Expression Analysis of Pluripotency Factors in the Undifferentiated Porcine Inner Cell Mass and Epiblast During In Vitro Culture

Authors: Blomberg, Le Ann; Schreier, Lori; Talbot, Neil

Submitted to: Molecular Reproduction and Development
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 4/23/2007
Publication Date: 3/1/2008
Citation: Blomberg, L., Schreier, L.L., Talbot, N.C. 2008. Expression Analysis of Pluripotency Factors in the Undifferentiated Porcine Inner Cell Mass and Epiblast During In Vitro Culture. Molecular Reproduction and Development. 75(3):450-63.

Interpretive Summary: The production of validated porcine embryonic stem cells (pESC) has numerous uses for the agricultural and biomedical fields that could provide great impact. A major impediment to the generation of pESC is the uncertainty of the molecular mechanisms that regulate stem cells in the porcine epiblast. Elucidation of the factor(s) required for the maintenance of pluripotency in pESC is important because species-specific differences have been identified among the stem cells of different organisms and it would also enable validation of current putative stem cell lines. Therefore, in the current study we 1) evaluated the usefulness of known mouse or human ESC markers as potential markers for porcine stem cells and 2) analyzed the influence of in vitro culture on the transcription of those markers, which may lend insight as to whether the in vitro process itself greatly hampers the establishment of cell lines. The expression profile of a panel of mES and hESC pluriopotency factors and a neuronal marker was evaluated by real-time RT-PCR in undifferentiated uncultured ICMs, undifferentiated cultured epiblast cells, and epiblast-derived primordial neuronal and endoderm cells. Exclusivity of the expression of the candidate genes within the stem cell population was determined by comparative gene expression with differentiated epiblast-derived somatic cell types and adult organ tissues via RT-PCR. Expression analysis of ESC associated factors common to mESC and hESC examined across all epiblast tissues, somatic cells and adult organ tissues indicated that, for the most part, POU5F1, TDGF1, and REXO1 mRNA was restricted to the undifferentiated epiblast cells and early primitive tissues (neuronal cells and endoderm) whereas Nanog was unbiquitous. Furthermore, in vitro culture induced alterations in the expression of ESC associated factors. Thus, our data suggested that thorough evaluations, 1) of the tissue distribution and context of the tissue examined are important considerations in the identification of putative pESC factors and 2) to elucidate the functional importance of in vitro culture transcriptional alterations are needed to improve and enable the establishment and validation of porcine ESC lines, respectively.

Technical Abstract: Factors associated with pluripotency have been defined in mouse embryonic stem cells (mESC) and human embryonic stem cells (hESC). Some of these factors were expressed in-common, i.e., in both mESC and hESC, or in a species-specific manner. However, in the pig there is a much less complete understanding of the expression of these factors and this impedes the establishment and validation of porcine ESC (pESC) lines. Therefore, a study to evaluate mESC and hESC marker expression in in vivo and in vitro-cultured porcine epiblast cells taken from zona pellucida-free 8-day porcine blastocysts was performed. In vivo epiblast cells, i.e., within the freshly immunodissected inner cell mass (ICM), and pure epiblast cells at points over a 96 h in vitro culture period, were assayed. In addition, primary cell cultures of epiblast-derived neuroprogenitor cells and epiblast-derived cell lines of various specialized somatic cell types were investigated. The expression profile of pluripoteny markers [POU domain 5 transcript factor 1(POU5F1), SRY- box containing gene 2 (SOX2), and Nanog homeobox (NANOG)], ESC species-specific markers [bone morphogenetic protein 4 (BMP-4), Noggin (NOG), and leukemia inhibitory factor receptor (LIFR)], ICM associated factors, and a neuronal marker was evaluated. The mRNA of uncultured ICMs and primary cultures of epiblast cells, epiblast-derived neuroprogenitor cells, and ICM-derived endoderm cells was amplified by Eberwine methodology. Transcript expression of the candidate marker genes was analyzed by real-time RT-PCR. In order to select factors whose expression correlated best with the undifferentiated porcine epiblast cells, the mRNA expression of the ESC/neuronal candidate markers and additional differentiation markers was also examined by RT-PCR in epiblast-derived somatic cell lines, porcine fetal fibroblasts, and three tissues of the adult pig. Real-time RT-PCR analysis revealed that the undifferentiated pig epiblast cells expressed in-common pluripotency markers (POU5F1, SOX2, NANOG), but that the species-specific markers, NOG and BMP-4, more closely resembled the expression observed in hESC. Across all the tissue types, POU5F1, teratocarcinoma-derived growth factor 1 (TDGF-1), and RNA exonuclease homolog 1 transcript (REXO1) expression was restricted primarily to undifferentiated ICMs and epiblasts, indicating they are good candidates for pluripotency evaluation in the pig. In contrast, NANOG expression was ubiquitous. Furthermore, alterations in the expression of ESC-associated factors were observed during the in vitro culture of the epiblast cells, and this may indicated the in vitro environment’s influence on the differentiation or developmental state of the epiblast cells. The data not only demonstrate the potential usefulness of some pluripotency factors for analyzing the “stemness” of porcine epiblast cells, but it also indicates that in vitro culture alters their transcription. This speaks to the need for further investigations to elucidate the functional importance the transcriptional alterations with respect to the establishment and validation of pESC lines and with respect to porcine developmental biology.