Utility of Lymphoblastoid Cell Lines for Induced Pluripotent Stem Cell Generation.

Satish Kumar,David C Glahn,Joanne E Curran,John Blangero

doi:10.1155/2016/2349261

Abstract

A large number of EBV immortalized LCLs have been generated and maintained in genetic/epidemiological studies as a perpetual source of DNA and as a surrogate in vitro cell model. Recent successes in reprograming LCLs into iPSCs have paved the way for generating more relevant in vitro disease models using this existing bioresource. However, the overall reprogramming efficiency and success rate remain poor and very little is known about the mechanistic changes that take place at the transcriptome and cellular functional level during LCL-to-iPSC reprogramming. Here, we report a new optimized LCL-to-iPSC reprogramming protocol using episomal plasmids encoding pluripotency transcription factors and mouse p53DD (p53 carboxy-terminal dominant-negative fragment) and commercially available reprogramming media. We achieved a consistently high reprogramming efficiency and 100% success rate using this optimized protocol. Further, we investigated the transcriptional changes in mRNA and miRNA levels, using FC-abs ≥ 2.0 and FDR ≤ 0.05 cutoffs; 5,228 mRNAs and 77 miRNAs were differentially expressed during LCL-to-iPSC reprogramming. The functional enrichment analysis of the upregulated genes and activation of human pluripotency pathways in the reprogrammed iPSCs showed that the generated iPSCs possess transcriptional and functional profiles very similar to those of human ESCs.

Highlights

Epstein-Barr virus (EBV) immortalized lymphoblastoid cell lines (LCLs) have been routinely used as surrogate in vitro cell models for various human primary tissues to study genetic influence on disease traits [1], drug response [2,3,4,5], and gene regulation [6, 7]
The molecular events leading to the maintenance of pluripotency in embryonic stem cells (ESCs) and reacquisition of a stem-like state in induced pluripotent stem cell (iPSC) during somatic reprogramming represent mechanistically distinct processes that converge on a set of remarkably similar transcriptional events that underpin the pluripotent state [17]
The nucleofected cells were cultured using the media and culture conditions described in the publication but after repeating the experiments twice on six different cell lines we did not achieve any success with this protocol

Summary

Introduction

Epstein-Barr virus (EBV) immortalized lymphoblastoid cell lines (LCLs) have been routinely used as surrogate in vitro cell models for various human primary tissues to study genetic influence on disease traits [1], drug response [2,3,4,5], and gene regulation [6, 7]. LCLs have clearly been a convenient and useful model in the absence of primary tissue accessibility and widely banked to study a variety of human diseases, including complex genetic disorders. The molecular events leading to the maintenance of pluripotency in embryonic stem cells (ESCs) and reacquisition of a stem-like state in iPSCs during somatic reprogramming represent mechanistically distinct processes that converge on a set of remarkably similar transcriptional events that underpin the pluripotent state [17] Both ESCs and iPSCs depend on fundamental transcription frameworks that are governed by a common set of “core” stem cell-specific transcription factors, namely, OCT4, SOX2, and NANOG [18]. A differential gene expression analysis was performed between LCLs and generated iPSC in combination with functional annotations and Ingenuity Pathway Analysis (IPA)

Materials and Methods

RNA Sequencing and Data Preprocessing

Results and Discussion

Conclusions