Rapid Generation of Induced Pluripotent Stem Cells from Lymphoblastoid Cell Lines Using an Episomal Plasmid Containing Multiple Reprogramming Factors in a Single Cassette (Matisse Reprogramming System)

Objective: Reprogramming of skin fibroblasts is increasingly used for modeling of human neurological diseases. Cell lines from many patients, however, have been generated using EBV-immortalized B-lymphocytes (EBV-B). In contrast to skin fibroblasts, blood lymphocytes and especially lymphoblastoid (LB) cells are difficult to reprogram and iPSC colonies are usually formed after weeks and with very low efficiency.

Background: B cells represent a larger fraction of the peripheral blood mononuclear cell population (~20%) and can be transformed in vitro by EBV to generate lymphoblastoid cell lines (LB), creating an unlimited proliferative source of cells for reprogramming trials. LB cells are a precious resource for immunologic, epidemiologic, and rare disease studies. Generating iPSCs from LB cells offers the advantage of working with minimal amounts of blood from living donors as well as frozen LB collections banked worldwide. Recently, iPSCs have been generated by delivering the reprogramming factors via oriP/EBNA-1-based plasmids in fibroblasts and peripheral blood CD34+cells.

Design/Methods: We have generated a novel episomal plasmid where RFs (c-MYC, KLF4, SOX2 and OCT3/4) are controlled by individually modified CMV promoters in a single cassette, designated pPuro(CMV_mt1)cMKSO (Matisse reprogramming system), which allows coordinated expression of the four RFs. EBV-B cells were electroporated with plasmid, pPuro(CMV_mt1)cMKSO and plated on matrigel coated plate with MEF-derived conditioned media (MEF-CM) containing basic fibroblast growth factor (bFGF).

Results: After 6-8 days of culture of electroporated EBV-B cells with MEF-CM, cells tend to attach and start to form colonies. EBV-B cell line-derived iPSCs showed ES cell morphology, stained with alkaline phosphatase (AP), and expressed pluripotent cell-specific genes. The ability to reprogram banked patient EBV-transformed cell lines efficiently will offer an unprecedented opportunity to rapidly generate genetic disease models and also provide a translational platform for therapeutic drug development. 

Conclusions: We developed a rapid method to generate iPSCs from EBV-immortalized B-lymphocyte cell lines (EBV-B cells) using a novel 4-factor vector.