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Gene expression in human embryonic stem cells (hESCs) upon the differentiation to neural lineages by timed delivery of retinoic acid

ABSTRACT: Gene expression in human embryonic stem cells (hESCs) upon the differentiation to neural lineages by timed delivery of retinoic acid

PROVIDER: PRJNA614495 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR11392730_1.fastq.gz	Fastqsanger.gz
	SRR11392730_2.fastq.gz	Fastqsanger.gz
	SRR11392731_1.fastq.gz	Fastqsanger.gz
	SRR11392731_2.fastq.gz	Fastqsanger.gz
	SRR11392732_1.fastq.gz	Fastqsanger.gz

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Project description:Purpose: We previously reported the generation of corneal endothelial cells (CECs) using the human peripheral blood mononuclear cells (PBMC)-originated induced pluripotent stem cells (iPSCs). Herein, we extend our analysis through RNA-Seq based transcriptome profiling of H9 human embryonic stem cells (hESCs)- and iPSCs-derived CECs. 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 subjected to the 20-day procedure according to our previously published CECs generation method. The differentiated CECs were characterized by immunocytochemistry. Total RNA from hESCs- and iPSCs-derived CECs was used for the preparation of RNA-Seq libraries, which were sequenced on HiSeq 2500 system. The raw RNA-Seq 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 hESCs and iPSCs were differentiated into CECs through multipotent neural crest cells (NCCs) using a 20-day procedure. The differentiating CECs on day 20 (D20) exhibited a tightly packed hexagonal/polygonal shape, which expressed CECs-associated markers, zona occludens-1 and N-cadherin at the cell boundaries. A total of 180.18, and 179.01 million reads were obtained for hESCs- and iPSCs-derived CECs, respectively. Of these, >97% reads aligned to the human reference genome resulting in >250x sequence coverage for both hESCs- and iPSCs-derived CECs. Additional analysis identified expression (≥ 0.659 RPKM) of 13,561 and 13,551 genes in hESCs- and iPSCs-derived CECs, respectively, representing ~68% of the total human protein-coding transcriptome expressed in these CECs. Finally, a comparative analysis of both hESCs- and iPSCs-derived CECs transcriptomes identified >95% similarity at the gene level. Conclusion: These analyses reveal an overall similar transcriptional profile in both hESCs- and iPSCs-derived CECs.

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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Gene expression in human embryonic stem cells (hESCs) upon the differentiation to neural lineages by timed delivery of retinoic acid

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