Production of interspecies somatic/pluripotent heterokaryons using polyethylene glycol (PEG) and selection by imaging flow cytometry for the study of nuclear reprogramming

Abstract

ABSTRACTFusion of somatic cells to pluripotent cells such as mouse embryonic stem (ES) cells induces reprogramming of the somatic nucleus, and can be used to study the effect of trans-acting factors from the pluripotent cell on the somatic nucleus. Moreover, fusion of cells from different species permits the identification of the transcriptome of each cell, so the gene expression changes can be monitored. However, fusion only happens in a small proportion of the cells exposed to fusogenic conditions, hence the need for a protocol that produces high fusion rate with minimal cell damage, coupled with a method capable of identifying and selecting fusion events from the bulk of the cells. Polyethylene glycol (PEG) is a polymer of repeated ethylene oxide units known to induce cell fusion within a certain range of molecular weight. Here, we describe a method to induce formation of bi-species heterokaryons from adherent mammalian cells, which can then be specifically labeled and selected using live cell immunostaining and a combination of imaging and traditional flow cytometry. First, we tested several PEG-based fusion conditions to optimize a protocol to consistently produce both mouse NIH/3T3 fibroblast and primary bovine fetal fibroblast (bFF) homokaryons. Initially, we obtained 7.28% of NIH/3T3 homokaryons when using 50% PEG 1500. Addition of 10% of DMSO to the PEG solution increased the percentage of NIH/3T3 homokaryons to 11.71%. In bFFs, treatment with 50% PEG 1500 plus 10% DMSO produced 11.05% of homokaryons. We then produced interspecies heterokaryons by fusing mouse embryonic stem (mES) cells to bFFs. To identify bi-species fusion products, heterokaryons were labeled using indirect immunostaining in live cells and selected using imaging (Amnis ImageStream Mark II) and traditional (BD FACSAria I) flow cytometry. Heterokaryons selected with this method produced ES cell-like colonies when placed back in culture. The method described here can also be combined with downstream applications such as nucleic acid isolation for RT-PCR and RNA-seq, and used as a tool to study cellular processes in which the effect of trans-acting factors is relevant, such as in nuclear reprogramming.