Simple Generation of Human Induced Pluripotent Stem Cells Using Poly-β-amino Esters As the Non-viral Gene Delivery System

Federico González,Núria Montserrat,Salvador Borrós,Jordán Gutiérrez,Elena Garreta,Juan Carlos Izpisua Belmonte,Cristina Eguizábal,Víctor Ramos

doi:10.1074/jbc.m110.168013

Abstract

Reprogramming of somatic cells to induced pluripotent stem (iPS) cells can be achieved by the delivery of a combination of transcription factors, including Oct4, Sox2, Klf4, and c-Myc. Retroviral and lentiviral vectors are commonly used to express these four reprogramming factors separately and obtain reprogrammed iPS cells. Although efficient and reproducible, these approaches involve the time-consuming and labor-intensive production of retroviral or lentiviral particles together with a high risk of working with potentially harmful viruses overexpressing potent oncogenes, such as c-Myc. Here, we describe a simple method to produce bona fide iPS cells from human fibroblasts using poly-β-amino esters as the transfection reagent for the delivery of a single CAG-driven polycistronic plasmid expressing Oct4, Sox2, Klf4, c-Myc, and a GFP reporter gene (OSKMG). We demonstrate for the first time that poly-β-amino esters can be used to deliver a single polycistronic reprogramming vector into human fibroblasts, achieving significantly higher transfection efficiency than with conventional transfection reagents. After a protocol of serial transfections using poly-β-amino esters, we report a simple methodology to generate human iPS cells from human fibroblasts avoiding the use of viral vectors.

Highlights

We have described for the first time the successful delivery of the large pCAG-OSKMG plasmid (11 kb) to human fibroblasts by using three different end-modified poly-␤-amino esters as non-viral delivery system
We chose them as a promising non-viral delivery system to develop an optimized plasmid transfection strategy to generate human iPS cells from human fibroblasts
We first demonstrated that poly-␤-amino esters could condense the pCAG-OSKMG plasmid-forming nanoparticles with appropriate biophysical properties to allow cellular uptake

Summary

EXPERIMENTAL PROCEDURES

Synthesis of the Poly-␤-amino Esters—Poly-␤-aminoesters were synthesized by the addition reaction of primary amines with excess of diol diacrylates. Human foreskin fibroblasts were transfected with pmaxGFP plasmid or pCAG-OSKMG plasmid in Opti-MEM medium using the three different poly-␤-amino esters. Polyplexes were formulated by mixing the poly-␤-amino esters (B1, B2, and B3) with the plasmids at different ratios (w/w) of DNA to polymer (between 1:20 to 1:100). The day cells were transfected with pCAG-OSKMG plasmid using poly-␤-amino esters. In Vitro Differentiation of the Generated iPS Cell Lines—After 3– 4 days, the embryoid bodies were transferred to 0.1% gelatin-coated glass chamber slides and cultured in differentiation medium (DMEM supplemented with 20% fetal bovine serum, 2 mM L-glutamine, 0.1 mM 2-mercaptoethanol, nonessential amino acids, and penicillin-streptomycin) for 2–3 weeks to allow spontaneous endoderm formation. Tumor samples were collected generally in 2 months and processed for paraffin embedding and hematoxylin and eosin staining following standard procedures

RESULTS

Particle size

GFP negative colonies

DISCUSSION