Unknown,Transcriptomics,Genomics,Proteomics

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Reprogramming LCLs to iPSCs Results in Recovery of Donor-Specific Gene Expression Signature

ABSTRACT: Renewable in vitro cell cultures, such as lymphoblastoid cell lines (LCLs), have facilitated studies that contributed to our understanding of genetic influence on human traits. However, the degree to which cell lines faithfully maintain differences in donor-specific phenotypes is still debated. We have previously reported that standard cell line maintenance practice results in a loss of donor-specific gene expression signatures in LCLs. An alternative to the LCL model is the induced pluripotent stem cell (iPSC) system, which carries the potential to model tissue-specific physiology through the use of differentiation protocols. Still, existing LCL banks represent an important source of starting material for iPSC generation, and it is possible that the disruptions in gene regulation associated with long-term LCL maintenance could persist through the reprogramming process. To address this concern, we studied the effect of reprogramming mature LCLs to iPSCs on the ensuing gene expression patterns within and between six unrelated donor individuals. We show that the reprogramming process results in a recovery of donor-specific gene regulatory signatures. Since environmental contributions are unlikely to be a source of individual variation in our system of highly passaged cultured cell lines, our observations suggest that the effect of genotype on gene regulation is more pronounced in the iPSCs than in the LCL precursors. Our observations indicate that iPSCs can be a powerful model system for studies of phenotypic variation across individuals in general, and the genetic association with variation in gene regulation in particular. We further conclude that LCLs are an appropriate starting material for iPSC generation. Whole genome gene expression data was collected for 34 samples including 17 iPSC lines and 17 LCL lines on the Illumina HT-12 v4 Expression BeadChip array platform. Three biological replicates of each individual were included in the study, except for one individual for which only two replicates were obtained.

ORGANISM(S): Homo sapiens

SUBMITTER: Samantha Thomas

PROVIDER: E-GEOD-64263 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description: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.

Project description:Epstein-Barr virus (EBV) transformed lymphoblastoid cell lines (LCLs) are a widely used renewable resource for functional genomic studies in humans. The ability to accumulate multidimensional data pertaining to the same individual cell lines, from complete genomic sequences to detailed gene regulatory profiles, further enhances the utility of LCLs as a model system. However, the extent to which LCLs are a faithful model system is relatively unknown. We have previously shown that gene expression profiles of newly established LCLs maintain a strong individual component. Here, we extend our study to investigate the effect of freeze-thaw cycles on gene expression patterns in mature LCLs, especially in the context of inter-individual variation in gene regulation. We found a profound difference in the gene expression profiles of newly established and mature LCLs. Once newly established LCLs undergo a freeze-thaw cycle, the individual specific gene expression signatures become much less pronounced as the gene regulatory programs in LCLs from different individuals converge to a more uniform profile, which reflects a mature transformed B cell phenotype. As expected, previously identified eQTLs are enriched among the relatively few genes whose regulations in mature LCLs maintain marked individual signatures. We thus conclude that findings and insight drawn from gene regulatory studies in mature LCLs are generally not affected by artificial nature of the LCL model system and are likely to faithfully reflect regulatory interactions in primary tissues. However, our data indicate that many aspects of primary B cell biology cannot be observed and studied in mature LCL cultures. We obtained unpurified buffy coats (of a unit of blood) from six unrelated healthy individuals of Caucasian ethnicity (age range: 20-45). We isolated CD20+ B cells and established six independent cultures of LCLs between October 2009 and January 2010. From each of these samples, we obtained genome wide gene expression data using Illumina HumanHT-12 v3 Expression BeadChip arrays. We refer to data from these experiments as M-bM-^@M-^\cycle 0M-bM-^@M-^] to acknowledge the fact that the LCLs from this cycle were newly established and therefore not frozen/thawed. Between February 2011 and October 2012, we thawed each of these LCL cultures every 3 months and cultured them until obtaining ~10 million cells (February 2011=cycle 1, June 2011=cycle 2, October 2011=cycle 3, February 2012=cycle 4, June 2012=cycle 5, and October 2012=cycle 6). We used Illumina HumanHT-12 v4 Expression BeadChip arrays and obtained genome wide gene expression data on cycle 2, cycle 4 and cycle 6 LCLs. At each hybridization batch we included a subset of RNA samples that were also hybridized in a previous batch. This allowed us to effectively consider the batch effect on gene expression profiles. Our final dataset included genome wide gene expression data from 187 samples collected at 4 different time points.

Project description:Human-induced pluripotent stem cells (iPSCs) are derived from differentiated somatic cells using defined factors and provide a renewable source of autologous cells for cell therapy. Many reprogramming methods have been employed to generate human iPSCs, including the use of integrating vectors and non-integrating vectors. Maintenance of the genomic integrity of iPSCs is highly desirable if the cells are to be used in clinical applications. Here, using the Affymetrix Cytoscan HD array, we investigated the genomic aberration profiles of 19 human cell lines: 5 embryonic stem cell (ESC) lines, 6 iPSC lines derived using integrating vectors (“integrating iPSC lines”), 6 iPSC lines derived using non-integrating vectors (“non-integrating iPSC lines”), and the 2 parental cell lines from which the iPSCs were derived. The genome-wide copy number variation (CNV), loss of heterozygosity (LOH) and mosaicism patterns of integrating and non-integrating iPSC lines were investigated. The maximum sizes of CNVs in the genomes of the integrating iPSC lines were 20 times higher than those of the non-integrating iPSC lines. Moreover, the total number of CNVs was much higher in integrating iPSC lines than in other cell lines. The average numbers of novel CNVs with a low degree of overlap with the DGV and of likely pathogenic CNVs with a high degree of overlap with the ISCA database were highest in integrating iPSC lines. Different SNP calls revealed that, using the parental cell genotype as a reference, integrating iPSC lines displayed more single nucleotide variations and mosaicism than did non-integrating iPSC lines. This study describes the genome stability of human iPSCs generated using either a DNA-integrating or non-integrating reprogramming method, of the corresponding somatic cells, and of hESCs. Our results highlight the importance of using a high-resolution method to monitor genomic aberrations in iPSCs intended for clinical applications to avoid any negative effects of reprogramming or cell culture. 19 human cell lines: 5 embryonic stem cell (ESC) lines, 6 iPSC lines derived using integrating vectors (“integrating iPSC lines”), 6 iPSC lines derived using non-integrating vectors (“non-integrating iPSC lines”), and the 2 parental cell lines from which the iPSCs were derived.

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Reprogramming LCLs to iPSCs Results in Recovery of Donor-Specific Gene Expression Signature

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