Generation of human induced pluripotent stem cells by simple transient transfection of plasmid DNA encoding reprogramming factors

Abstract

BackgroundThe use of lentiviruses to reprogram human somatic cells into induced pluripotent stem (iPS) cells could limit their therapeutic usefulness due to the integration of viral DNA sequences into the genome of the recipient cell. Recent work has demonstrated that human iPS cells can be generated using episomal plasmids, excisable transposons, adeno or sendai viruses, mRNA, or recombinant proteins. While these approaches offer an advance, the protocols have some drawbacks. Commonly the procedures require either subcloning to identify human iPS cells that are free of exogenous DNA, a knowledge of virology and safe handling procedures, or a detailed understanding of protein biochemistry.ResultsHere we report a simple approach that facilitates the reprogramming of human somatic cells using standard techniques to transfect expression plasmids that encode OCT4, NANOG, SOX2, and LIN28 without the need for episomal stability or selection. The resulting human iPS cells are free of DNA integration, express pluripotent markers, and form teratomas in immunodeficient animals. These iPS cells were also able to undergo directed differentiation into hepatocyte-like and cardiac myocyte-like cells in culture.ConclusionsSimple transient transfection of plasmid DNA encoding reprogramming factors is sufficient to generate human iPS cells from primary fibroblasts that are free of exogenous DNA integrations. This approach is highly accessible and could expand the use of iPS cells in the study of human disease and development.

A small-molecule screen reveals that HSP90β promotes the conversion of induced pluripotent stem cell-derived endoderm to a hepatic fate and regulates HNF4A turnover

“…Human K3 iPSCs were plated on 96-well plates (day 0) and induced to differentiate into hepatic progenitor cells. After (Si-Tayeb et al, 2010c) and HNF4A-depleted iPSCs (Delaforest et al, 2011). (B) Graphs showing the result of quantification of HNF4A immunostaining by measuring average pixel intensity using ImageJ software in each of 30 independent wells of differentiated control (blue diamonds) or HNF4A-depleted cells (red circles).…”

Concise Review: Alchemy of Biology: Generating Desired Cell Types from Abundant and Accessible Cells

“…Some key advances aimed at overcoming these safety concerns have been achieved for the production of iPS cells [11,12,68], and these may be applicable to iTD. Such advances include using nonintegrating viruses (such as adenovirus or episomal plasmid transfection) [69][70][71], treatment of cells with cell-penetrating recombinant reprogramming factor proteins [72], transposon-based systems [73] and the delivery of reprogramming factors in plasmids [74][75][76], repeated transfection of modified mRNA encoding reprogramming factors [77], and miRNAs [78,79].…”

TATκ Fusion Protein of OCT-3/4 and KLF-4: Stable Mixed Population Cell Lines Capable of Delivering Fusion Proteins to Target Cells

“…There are various other strategies to minimise the permanent genetic modification of cell-based therapies have successfully generated iPSCs in both human and mouse system. These include adenoviral and direct plasmid transfection has been successfully used to generate iPSCs in the mouse system [1][2][3]. Also, piggyback transposon vectors [4,5], and most recently, the use of non-integrating episomal vectors [6][7][8], have confirmed the reprogramming of iPSCs from either human or mouse fibroblasts.…”

“…These studies show that genomic integration of viral vectors is not essential for successful reprogramming of iPSCs. This is achievable with episomal plasmids, excisable transposons, adeno or sendai viruses, mRNA, transient plasmid transfection, or recombinant proteins [3]. Another recently published data demonstrated the generation of iPSCs from murine embryonic fibroblasts using recombinant cell penetratingreprogramming fusion proteins [10].…”