Final Report Summary - PARTNERS (Comparative embryonic stem cell research in mammalians) The PARTNERS project was aimed to generate new comparative information on the generation and maintenance of embryonic stem cells (ESCs) in mouse, pig and human, and on their differentiation towards cardiac and/or neural lineages. A new mouse ESC line from C57Bl/6J genetic background was established and characterised morphologically, enzymatically (alkaline phosphatase staining), immunologically using antibodies against pluripotency markers including Oct4/Pou5f, Nanog, SSEA1 and by RT-PCR verifying the expression pattern of Oct4/Pou5f, Nanog, Sox2, Klf4 and c-Myc by Biotalentum Ltd. The pluripotency stage of this new cell line was verified in vitro, by performing cardiac and neural differentiation, and in vivo by blastocyst injection with ESCs resulting hair coat and germline chimeras. Comparative studies of 2D cardiac and neural differentiation was performed for mESCs from C57Bl/6J and 129SV/Ola (HM1) genetic backgrounds, showing that the newly generated C57Bl/6J ESC line behaved similarly to the well characterised ESC line. Three-dimensional (3D) differentiation, using an air-liquid interface based culture which allows three dimensional cell expansion and neural differentiation in the absence of added growth factors (engineered neural tissue (ENT)), was performed for the ESC lines from both genetic backgrounds. Comparing the 3D structures for the two cell lines differences could be detected. The tissue obtained from HM1 ESCs had a compact, homogenous structure. In contrast to this, the tissue obtained from C57Bl/6J genetic background ESCs showed tubular structures. University of Copenhagen has aimed at generating pluripotent porcine stem cell lines and performing neural differentiation of these lines. The pluripotent stem cell types, which have been studied, were ESCs, embryonic germ cells (EGCs), and induced pluripotent stem cells (iPSCs). In order to characterise the biology of and find markers for pluripotency in the pig, studies of the pluripotent cell types of the embryo (inner cell mass (ICM) and epiblasts) have been performed with respect to effects of transcription factors and epigenetic changes. Expression profiling of the porcine embryo with particular focus on the development of the ICM to the epiblast has been investigated (Hall et al., 2010). Likewise, epigenetic characteristics with respect to DNA methylation and histone modifications during initial embryonic development in the pig were studied (Deshmukh et al., 2011, 2012; Gao et al., 2010, 2011a,b). This work forms a solid future basis for the use of pluripotency markers on porcine cell cultures. Elaborate experiments have been performed in order to establish pluripotent porcine ESCs. Although well-defined methods for establishing outgrowth of pluripotent cells from the ICM and epiblast were tested, the cells lost their pluripotency over a few passages (Wolf et al., 2010). Other laboratories have obtained similar results. Due to the fact that porcine ESCs could not be established, establishment of alternative types of porcine pluripotent stem cells were pursued including EGCs and iPSCs. Refined methods for deriving EGCs from individual embryos (first report in the World) were developed. The characterisation of the cells, including RNA sequencing, demonstrated that porcine EGCs are multipotent rather than pluripotent (Petkov et al., 2011). Employing the human pluripotency factors hOCT4, hNANOG, and hcMYC introduced into somatic cells under doxycycline-regulated conditions resulted in partially reprogrammed porcine iPSC-like (piPSC) cell lines (Hall et al., 2012). Despite of the expression of some endogenous genes, the piPSC-like cells still cannot be maintained without doxycycline. Recently, using a polycistronic lentivirus construct containing the four porcine pluripotency markers, piPSC from both porcine fibroblasts and neural progenitor cells were established. These cells have a robust up regulation of endogenous OCT4, SOX2, and NANOG, are alkaline phosphatase positive, and can be differentiated into all three germlayers. The in vivo pluripotency test is ongoing. Neural progenitor cells (NPCs) have been generated from porcine epiblasts by co-culture with mouse MS5 stromal feeder cells and characterised. The NPCs were indeed multipotent and formed both neurons and glia, and they have had the capacity of self-renewal (Rasmussen et al., 2011). Also, porcine iPSCs derived from porcine NPCs have been subjected to neural differentiation by co-culture with MS5 stromal cells. Mature neurons were detected after 21 days of culture and two NPC lines were established. Two-dimensional (2D) versus 3D neural differentiation was performed with porcine epiblast derived NPCs, which were cultured for up to two weeks as neurospheres (NSs). Day seven NSs were also transferred to hydrophilic membranes for ENT culture. The ENTs were composed of densely packed cellular material with sparse intercellular spaces. Neuron-like cells presented large spherical euchromatic nuclei with prominent nucleoli, and abundant mitochondria, Golgi and lysosomes in the perinuclear cytoplasm. Their elongated projections resembled non-myelinated dendrites and axons, and early synapse formation was indicated by the presence of vesicles located in their bulbous endings. The Chulalongkorn University (Thailand) team, as a first step in improving the efficiency and functionality of human neural cells, adapted the hESC to xeno free culture condition. During this process, the effect of Rho-kinase (Rock) inhibitor, Y-26732 on cell survival and apoptosis following enzymatic passaging was also evaluated. By transferring the knowledge gained from the 2D and 3D neural differentiation of mESC lines, the methods were successfully used for hESC differentiation towards neural lineages. The direct outcome of the project is the generation of standardised culture and differentiation protocols for mouse, pig and human embryonic and induced pluripotent stem cells.
