Submitted by Satish Kumar (University of Texas Rio Grande Valley, USA) on Oct 23 2015
Platform: ngs – Illumina HiSeq 2500 (Homo sapiens)
Summary Purpose: There exists a rich bio-resource of numerous lymphoblastoid cell line (LCL) repositories generated from a wide array of patients, many of them with extensive genotypic and phenotypic data already generated. We have developed a highly efficient LCL to induced pluripotent stem cells (iPSC) reprogramming method and performed whole genome mRNA and miRNA analysis to understand mechanistic changes that take place at the transcriptome and cellular functional level during reprogramming of LCLs into iPSCs and further differentiation.
Methods: Applying our optimized protocol which utilizes episomal plasmids encoding pluripotency transcription factors and mouse p53DD - p53 carboxy-terminal dominant-negative fragment and commercially available reprogramming media, we reprogrammed six LCLs into iPSCs and then differentiated them into neural stem cells (NSC). The LCLs, their reprogrammed iPSCs and differentiated NSC (n=18) were sequenced for mRNA and smallRNA on an illumina HiSeq 2500. Differential gene expression analysis was performed between LCL-iPSC and iPSC-NSC pairs in combination with functional annotations and Ingenuity® Pathway Analysis (IPA).
Results: Our LCL reprogrammed iPSCs express the majority of genes and miRNAs known to contribute to stemness in human ESCs. The functional enrichment analysis of the up-regulated genes and activation of human pluripotency pathways in the reprogrammed iPSCs showed that the generated iPSCs have a transcriptional and functional profile very similar to that of human ESCs. The reprogrammed iPSCs also showed the potential to differentiate into cells of all three germ layers. Significantly, the transcriptomic effect of EBV encoded oncoproteins which were very pronounced in LCLs, were significantly inhibited in reprogrammed iPSCs. The transcriptomic and functional enrichment analysis of the NSC differentiated from the reprogrammed iPSCs showed that they share a functional profile of self-renewing NSCs.
Conclusions: We have been able to develop a MEF feeder free protocol for efficient and reproducible reprogramming of LCLs into iPSC. In addition our comprehensive analysis of genome wide miRNA and mRNA of LCLs, their reprogrammed iPSC and differentiated NSCs provides important documentation of differentially expressed genes and miRNAs and their functional consequences during LCL to iPSC reprogramming and NSC differentiations that were previously unknown.
ID | Title | Cell Type | Timepoint | Reported Virus | Virus Species | Exclusion Reason |
---|---|---|---|---|---|---|
GSM1916404 | LCL-1 (mRNA) | lymphoblastoid cell line ![]() |
Human gammaherpesvirus 4 | |||
GSM1916405 | LCL-2 (mRNA) | lymphoblastoid cell line ![]() |
Human gammaherpesvirus 4 | |||
GSM1916406 | LCL-3 (mRNA) | lymphoblastoid cell line ![]() |
Human gammaherpesvirus 4 | |||
GSM1916407 | LCL-4 (mRNA) | lymphoblastoid cell line ![]() |
Human gammaherpesvirus 4 | |||
GSM1916408 | LCL-5 (mRNA) | lymphoblastoid cell line ![]() |
Human gammaherpesvirus 4 | |||
GSM1916409 | LCL-6 (mRNA) | lymphoblastoid cell line ![]() |
Human gammaherpesvirus 4 | |||
GSM1916422 | LCL-1 (miRNA) | lymphoblastoid cell line ![]() |
Wrong technology small RNA seq |
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GSM1916423 | LCL-2 (miRNA) | lymphoblastoid cell line ![]() |
Wrong technology small RNA seq |
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GSM1916424 | LCL-3 (miRNA) | lymphoblastoid cell line ![]() |
Wrong technology small RNA seq |
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GSM1916425 | LCL-4 (miRNA) | lymphoblastoid cell line ![]() |
Wrong technology small RNA seq |
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GSM1916426 | LCL-5 (miRNA) | lymphoblastoid cell line ![]() |
Wrong technology small RNA seq |
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GSM1916427 | LCL-6 (miRNA) | lymphoblastoid cell line ![]() |
Wrong technology small RNA seq |