Project description:Human liver progenitor cells (LPCs) show therapeutic potential, however, their in vitro culture results in inadequate function and phenotypic instability reflecting incomplete understanding of in vivo processes. Foetal LPCs capable of differentiation to a hepatocyte phenotype were isolated and mRNA expression profiling carried out using Affymetrix HGU133plus2 microarrays. This was compared to profiles from mature human hepatocytes and human embryonic stem cells undergoing hepatocytic differentiation. Foetal LPCs exhibit a distinct molecular profile consistent with a stem cell signature, cell division, and some liver-specific functions. 9 independent samples of second trimester liver progenitor cells were profiled along with 3 independent mature hepatocyte samples and 3 each of embryonic stem cells at day 9 and day 17 of differentiation to hepatocyte like cells.
Project description:Epithelial to mesenchymal transition (EMT) in cancer cells has been associated with metastasis, stemness and resistance to therapy. The reason why some tumors undergo EMT and other not might reflect intrinsic properties of their cell of origin, although this possibility is largely unexplored. By targeting the same oncogenic mutations to discrete skin compartments, we show cell type-specific chromatin and transcriptional states differentially prime tumors to EMT. Squamous cell carcinomas (SCCs) derived from intrafollicular epidermis (IFE) are generally well-differentiated, while hair follicle (HF) stem cell-derived SCCs frequently exhibit EMT, efficiently form secondary tumors, and possess increased metastatic potential. Transcriptional and epigenomic profiling revealed IFE and HF tumor-initiating cells possess distinct chromatin landscapes and gene regulatory networks associated with tumorigenesis and EMT that correlate with accessibility of key epithelial and EMT transcription factor binding sites. These findings highlight the importance of chromatin states and transcriptional priming in dictating tumor phenotypes and EMT. Accessible chromatin regions were profiled using ATAC-seq, enriched peak regions were used to infer transcription factor binding sites.
Project description:To identify microRNAs which differentially expressed in the BMSCs of aged and young mice and and investigate its influences on BMSCs differentiation with ageing. The microRNAs expressions of BMSCs from 3 aged mice and 3 young mice were measured.
Project description:Factor induced reprogramming is a slow and inefficient process with only rare cells progressing towards induced pluripotent stem cells (iPSCs). Owing to these restraints, mechanistic studies have been limited to analyses of heterogeneous bulk populations undergoing reprogramming and partially reprogrammed cell lines. Here, by combining surface markers (Thy1, SSEA1) and an Oct4-GFP fluorescent reporter allele, we analyzed defined intermediate cell populations poised to becoming iPSCs at the transcriptional and epigenetic levels using genome-wide and single cell technologies. We found that factor-induced reprogramming elicits two discernible transcriptional waves that are characterized by the initial extinction of the somatic gene expression program and the concomitant acquisition of an ESC-like proliferative and metabolic state, followed by the activation of an embryonic pluripotent state primed for differentiation. The first wave is mostly driven by gene activation through c-Myc and gene repression by Klf4, whereas the second wave is a result of gradually activated Oct4/Sox2 targets in cooperation with Klf4 targets and other downstream regulators. While microRNA expression and enrichment for individual histone modifications (H3K4me3 or H3K27me3 enriched promoters) mirrored the observed biphasic transcriptional pattern, the establishment of bivalent domains (H3K4me3/H3K27me3 enriched promoters) occurred more gradually. In contrast, changes in DNA methylation took place predominantly at the end of reprogramming when cells assumed a stable pluripotent state. Cells that became refractory to reprogramming activated the first but failed to initiate the second transcriptional wave. However, introduction of additional copies of the reprogramming transgenes into these cells rescued their ability to form iPSCs, indicating that suboptimal transcription factor levels are a limiting factor for efficient iPSC formation. This integrative analysis allowed us to identify novel genes and microRNAs that enhance reprogramming and surface markers that further subdivide intermediate cell populations. Collectively, our data offer new mechanistic insights into the nature and sequence of molecular events inherent to cellular reprogramming and provide a valuable resource of molecules that may act as roadblocks during iPSC formation. Time series design with samples sorted into subpopulations according to surface markers.
Project description:Specific surface marker for NKX2-1+ VAFECs may be helpful for isolating a homogeneous population of alveolar epithelial progenitor cells and distinguishing the differentiation from a thyroid lineage to a lung lineage. In order to identify specific markers of VAFECs, a microarray analysis was performed to compare the global gene expression patterns between AFECs and VAFECs in 201B7 hiPSCs. We hypothesized that NKX2-1+ cells could be purified by sorting CPM+ VAFECs. After dissociating VAFECs cells on day 14 with Accutase, FACS was performed using anti-EPCAM and anti-CPM antibodies. EPCAM+CPM+ and EPCAM+CPM- cells were then sorted, and the global gene expression patterns of these two populations were examined using a microarray analysis. In addition, MACS was performed to obtain CPM+ cells for comparison. We extracted total RNA from hiPSCs-derived AFECs, VAFECs, EPCAM+CPM+ and EPCAM+CPM- VAFECs and CPM+ VAFECs and hybridized them to Affymetrix microarrays.
Project description:Purpose: In all vertebrates, the thymus is necessary and sufficient for production of classic adaptive T cells. The key components of the thymus are cortical and medullary thymic epithelial cells (cTECs and mTECs). Despite the capital role of TECs, our understanding of TEC biology is quite rudimentary. For instance, we ignore what might be the extent of divergence in the functional program of these two TECs populations. It also remains unclear why the number of TECs decreases rapidly with age, thereby leading to progressive thymic insufficiency. Methods: Systems level understanding of cell function begins with gene expression profiling, and the transcriptome is currently the only '-ome' that can be reliably tackled in its entirety in freshly harvested primary cells. In order to gain novel insights into TEC biology, we therefore decided to analyse the whole transcriptome of cTECs, mTECs and skin epithelial cells. We elected to analyse gene expression using RNA-seq rather microarrays because RNA-seq has higher sensitivity and dynamic range coupled to lower technical variations. Results: Our deep sequencing approach provides a unique perspective into the transcriptome of TECs. Consistent with their ability to express ectopic genes, we found that mTECs expressed more genes than other cell populations. Out of a total of 15,069 genes expressed in TECs, 25% were differentially expressed by at least 5-fold in cTECs vs. mTECs. Genes expressed at higher levels in cTECs than mTECs regulate numerous cell functions including cell differentiation, cell movement and microtubule dynamics. Almost all positive regulators of the cell cycle were overexpressed in skin ECs relative to TECs. Conclusions: Our RNA-seq data provide novel insights into the transcriptional landscape of TECs, highlight substantial divergences in the transcriptome of TEC subsets and suggest that cell cycle progression is differentially regulated in TECS and skinECs. We believe that our work will therefore represent a valuable resource and will be of great interest to readers working in biological sciences, particularly in the areas of immunology and systems biology. The mRNA profiles of cTEC, mTEC (from 14 thymi of 7-days old C57BL/6 mice) and skinEC (from the trunk and dorsum of seven newborn mice) were generated by RNA-sequencing using Illumina HiSeq2000.
Project description:Quiescent hepatic stem cells (HSCs) can be activated when hepatocyte proliferation is compromised. Chemical injury rodent models have been widely used to study the locazation, biomarkers, and signaling pathways in HSCs, but these models usually exhibit severe promiscuous toxicity and fail to distinguish damaged and non-damaged cells. Our goal is to establish new animal models to overcome these limitations, thereby providing new insights into HSC biology and application. We generated mutant mice with constitutive or inducible deletion of Damaged DNA Binding protein 1 (DDB1), an E3 ubiquitin ligase, in hepatocytes. We show that deletion of DDB1 abolishes self-renewal capacity of mouse hepatocytes in vivo, leading to compensatory activation and proliferation of DDB1-expressing OCs. Importantly, the DDB1 mutant mice exhibit very minor liver damage, compared to a chemical injury model. Microarray analysis reveals several previously unrecognized markers, enriched in oval cells. This genetic model in which irreversible inhibition of hepatocyte duplication results in HSC-driven liver regeneration. The DDB1 mutant mice can be broadly applied to studies of HSC differentiation, HSC niche and HSCs as origin of liver cancer. Total RNA obtained from isolated EpCAM+ cells from DDB1 mutant mice compared to wild type hepatocytes
Project description:Clusters of circulating tumor cells (CTC-clusters) are present in the blood of patients with cancer but their contribution to metastasis is not well defined. Here, we first use mouse models to demonstrate that breast cancer cells injected intravascularly as clusters are more prone to survive and colonize the lungs than single cells. Primary mammary tumors comprised of tagged cells give rise to oligoclonal CTC-clusters, with 50-fold increased metastatic potential, compared with single CTCs. Using intravital imaging and in vivo flow cytometry, CTC-clusters are visualized in the tumor circulation, and they demonstrate rapid clearance in peripheral vessels. In patients with breast cancer, presence of CTC-clusters is correlated with decreased progression-free survival. RNA sequencing identifies the cell junction protein plakoglobin as most differentially expressed between clusters and single human breast CTCs. Expression of plakoglobin is required for efficient CTC-cluster formation and breast cancer metastasis in mice, while its expression is associated with diminished metastasis-free survival in breast cancer patients. Together, these observations suggest that plakoglobin-enriched primary tumor cells break off into the vasculature as CTC-clusters, with greatly enhanced metastasis propensity. RNA-seq from 29 samples (15 pools of single CTCs and 14 CTC-clusters) isolated from 10 breast cancer patients
Project description:Tumour tissue was immediately obtained post-surgery along with paired healthy tissue near (<2cm from tumour margin) and far (>10cm) from the tumour site. Each tissue was dissociated and stained with flow cytometry antibodies and sorted by FACS into Live/Dead-negative EpCAM-positive CD3-negative populations. An additional sample was passed through the FACS however no sorting was applied (control for the effect of sorting). Total RNA was purified and tissues compared for gene expression to identify potential hits that were assessed for protein expression and immunogenicity in later experiments.