Production of interspecies somatic/pluripotent heterokaryons using polyethylene glycol (PEG) and selection by imaging fl
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Production of interspecies somatic/pluripotent heterokaryons using polyethylene glycol (PEG) and selection by imaging flow cytometry for the study of nuclear reprogramming Cristina Villafranca . Melissa R. Makris . Maria Jesus Garrido Bauerle . Roderick V. Jensen . Willard H. Eyestone
Received: 27 August 2019 / Accepted: 29 August 2020 Ó Springer Nature B.V. 2020
Abstract Fusion of somatic cells to embryonic stem cells induces reprogramming of the somatic nucleus and can be used to study the effect of trans-acting factors from the pluripotent cell over the differentiated nucleus. However, fusion only occurs in a small fraction 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 these rare events. Here, we describe a protocol to induce formation of bispecies mouse pluripotent/bovine somatic heterokaryons, as well as same-species homokaryons, using polyethylene glycol (PEG). To identify bi-species fusion products, heterokaryons were labeled using cell type-specific fluorescent antibodies 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 in vitro. This procedure can be combined with downstream applications such as nucleic acid
C. Villafranca (&) M. R. Makris M. J. Garrido Bauerle W. H. Eyestone Department of Large Animal Clinical Sciences, VirginiaMaryland College of Veterinary Medicine, Virginia Tech, Blacksburg, VA, USA e-mail: [email protected] R. V. Jensen Department of Biological Sciences, Virginia Tech, Blacksburg, VA, USA
isolation for RT-PCR and RNA-Seq, and used as a tool to study somatic cell nuclear reprogramming. Keywords Cell fusion Nuclear reprogramming Polyethylene glycol Homokaryon Heterokaryon Indirect immunofluorescence Imaging Flow cytometry ImageStream
Introduction Cell fusion is a process that involves combining the cytoplasmic membrane of two or more cells to form one single multinucleated cell (Yamanaka and Blau 2010). Nuclear reprogramming can be achieved by fusion of somatic cells to pluripotent cells such as embryonic stem (ES) cells (Tada et al. 2001; Silva et al. 2006; Sullivan et al. 2006), embryonal germ (EG) cells (Tada et al. 1997), or embryonal carcinoma (EC) cells (McBurney and Strutt 1979), suggesting the reprogramming activity of the pluripotent cell is predominant over the gene expression pattern of the somatic cell. The nuclei of somatic cells fused to ES cells reprogram faster (1 to 2 days) and with greater efficiency (up to 70%) than cells reprogrammed by transcription factor induction (Yamanaka and Blau 2010). Fused cells can become hybrids or heterokaryons depending if their nuclei fuse or remain intact, respectively. Gene expression changes in heterokaryons happen in the absence of cell division, without
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Cytotechnology
genom
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