAT) of the mammalian Tip60/NuA4 complex. While Tip60 is important for early mouse development and mouse embryonic stem cell (mESC) pluripotency, the function of Tip60 as reflected in a genome-wide context is not yet well understood.ResultsGel filtration of nuclear mESCs extracts indicate incorporation of Tip60 into large molecular complexes and exclude the existence of large quantities of “free” Tip60 within the nuclei of ESCs. Thus, monitoring of Tip60 binding to the genome should reflect the behaviour of Tip60-containing complexes. The genome-wide mapping of Tip60 binding in mESCs by chromatin immunoprecipitation (ChIP) coupled with high-throughput sequencing (ChIP-seq) shows that the Tip60 complex is present at promoter regions of predominantly active genes that are bound by RNA polymerase II (Pol II) and contain the H3K4me3 histone mark. The coactivator HAT complexes, Tip60- and Mof (KAT8)-containing (NSL and MSL), show a global overlap at promoters, whereas distinct binding profiles at enhancers suggest different regulatory functions of each essential HAT complex. Interestingly, Tip60 enrichment peaks at about 200 bp downstream of the transcription start sites suggesting a function for the Tip60 complexes in addition to histone acetylation. The comparison of genome-wide binding profiles of Tip60 and c-Myc, a somatic cell reprogramming factor that binds predominantly to active genes in mESCs, demonstrate that Tip60 and c-Myc co-bind at 50–60 % of their binding sites. We also show that the Tip60 complex binds to a subset of bivalent developmental genes and defines a set of mESC-specific enhancer as well as super-enhancer regions.ConclusionsOur study suggests that the Tip60 complex functions as a global transcriptional co-activator at most active Pol II promoters, co-regulates the ESC-specific c-Myc network, important for ESC self-renewal and cell metabolism and acts at a subset of active distal regulatory elements, or super enhancers, in mESCs.Electronic supplementary materialThe online version of this article (doi:10.1186/s13072-015-0039-z) contains supplementary material, which is available to authorized users.

Results for a New SOFC-Stack Design with Parallel-Connected Cells

The TU Clausthal and CUTEC institute have teamed up to design, build and test a new stack architecture, based on repeating units with cells connected in parallel. This approach allows the use of electrically conducting seals like brazes and can be realized without any glass sealing. Unlike conventional (serially connected) stacks, where all cells operate at the same current but individual voltage, in this setup each cell contributes as much current as possible at a common voltage. Thereby accelerated degeneration caused by undervoltage and the associated oxidation of the nickel catalyst can be completely overcome. The concept uses two cells in electrical parallel connection with the anodes facing each other to form a repeating unit. Two designs have been realized: An all ceramic housing made from 3YSZ, sealed with reactive air brazing (RAB) and a metallic one with a Crofer 22 APU housing, joined by laser beam welding. The housing materials have been chosen with respect to their thermal expansion coefficient (TEC) matching the TEC of the used ESC cells. Special interconnectors have been produced via hydroforming to create an even flow field while providing sufficient contact area. The paper will focus on the test results of the repeating units under various operation conditions and the analysis of the degradation of components and joints. Polarization curves for the twincell repeating unit and the individuell cells under H2 and air at 880°C are depicted in figure one. Figure 1

Ionizing radiation is a potent inducer of mitotic recombination in mouse embryonic stem cells

Maintenance of genomic integrity in embryonic cells is pivotal to proper embryogenesis, organogenesis and to the continuity of species. Cultured mouse embryonic stem cells (mESCs), a model for early embryonic cells, differ from cultured somatic cells in their capacity to remodel chromatin, in their repertoire of DNA repair enzymes, and in the regulation of cell cycle checkpoints. Using 129XC3HF1 mESCs heterozygous for Aprt, we characterized loss of Aprt heterozygosity after exposure to ionizing radiation. We report here that the frequency of loss of heterozygosity mutants in mESCs can be induced several hundred-fold by exposure to 5–10 Gy of X-rays. This induction is 50–100-fold higher than the induction reported for mouse adult or embryonic fibroblasts. The primary mechanism underlying the elevated loss of heterozygosity after irradiation is mitotic recombination, with lesser contributions from deletions and gene conversions that span Aprt. Aprt point mutations and epigenetic inactivation are very rare in mESCs compared to fibroblasts. Mouse ESCs, therefore, are distinctive in their response to ionizing radiation and studies of differentiated cells may underestimate the mutagenic effects of ionizing radiation on ESC or other stem cells. Our findings are important to understanding the biological effects of ionizing radiation on early development and carcinogenesis.

Stem Cells in Heart Failure

Stem Cells in Heart Failure

Vitrification of mouse embryo derived ICM cells: a tool for preserving embryonic stem cell potential?

OBJECTIVE: Vitrification technology presents new opportunities for preservation of embryo derived stem cells without first establishing a viable ESC cell line. This study explores the possibility of cryopreserving the inner cell mass (ICM) from blastocysts using vitrification.