Reprogramming of somatic cells induced by fusion of embryonic stem cells using hemagglutinating virus of Japan envelope (HVJ-E)

Abstract

In this research, hemagglutinating virus of Japan envelope (HVJ-E) was used to reprogram somatic cells by fusion with mouse embryonic stem (ES) cells. Neomycin-resistant mouse embryonic fibroblasts (MEFs) were used as somatic cells. Nanog-overexpressing puromycin-resistant EB3 cells were used as mouse ES cells. These two cells were fused by exposing to HVJ-E and the generated fusion cells were selected by puromycin and G418 to get the stable fusion cell line. The fusion cells form colonies in feeder-free culture system. Microsatellite analysis of the fusion cells showed that they possessed genes from both ES cells and fibroblasts. The fusion cells were tetraploid, had alkali phosphatase activity, and expressed stem cell marker genes such as Pou5f1, Nanog, and Sox2, but not the fibroblast cell marker genes such as Col1a1 and Col1a2. The pluripotency of fusion cells was confirmed by their expression of marker genes for all the three germ layers after differentiation induction, and by their ability to form teratoma which contained all the three primary layers. Our results show that HVJ-E can be used as a fusion reagent for reprogramming of somatic cells.

Introduction

Hemagglutinating virus of Japan (HVJ) was the first virus isolated in Japan in the early 1950s. As a mouse parainfluenza virus belonging to the Paramyxoviridae genus, HVJ is 150–600 nm in diameter and contains negative-strand RNA (15,383 bases) inside its viral envelope. Two glycoproteins, fusion (F) and hemagglutinin-neuraminidase (HN), are present on the viral envelope. HN binds to acetylated sialic acid as a receptor on cell membrane and induces cell fusion through F protein [1], [2], [3]. Based on these properties, inactivated HVJ-envelope (HVJ-E) was developed as a drug-delivery vector or a cell fusion agent [4], [5].

New methods for reprogramming somatic cells have been developed in the field of regenerative medicine. One method is somatic cell nuclear transfer, in which a somatic nucleus is transplanted into an unfertilized egg whose nucleus is removed in advance [6], [7]. In 2006, induced pluripotent stem (iPS) cells [8] were established by transfection with four genes, Oct3/4 (Pou5f1), Sox2, Klf4, and c-Myc. In addition to these methods, a lot of researchers also tried to reprogram somatic cells by fusion with ES cells [9], [10], [11], [12].

However, in previous studies, cell fusion was mostly induced by polyethylene glycol or with electrical stimuli. These methods normally could induce severe toxicity or require special electrical instruments. Since HVJ-E is considered less toxic and can be much more conveniently used without special requirements, in this study, we used HVJ-E for cell fusion and induced reprogramming of somatic cells.