Project description:To search for factors regulating paternally imprinted genes (PEGs), we performed a genome-wide CRISPR/Cas9 screen in haploid hpESCs, and further analyzed the molecular phenotype upon perturbation of candidate PEGs regulators.
Project description:In this study, we investigate the impact of ladostigil on neuronal-like SH-SY5Y cells. As ladostigil may attenuate neurotoxicity and cell death that signify chronic stress conditions of an aging brain.
Project description:Forced expression of transcription factors for lineage reprogramming brings hope to cell-based therapy. However, its application is hampered by risks of potential genetic aberrations and tumorigenicity. Using defined small molecules in presence of gastric stromal cells as feeders, we reprogramed human gastric epithelia into induced multipotent endodermal progenitors (hiMEPs) with efficiency of up-to-6%. The hiMEPs expressed genes relative to endodermal lineages but not associating with pluripotency, and could be expanded clonogenically remaining as undifferentiated colonies. Upon induction, hiMEPs were able to give rise to multiple functional endodermal cell types, apart from ectodermal or mesodermal lineages. TGFβ inhibition and particular Wnt signaling activation were crucial in reprogramming process. Collective advantages of availability from donors without age restriction, capabilities in expansion and differentiation, and no concern of tumorigenesis, let hiMEPs have the considerable application potentials on cell therapies of diseases such as liver failure and diabetes, as well as personalized drug-screenings. Gastric epithelial cells (GECs) were isolated from human stomach. Human induced multipotent endodermal progenitors (hiMEPs) were reprogrammed from GECs by small molecules. The hiMEP-Heps were differentiated from hiMEPs under hepatic differentiation protocol. Fetal-Heps were isolated from aborted fetal liver. Definitve endoderm (DE), primitive gut tube (PGT), and posterior foregut (PFG) were endodermal stem cells derived form human enbryonic stem cells (hESCs).We used RNA sequencing and DNA methylation analysis to detail the global gene expression profile of GECs, hiMEPs, hiMEP-Heps, Fetal-Heps, DE, PGT and PFG to delineate the difference of these cells.
Project description:During mammalian pre-implantation development, the cells of the blastocyst’s inner cell mass differentiate into the epiblast and primitive endoderm lineages, which give rise to the fetus and extra-embryonic tissues, respectively. Extra-embryonic endoderm differentiation can be modeled in vitro by induced expression of GATA transcription factors in mouse embryonic stem cells. Here we use this GATA-inducible system to quantitatively monitor the dynamics of global proteomic changes during the early stages of this differentiation event and also investigate the fully differentiated phenotype, as represented by embryo-derived extra-embryonic endoderm (XEN) cells. Using mass spectrometry-based quantitative proteomic profiling with multivariate data analysis tools, we reproducibly quantified 2,336 proteins across three biological replicates and have identified clusters of proteins characterized by distinct, dynamic temporal abundance profiles. We first used this approach to highlight novel marker candidates of the pluripotent state and extra-embryonic endoderm differentiation. Through functional annotation enrichment analysis, we have shown that the downregulation of chromatin-modifying enzymes, the re-organization of membrane trafficking machinery and the breakdown of cell-cell adhesion are successive steps of the extra-embryonic differentiation process. Thus, applying a range of sophisticated clustering approaches to a time-resolved proteomic dataset has allowed the elucidation of complex biological processes which characterize stem cell differentiation and could establish a general paradigm for the investigation of these processes.
Project description:MicroRNAs (miRNAs), small non-coding RNAs that fine-tune gene expression, play multiple roles in the cell, including cell fate specification. We have analyzed the differential expression of miRNAs during fibroblast reprogramming into induced pluripotent stem cells (iPSCs) and endoderm induction in iPSCs upon treatment with high concentrations of Activin-A in reduced serum. During reprogramming, adult mouse fibroblasts are converted into cells that resemble embryonic stem cells (ESCs) according to standard molecular and functional assays for pluripotency. The reprogrammed iPSCs assume an ESC-like miRNA signature, marked by the strong induction of pluripotency clusters miR-290-295 and miR-302/367 and conversely the downregulation of the let-7 family. On the other hand, endoderm induction in iPSCs results in the upregulation of 13 miRNAs. Given that the liver and the pancreas are common derivatives of the endoderm, the comparison of the expression levels of these 13 upregulated miRNAs with those in hepatocytes and pancreatic islets suggests a trend of miRNA upregulation in the endoderm tending towards an islet phenotype rather than that of a hepatocyte. These observations provide insights into how differentiation may be guided more efficiently towards the endoderm and further into the liver or pancreas. Moreover, we also report novel miRNAs enriched for each of the cell types analyzed. Stemloop RT-qPCR gene expression profiling. REPROGRAMMING: Differentially expressed miRNAs were determined between iPSCs (n=5 clones) and parent tail-tip fibroblasts (n=5) using mESCs R1 (n=3) and D3 (n=3). DIFFERENTIATION: Differentially expressed miRNAs were also analyzed in two iPSC clones upon treatment with Activin-A (n=2 each), and between primary mouse hepatocytes (n=3) and pancreatic islets (n=3).