Project description:We report the interaction between HEB and PRC2 components in mouse embryonic stem cells (ESCs) ChIP-Seq of HEB and SMAD2/3 in mouse ESC and derived endoderm
Project description:We report the interaction between HEB and PRC2 components in mouse embryonic stem cells (ESCs) We profiled gene expression for biological triplicate shControl- and shHEB-infected ESCs and differentiated embryoid body (EB) in serum-free defined culture system with or without activin treatment
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:Generation of oligodendrocytes (OLs) is a sophisticated multistep process, mechanistic underpinnings of which are not fully understood and demand further investigation. To systematically profile proteome dynamics during human embryonic stem cell (hESC) differentiation into OLs, we applied in-depth quantitative proteomics at different developmental stages and monitored changes in protein abundance using a multiplexed tandem mass tag (TMT) based proteomics approach. Findings: Our proteome data provided a comprehensive protein expression profile that highlighted specific expression clusters based on the protein abundances over the course of human OL lineage differentiation. The proteome profile of OL lineage cells revealed 378 proteins that were specifically up-regulated only in one differentiation stage. In addition, comparative pairwise analysis of differentiation stages demonstrated that abundances of 352 proteins differentially changed between consecutive differentiation time points. Our results highlighted the eminence of the planar cell polarity (PCP) signaling and autophagy (particularly macroautophagy) in the progression of OL lineage differentiation
Project description:During mammalian embryonic development, the primitive streak is initiates the differentiation of pluripotent epiblast cells into germ layers. Pluripotency can be reacquired in committed somatic cells using a combination of handful transcription factors, such as OCT3/4, SOX2, KLF4 and c-MYC (hereafter referred to as OSKM), albeit with low efficiency . Here we show that, during the OSKM-induced reprogramming process toward pluripotency in human cells, intermediate cells transiently show gene expression profiles resembling mesendoderm, which is a major component of the primitive streak. Based on these findings, we discover that forkhead box H1 (FOXH1), a transcription factor required for anterior primitive streak specification during early development, significantly enhances the reprogramming efficiency of human fibroblasts by promoting their maturation, including the mesenchymal to epithelial transition and the activation of late pluripotent markers. These results demonstrate that during the reprogramming process, human somatic cells go through a transient state that resembles mesendoderm. Human differentiated progeny derived from pluripotent stem cells, N=13 Human undifferentiated pluripotent stem cells, N=6 Transgenic ESC line, N=6 Human tissues, N=29 Human tissue-derived cells, N=20 Human nascent reprogrammed cells, N=95 Mouse cells, N=12
Project description:Human lung organoids (HLOs) were derived from human embryonic stem cell line H9 NIH registry #0062. Sequencing of HLOs was performed by the UM DNA Sequencing Core, using Illumina Hi-Seq platform and single-end 50 bp reads. The UM Bioinformatics Core downloaded the reads files from the Sequencing Core storage, and concatenated those into a single .fastq file for each sample. 3 HLOs were cultured for 65 days and 3 HLOs were cultured in vitro for 110 days. HLO Culture Protocol 4-day Activin A (R&D systems) differentiation protocol was used to derive definitive endoderm from human pluripotent stem cells (hPSCs). Cells were treated with Activin A (100ngml-1) for three consecutive days in RPMI 1640 media (Life Technologies) with increasing concentrations of 0%, 0.2% and 2% HyClone defined fetal bovine serum (dFBS, Thermo Scientific). The fourth day was a repeat of day 3 media (2% HyClone FBS). After differentiation into definitive endoderm, cells were incubated in foregut media (advanced DMEM/F12 plus N-2 and B27 supplement, 10mM Hepes, 1x L-Glutamine (200mM), 1x Penicillin-streptomycin (5,000 U/mL, all from Life Technologies)) with 200ng/mL Noggin (NOG, R&D Systems) and 10uM SB431542 (SB, Stemgent) for 4 days, SB, 500ng/mL FGF4 (R&D Systems), and 2 uM CHIR99021 (Chiron, Stemgent), and 1uM SAG (Enzo Life Sciences) for 4-6 days. After 4 days with treatment of growth factors, three-dimensional floating spheroids were present in the culture. Three-dimensional spheroids were transferred into Matrigel to support 3D growth. Spheroids were embedded in a droplet of Matrigel (BD Bioscience #356237) in one well of a 24 well plate, and incubated at room temperature for 10 minutes. After the Matrigel solidified, foregut media with 1% Fetal bovine serum (FBS, CAT#: 16000-044, Life Technologies) and 500ng/mL FGF10 (R&D Systems) were overlaid and replaced every 4 days. Organoids were transferred into new Matrigel droplets every 10 to 15 days.
Project description:Tumor formation constitutes a major obstacle to the clinical application of embryonic stem cells (ESCs). As P-RPCs could successfully integrate into host eyes without development of teratomas or NOG, we sought to identify differentially expressed genes between P-RPCs and ESC-RPCs through genome-wide transcript profiling. Inhibition of Wnt signaling by DKK1 promotes the commitment of ESC-RPCs to more mature retinal cells and reduces the occurrence of NOG to 3%. DKK1-treated ESC-RPCs efficiently integrate to the host retina, form synaptic connections and restore visual function. Here, we report that further differentiation of ESC-derived neural progenitors into retinal progenitor cells (ESC-RPCs) completely eliminates teratomas in ocular transplantation. However, tumor-like neural overgrowth (NOG) occurs in 61% of transplanted eyes. ESC-RPCs were divided into two groups according to the differentiation stages for RNA extraction and hybridization on Affymetrix microarrays. Normal control ESC-RPCs (N) were represented the homogeneous populations of early stage expression profiles of immature ESC-RPCs. DKK1 treated ESC-RPCs (D) were represented the homogeneous populations of late stage expression profiles of further differetiated mature ESC-RPCs. To sought the pathways involved in the proliferation and oncogenesis of ESC-RPCs, the newborn C57 mice reitinal progenitor cells (R) were applied as negative control. Each group above had three independent biological repeats.
Project description:Genome-wide analysis of miRNA expression was performed in Activin A and Wnt3a-treated mouse ESCs during the different stages of DE differentiation to identify candidate miRNAs likely to be involved in Wnt3a and Activin A induced DE formation. Our analysis exhibited a distinct miRNA expression finger print. Furthermore, we found that forced expression of a subset of synergistically regulated miRNAs could partially mimic the roles of Wnt3a and Activin A. Pathway analyses also revealed the involvement of histone acetylation in Activin A/Wnt3a-driven DE differentiation, which is further confirmed by treating the cells with small molecular weight HDAC inhibitors as well as ChIP experiments. Our study established a regulatory cascade from extracellular cytokine treatment to miRNA expression to histone modification in cell nucleus during DE differentiation. ESCs were treated with 100ng/ml Activin A, or 50ng/ml Wnt3a, or 100ng/ml Activin A plus 50ng/ml Wnt3a, and the samples were collected at 1 day, 3 days, and 5 days after treatment, respectively. Cells treated with the same medium without growth factors were used as the negative controls for each time point.
Project description:H3K4me3 ChIP-Seq profiling of a directed differentiation time course converting human embryonic stem cells (hES) into immature pancreatic beta cell precursors. CyT49, a Viacyte proprietary male hESC line with normal karyotype was used.