Human lymphoblastoid B-cell lines reprogrammed to EBV-free induced pluripotent stem cells

Deepika Rajesh, Sarah J. Dickerson, Junying Yu, Matthew E. Brown, James A. Thomson, and Nicholas J. Seay
Source: Blood
Publication Date: (2011)
Issue: 118 (7): 1797-1800
Research Area:
Immunotherapy / Hematology
Gene Expression
Basic Research
Cells used in publication:
Species: human
Tissue Origin: blood
4D-Nucleofector® 96-well Systems
Human B Cell 96-well Nucleofector Kit and program E0-100 with 1–2µg of DNA per reaction; (~1.0E+06 cells per condition) were allowed to recover a few hours and directly plated to Matrigel-coated 6-well plates in reprogramming medium (DMEM:F12). Colonies with morphology similar to iPSC colonies were readily visible between days 14–20 post-nucleofection. The presence of true-iPSC colonies was confirmed by morphology and live-cell staining with Tra-1–60 antibody. The iPSC-like colonies were picked and propagated using TeSR-2 medium on Matrigel-coated plates
Generation of patient-specific induced pluripotent cells (iPSCs) holds great promise for regenerative medicine. Epstein-Barr virus immortalized lymphoblastoid B-cell lines (LCLs) can be generated from a minimal amount of blood and are banked worldwide as cellular reference material for immunologic or genetic analysis of pedigreed study populations. We report the generation of iPSCs from 2 LCLs (LCL-iPSCs) via a feeder-free episomal method using a cocktail of transcription factors and small molecules. LCL-derived iPSCs exhibited normal karyotype, expressed pluripotency markers, lost oriP/EBNA-1 episomal vectors, generated teratomas, retained donor identity, and differentiated in vitro into hematopoietic, cardiac, neural, and hepatocyte-like lineages. Significantly, although the parental LCLs express viral EBNA-1 and other Epstein-Barr virus latency-related elements for their survival, their presence was not detectable in LCL-iPSCs. Thus, reprogramming LCLs could offer an unlimited source for patient-specific iPSCs.