Epigenetic reprogramming of somatic genomes by electrofusion with embryonic stem cells

Abstract

Cell fusion is an approach for combining genetic and epigenetic information between two different types of cells. Electrofusion for generating hybrid cells between mouse embryonic stem cells and somatic cells, which is a type of nonchemically induced and nonvirus-mediated cell fusion, is introduced here as a highly effective, reproducible, and biomedically safe in vitro system. Under optimized electrofusion conditions, cells are ligned and form pearl chains between electrodes in response to AC pulse stimulation, and subsequently adjacent cytoplasmic membranes are fused by DC pulse stimulation. Hybrid cells survive as drug-resistant colonies in selection medium. Cell fusion is a technique that is applied widely in the life sciences. A recent topic of great interest in the field of stem cell research is the successful production of cloned animals via epigenetic reprogramming of somatic nuclei. Interestingly, nuclear reprogramming for conferring pluripotency on somatic nuclei also occurs via cell fusion between pluripotential stem cells and somatic cells. Furthermore, it has been shown that spontaneous cell fusion contributes to generating the intrinsic plasticity of tissue stem cells. Cell fusion technology may make important contributions to the fields of regenerative medicine and epigenetic reprogramming.

Fig 1.

Scheme of electrofusion system. The cell mixture of ES cells and somatic cells suspended in nonelectrolyte 0.3 M mannitol is placed into the 1-mm gap between two electrodes fixed on a Microslide. Presettings of optimized AC voltage, AC duration, DC pulse voltage, and DC pulse number are required. The function of AUTOMATIC START in ECM 2001 initiates cell alignment through AC application and reversible breakage of cytoplasmic membranes by sequential DC electroporation pulse, leading to the fusion of cell membranes between two different cells. After the cell fusion procedure, ES cells and ES-ES hybrid cells are selectively killed using selection medium, while thymocytes are nonadherent. ES-somatic hybrid cells are selectively grown in the selection medium and isolated in independent culture dishes.

Fig 2.

(A) Setup of AC/DC pulse generator ECM2001 (BTX) (B) Microslide chamber in a 100-mm plastic dish made using a bacterial dish and Micrograbber cable. (C) Cell mixture of ES cells and thymocytes applied between electrodes. (D) Pearl chain formation during AC application. (E) Instruments for dissociating mouse thymus into single cells. (F) Culture dishes with inactivated PEFs prepared 1 d before cell fusion. (G) The recommended fusion parameters.