Project description:In order to examine the transcriptomes alteration of LN511-based lens epithelial stem cells induction, human embryonic stem cells (hESCs) were induced into lentoid body followed the protocol from Yang et al (FASEB J. 2010), on Matrigel-coated or LN511-coated culture. Each group of cells were collected at day 12 and day 18, during the time when lens epithelial stem cells were generated. The samples were named as Ctrl-d12, Ctrl-d18, LN-511-d12 and LN-511-d18, respectively, (3 replicates, -1/2/3) and sent for RNA-sequence using Illumina HiSeq2500. It turned out that LN511 acted as a robust signaling factor for lens epithelial stem cells induction via enhancing the expression of lens specification related transcription factor, promoting cell proliferation, and suppressing Hippo/Yap signaling. These results provided a valuable resource for studying the mechanisms regulating in vitro lens epithelial stem cells induction.
Project description:Purpose: To investigate the transcriptomes of H9 human embryonic stem cells (hESCs)- and peripheral blood mononuclear cells (PBMC) originated induced pluripotent stem cells (iPSCs)-derived early stage lentoid bodies at day 24 through RNA-Seq based whole transcriptome sequencing. Methods: The PBMC obtained from a healthy donor were subjected to generate iPSCs using Sendai-virus delivery system Cytotune 2.0 whereas the H9 hESCs were obtained commercially. Both hESCs and iPSCs were differentiated into lentoid bodies using “fried egg” method with feeder-free conditions as described previously. The differentiating lentoid bodies were examined for the expression of lens-specific and pluripotency markers at days 0, 6, 10, 15 and 24 by quantitative real-time PCR (qRT-PCR). Briefly, four biological replicates for each hESCs- and iPSC-derived lentoid bodies at day 24 were used for the RNA-Seq library preparation followed by sequencing on a single lane of HiSeq 2500. The raw reads were processed and analyzed using Lasergene Genomics Suite and the expression profiles were examined for differential expression using Spotfire DecisionSite with Functional Genomics. Results: The differentiating lentoid bodies at day 24 revealed transparent lens like morphological features with an increased expression of lens-specific markers including CRYGC. A total of 193.41, and 170.00 million reads were obtained for hESCs- and iPSCs-derived lentoid bodies, respectively. Of these, >96% reads aligned to the human reference genome resulting in >200x sequence coverage for both hESCs- and iPSCs-derived lentoid bodies. Additional analysis identified expression (≥ 0.659 RPKM) of 13,991 and 14,018 genes in hESCs- and iPSCs-derived lentoid bodies, respectively, representing ~70% of the total human protein-coding transcriptome expressed in lentoid bodies. Finally, a comparative analysis of both hESCs- and iPSCs-derived lentoid bodies transcriptomes identified >96% similarity at the gene level. Conclusion: The transcriptome analysis revealed an overall similar transcriptional profile in both hESCs- and iPSCs-derived lentoid bodies during differentiation at day 24.
Project description:To understand the molecular process associated with tissue morphogenesis of pancreatic epithelial cells, we profiled the transcriptomes of normal or malignant pancreatic organoids formed in three-dimenstional reconsitututed basement membrance (rBM). Cell monolayers cultured on rBM-coated culture plastics were used as controls.
Project description:To understand the molecular process associated with tissue morphogenesis of pancreatic epithelial cells, we profiled the transcriptomes of normal or malignant pancreatic organoids formed in three-dimenstional reconsitututed basement membrance (rBM). Cell monolayers cultured on rBM-coated culture plastics were used as controls. HPDE (immortalized pancreatic epithelial cells) and PANC-1 cells (pancreatic cancer cells) were seeded on reconstituted basement membrane (rBM)-coated culture plastics or cultured on top of three-dimensional (3D) rBM gels. Total RNA samples were collected from cell monolayers or 3D cell clusters (formed at day 2), pancreatic tubules or tumor spheroids (formed at day 6) in the rBM culture, followed by global gene expression profiling.
Project description:Some mouse embryonic stem cell (mESC) lines need to be maintained on feeder cells in gelatin/Std condition. To eliminate the need for feeder cells, we decided to maintain the C57BL6/J mESCs on dishes coated with Laminin-511 (LN511) enabling maintenance of mESCs without feeder cells in Std condition (Domogatskaya et al., 2008). To compare the transcriptomes of mESCs cultured on gelatin-coated dish and those cultured on Laminin-511 coated dish, we performed RNA-Seq analysis. We found that the transcriptomes of mESCs cultured on gelatin-coated dish and those cultured on LN511 coated dish showed no considerable difference in expression patterns.
Project description:SVG-A cells are immortalized fetal astrocytes that respond to Notch signaling. We characterized the transcriptional response to Notch activation in this cell line by performing RNA-seq on cells grown on tissue culture dishes coated with JAGGED1, a Notch ligand. Differential gene expression analysis shows induction of both canonical Notch responsive genes, as well as cell-type specific Notch responsive genes.