Highly efficient induced pluripotent stem cell reprogramming of cryopreserved lymphoblastoid cell lines

Authors:
Satish Kumar , Joanne E Curran , Erika C Espinosa , David C Glahn , John Blangero 
In:
Source: J Bio Med Opt
Publication Date: (2020)
Issue: 7(1): 124
Research Area:
Cancer Research/Cell Biology
Immunotherapy / Hematology
Stem Cells
Regenerative medicine
Cells used in publication:
BLCL
Species: human
Tissue Origin: blood
LCL
Species: human
Tissue Origin: blood
Platform:
4D-Nucleofector™ X-Unit
Experiment

Start 4D-Nucleofector system and create or upload the file with following experimental parameters:
SE Cell Line solution and 4D-Nucleofector DN-100 program.
 Add 3.6 µl of nucleofector supplement to 16.4 µl of nucleofector solution per sample nucleofected. Alternatively, add entire supplement supplied in the kit to the nucleofector solution, and use 20 µl of this mix per sample nucleofected. When stored at 4°C, the nucleofector solution and supplement mix is stable for 3 months. Thaw plasmid DNA on ice and prepare plasmid mix by adding equal amounts of pCEh-OCT3/4, pCE-hSK, pCE-hUL, and pCE-mp53DD plasmid DNA (250 ng each/sample) in a 0.5 ml tube. Keep the tube on ice. To prepare LCLs for nucleofection, gently pipette LCL culture 3–5 times to make single cell suspension, then collect 5 ml of cell suspension in a 15 ml tube and centrifuge at 500 × g for 5 min at room temperature. Aspirate the supernatant and resuspend cells in 3–5 ml of pre-warmed PBS without CaCl2 and MgCl2. Determine the cell density using preferred cell-counting method and aliquot 4 × 105 cells in a 1.5 ml microcentrifuge tube. Centrifuge the LCL aliquot at 500 × g for 5 min at room temperature. Remove supernatant completely. Add 1 µg of the plasmid DNA mix prepared in step 3.5 and resuspend the cell pellet gently in 20 µl of room temperature nucleofector solution mix prepared in step 3.4. The volume of plasmid DNA mix should not exceed 10% of the total nucleofection reaction volume. Transfer 20 µl of the cells and plasmid DNA mix into a well of 16-well nucleocuvette strip (supplied with nucleofection kit). Gently tap the nucleocuvette strip to make sure the sample covers the bottom of the cuvette. Place nucleocuvette strip into the retainer of the 4D-Nucleofector X Unit and start nucleofection. After run completion, remove the nucleocuvette strip from the retainer, add 80 µl of prewarmed (37°C) RPMI complete medium, and incubate for 5 min at room temperature. Mix cells by gently pipetting 2–3 times and transfer to the 35 mm cell culture dish prepared in step 3.1. Transfer the culture dish to a CO2 incubator at 37°C, 5% CO2 and atmospheric O2 and allow the cells to recover overnight (16–18 h).

Abstract

Tissue culture based in-vitro experimental modeling of human inherited disorders provides insight into the cellular and molecular mechanisms involved and the underlying genetic component influencing the disease phenotype. The breakthrough development of induced pluripotent stem cell (iPSC) technology represents a quantum leap in experimental modeling of human diseases, providing investigators with a self-renewing and thus unlimited source of pluripotent cells for targeted differentiation into functionally relevant disease specific tissue/cell types. The existing rich bio-resource of Epstein-Barr virus (EBV) immortalized lymphoblastoid cell line (LCL) repositories generated from a wide array of patients in genetic and epidemiological studies worldwide, many of them with extensive genotypic, genomic and phenotypic data already existing, provides a great opportunity to reprogram iPSCs from any of these LCL donors in the context of their own genetic identity for disease modeling and disease gene identification. However, due to the low reprogramming efficiency and poor success rate of LCL to iPSC reprogramming, these LCL resources remain severely underused for this purpose. Here, we detailed step-by-step instructions to perform our highly efficient LCL-to-iPSC reprogramming protocol using EBNA1/OriP episomal plasmids encoding pluripotency transcription factors (i.e., OCT3/4, SOX2, KLF4, L-MYC, and LIN28), mouse p53DD (p53 carboxy-terminal dominant-negative fragment) and commercially available reprogramming media. We achieved a consistently high reprogramming efficiency and 100% success rate (> 200 reprogrammed iPSC lines) using this protocol.