Reprogramming of EBV-immortalized B-lymphocyte cell lines into induced pluripotent stem cells

Authors:
Choi SM, Liu H, Chaudhari P, Kim Y, Cheng L, Feng J, Sharkis S, Ye Z, Jang YY
In:
Source: Blood
Publication Date: (2011)
Issue: 118(7): 1801-5
Research Area:
Cancer Research/Cell Biology
Immunotherapy / Hematology
Stem Cells
Cells used in publication:
Fibroblast, dermal(NHDF-Ad), human adult
Species: human
Tissue Origin: dermal
B-cell lymphoma cell line
Species: human
Tissue Origin:
Platform:
Nucleofector™ I/II/2b
96-well Shuttle™ System
Experiment
- 96well nucleofection of human fibroblasts according to the basic protocol: 1x10e5 cells, P2 solution, program 96-CA-137 DNA per reaction: 0.7µg EN2L, 0.5µg ET2K, 0.8µg EM2K 97% transfection efficiency after 24h plated in hESC media; - EBV transformed B-cell lines: cell line Nucleofector kit V, program X-005, DNA per reaction: 3.5µg EN2L, 2.5µg ET2K, 4.0µg EM2K; 58% transfection efficiency; plated in modified hESC media
Abstract
EBV-immortalized B lymphocyte cell lines have been widely banked for studying a variety of diseases, including rare genetic disorders. These cell lines represent an important resource for disease modeling with the induced pluripotent stem cell (iPSC) technology. Here we report the generation of iPSCs from EBV-immortalized B-cell lines derived from multiple inherited disease patients via a nonviral method. The reprogramming method for the EBV cell lines involves a distinct protocol compared with that of patient fibroblasts. The B-cell line-derived iPSCs expressed pluripotency markers, retained the inherited mutation and the parental V(D)J rearrangement profile, and differentiated into all 3 germ layer cell types. There was no integration of the reprogramming-related transgenes or the EBV-associated genes in these iPSCs. The ability to reprogram the widely banked patient B-cell lines will offer an unprecedented opportunity to generate human disease models and provide novel drug therapies