LncRNA profile study reveals massive lncRNAs associated with lentoid body differentiation derived from human iPSCs
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ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE97683 | GEO | 2017/11/30
SECONDARY ACCESSION(S): PRJNA382642
REPOSITORIES: GEO
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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.
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Project description:Corneal endothelial cells (CECs) are critical to maintaining clarity of the cornea. This study was initiated to develop peripheral blood mononuclear cells (PBMC)-originated induced pluripotent stem cells (iPSCs)-derived CECs. We isolated PBMC and programmed the mononuclear cells to generate iPSCs. Subsequently, the PBMC-originated iPSCs were differentiated to CECs. The morphology of differentiating iPSCs was examined at regular intervals by phase contrast microscopy. In parallel, the expression of pluripotent, and CECs-associated markers was investigated by quantitative real-time PCR (qRT-PCR). The molecular architecture of the iPSCs-derived CECs and human corneal endothelium (CE) were examined by mass spectrometry-based proteome sequencing. The PBMC-originated iPSCs expressed pluripotent-specific markers at levels similar to expression in H9 human embryonic stem cells (hESCs). Phase contrast microscopy illustrated that iPSCs-derived CECs are tightly adherent, exhibiting a hexagonal-like shape, one of the cardinal characteristics of CECs. The CECs-associated markers were expressed at many orders of magnitude higher in iPSCs-derived CECs at days 13, 20, and 30 compared to their respective levels in iPSCs. Importantly, only residual expression levels of pluripotency markers were detected in iPSCs-derived CECs. Mass spectrometry-based proteome profiling identified 10,575 proteins in iPSCs-derived CECs. In parallel, we completed proteome profiling of the human CE identifying 6345 proteins. Of these, 5763 proteins were identified in the iPSCs-derived CECs suggesting a 90.82% overlap between the iPSCs-derived CECs and human CE proteomes. Importantly, cryopreservation of iPSCs-derived CECs did not affect the tight adherence of CECs, and their hexagonal-like shape while expressing high levels of CECs-associated markers. We have successfully developed a personalized approach to generate CECs that closely mimic the molecular architecture of the human CE. To the best of our knowledge, this is the first report describing the development of PBMC-originated, iPSCs-derived CECs.
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Project description:Differentiation of pluripotent stem cells into lentoid bodies is important for the understanding of the lens development and investigating the processes critical for lens morphogenesis. This Study was initiated to investigate a comprehensive proteome profiling of the peripheral blood mononuclear cell (PBMC)-originated, induced pluripotent stem cell (iPSC)-derived lentoid bodies through mass spectrometry-based protein sequencing. Briefly, a small aliquot of blood sample was ascertained to collect PBMCs that were reprogrammed to iPSCs using the Sendai-virus delivery system. The PBMC-originated, iPSCs were differentiated into lentoid bodies employing the “fried egg” method using feeder-free conditions. The quantitative real-time PCR (qRT-PCR) confirmed the expression of lens-associated markers, which exhibited at least an order magnitude increased expression in lentoid bodies at differentiation day 35. Subsequently, the total cellular protein was extracted from lentoid bodies at day 35, digested with trypsin, fractionated into 96 fractions and subjected to an mass spectrometry-based label-free quantitative proteomics. mass spectrometry-based proteome profiling revealed 9,717 proteins in iPSC-derived lentoid bodies at differentiation day 35. In here, we report a comprehensive proteome of PBMC-originated, iPSC-derived lentoid bodies at day 35, which will help in better understanding processes critical for the development of the ocular lens.
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Project description:Differentiation of pluripotent cells to generate lentoid bodies is important for the understanding of the lens development and investigating the processes critical for lens morphogenesis. This Study was initiated to investigate a comprehensive proteome profiling of the peripheral blood mononuclear cell (PBMC)-originated, induced pluripotent stem cell (iPSC)-derived lentoid bodies through mass spectrometry-based protein sequencing. Briefly, a small aliquot of blood sample was ascertained to collect PBMCs that were reprogrammed to iPSCs using the Sendai-virus delivery system. The PBMC-originated, iPSCs were differentiated into lentoid bodies employing the “fried egg” method using feeder-free conditions. The quantitative real-time PCR (qRT-PCR) confirmed the expression of lens-associated markers, which exhibited at least an order magnitude increased expression in lentoid bodies at differentiation day 25. Subsequently, the total cellular protein was extracted from lentoid bodies at day 25, digested with trypsin, fractionated into 24 fractions and subjected to an mass spectrometry-based label-free quantitative proteomics. mass spectrometry-based proteome profiling revealed 9,473 proteins in iPSC-derived lentoid bodies at differentiation day 25. In here, we report a comprehensive proteome of PBMC-originated, iPSC-derived lentoid bodies at day 25, which will help in better understanding processes critical for the development of the ocular lens.
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Project description:Leptin receptors (Lepr) are expressed by various types of stem cells including mesenchymal stem cells, hematopoietic stem cells, embryonic stem cells, and induced pluripotent stem cells. Leptin/lepr signaling is also a central regulator of metabolism. However, the role of Lepr in pluripotency, metabolic disease progression and growth development is still controversial and poorly understood. In the present study, we explored the Lepr function in disease progression, pluripotency and metabolism using day 14.5 mouse embryonic fibroblasts (MEFs) and their reprogrammed induced pluripotent stem cells (iPSCs) as model system. We successfully reprogrammed mouse embryonic fibroblasts into iPSCs from control and db/db (Lepr deficient) mice. Using a global quantitative proteomic approach, we identified key pathways regulating pluripotency, metabolic homeostasis and protein synthesis during fetal growth and development. The Lepr MEFs show abnormal metabolic abnormalities and mitochondrial dysfunction as compared to control MEFs, while Lepr iPSCs show upregulated elongated factor 4 e (eIF4e) protein synthesis pathway and altered Oct4 and Stat3 pathways which are involved in normal fetal growth development. Furthermore, chip analysis revealed that higher Stat3 binding on the promoter of eIF4e in Lepr iPSCs leads to higher protein synthesis in these cell types as compared to control iPSCs. Finally, point mutation corrected Lepr iPSCs using CRISPR/Cas9 gene editing method showed recovered pluripotency, metabolic and protein synthesis pathways. In conclusion, we have shown that Lepr signaling is involved in the regulation of the metabolic properties and key developmental pathways in MEFs and stemness of pluripotent stem cells. Disruption of Lepr signaling has been shown to involve in the pathophysiology of various diseases including obesity and diabetes. The generated MEFs and iPSCs in this present study provide valuable tools to explore the role of Lepr in the progression of obesity, diabetes and metabolic abnormalities, and to find the putative targets of Lepr signaling during the development of these diseases.
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