To derive porcine embryonic stem (ES) cell lines, the time window during which porcine embryos contain pluripotent cells that are predisposed to undifferentiated self-renewal in vitro must be... Show moreTo derive porcine embryonic stem (ES) cell lines, the time window during which porcine embryos contain pluripotent cells that are predisposed to undifferentiated self-renewal in vitro must be identified. Therefore we first studied the spatial and temporal expression pattern of key factors in pluripotency and lineage segregation of blastocyst-stage porcine embryos between embryonic days (E) 6.5 and E10.5 using whole mount in situ hybridization, quantitative reverse transcription (RT)-PCR and whole mount immunofluorescence. Expression of NANOG and SOX2 was detected in both the ICM and epiblast, while OCT4 expression became restricted to the epiblast at E9.5. Surprisingly ICM and epiblast cells also expressed CK18. Consequently, growth factors which sustain the undifferentiated growth of human ES cells and mouse epiblast stem cells (EpiSCs) were tested for their ability to sustain undifferentiated self-renewal of porcine ICM and epiblast cells in vitro. Cultures of ICM cells resulted in a higher percentage of primary colonies with an ES-like morphology compared to primary cultures derived from epiblast cells. These undifferentiated colonies sustained expression of OCT4, NANOG, SOX2 and CK18. The expression of CK18 suggests that these cells are more similar to human ES cells and mouse EpiSCs than to mouse ES cells. Although undifferentiated cultures were maintained for limited passages, ICM and epiblast cultures rapidly differentiated into cell types of mesodermal, ectodermal, and endodermal origin, as characterized by RT-PCR. These results demonstrate that porcine ICM and epiblast cells can not be cultured in vitro with currently used human ES cell culture conditions. Importantly however, the trio of OCT4, NANOG and SOX2, which are known to form an autoregulatory network for pluripotency in other systems, are co-expressed also by porcine epiblasts, and by undifferentiated primary colonies in culture. (C) 2011 Elsevier Inc. All rights reserved. Show less
Puy, L. du; Lopes, S.M.C.D.; Haagsman, H.P.; Roelen, B.A.J. 2010
Here we report the culture and differentiation of porcine neural cells derived from the inner cell masses (ICMs) of blastocyst-stage embryos. Manually dissected ICMs were cultured on feeder layers... Show moreHere we report the culture and differentiation of porcine neural cells derived from the inner cell masses (ICMs) of blastocyst-stage embryos. Manually dissected ICMs were cultured on feeder layers of inactivated mouse embryonic fibroblasts (MEFs). Neural rosette-like structures were selected and passaged mechanically. These structures were transient and disappeared after six passages. After transfer to Matrigel-coated dishes, the cells proliferated for similar to 2 months. Expression of neural progenitor cell (NPC)-specific genes NESTIN, SOX1, SOX2, and MUSASHI as detected by RT-PCR suggests that the cell culture contained neural progenitor-like cells. To further confirm the culture of neural progenitor-like cells, we analyzed their differentiation potential in vitro. The porcine neural cells were able to differentiate into glial fibrillary acidic protein (GFAP)-positive astrocytes and O4-positive oligodendrocytes, identified by quantitative RT-PCR and immunofluorescence. Differentiated cells expressed microtubule-associated protein 2 (MAP2) RNA but were unable to differentiate into neurons. It is concluded that porcine blastocysts have the ability to provide an expandable source of neural cells that can develop into glial subtypes. Show less
