Biochemical and Biophysical Research Communications
Reprogramming of somatic cells induced by fusion of embryonic stem cells using hemagglutinating virus of Japan envelope (HVJ-E)
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.
Section snippets
Cell culture
The mouse ES (mES) cell line, feeder-free EB3 cells (129/Ola-derived EB3 ES cells), which was kindly supplied by Dr. Niwa (RIKEN CBD), was cultured in GMEM (Glasgow minimum essential medium), supplemented with 15% fetal bovine serum (FBS), 1 mM sodium pyruvate, 0.1 mM 2-mercaptoethanol, 1× nonessential amino acids, penicillin/streptomycin, and 1000 U/mL of leukemia inhibitory factor (LIF), in gelatin-coated dishes [13]. As a mouse somatic cell line, neomycin-resistant primary MEFs (Chemicon) were
Cell fusion and reprogramming of MEFs
In 2001, using thymus cells as somatic cells, Tada et al. reported that reprogramming of somatic cells occurred by fusion with ES cells [9]. However, they also reported that the reprogramming efficiency of thymus cells is very low with the cell fusion method. Silva et al. then reported that overexpression of Nanog in ES cells might increase the efficiency of cell reprogramming [17]. Huangfu et al. reported recently that valproic acid (VPA), a histone deacetylase inhibitor, enhanced the
Conclusion
MEFs were fused with mES cells using HVJ-E. The fusion cells were colony-forming tetraploid cells, had genes from both mES cells and MEFs, exhibited alkali phosphatase activity, expressed stem cell marker genes like Pou5f1, Nanog and Sox2, and was pluripotent cells which could differentiate into tissues derived from all the three primary layers. These results suggest that HVJ-E can be used as a cell fusion reagent for cell reprogramming.
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Cytoplasmic fusion between an enlarged embryonic stem cell and a somatic cell using a microtunnel device
2019, Biochemical and Biophysical Research CommunicationsCitation Excerpt :Washizu and colleagues developed a microfluidic device that utilizes an electrofusion method for efficient cell fusion and applied the device for cytoplasmic transfer [6–8]. We previously found that the hemagglutinating virus of Japan (HVJ) envelope can be applied for cell fusion to reprogram a somatic cell [9], and can be further combined with microfabricated apertures for cytoplasmic fusion or organelle transfer in cells [10–12]. However, when the cytosol fusion system is used for somatic cell reprogramming, the ES cell is relatively smaller than the normal somatic cell and cannot be adequately trapped within the cell-pairing structure (CPS) to allow pairing with the somatic cell.
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