Stem cells are an immortal cell population capable of self-renewal; they are essential for human development and ageing and are a major focus of research in regenerative medicine. Despite considerable progress in differentiation of stem cells in vitro, culture conditions require further optimization to maximize the potential for multicellular differentiation during expansion. The aim of this study was to develop a feeder-free, serum-free culture method for human embryonic stem cells (hESCs), to establish optimal conditions for hESC proliferation, and to determine the biological characteristics of the resulting hESCs. The H9 hESC line was cultured using a homemade serum-free, feeder-free culture system, and growth was observed. The expression of pluripotency proteins (OCT4, NANOG, SOX2, LIN28, SSEA-3, SSEA-4, TRA-1-60, and TRA-1-81) in hESCs was determined by immunofluorescence and western blotting. The mRNA expression levels of genes encoding nestin, brachyury and α-fetoprotein in differentiated H9 cells were determined by RT-PCR. The newly developed culture system resulted in classical hESC colonies that were round or elliptical in shape, with clear and neat boundaries. The expression of pluripotency proteins was increased, and the genes encoding nestin, brachyury, and α-fetoprotein were expressed in H9 cells, suggesting that the cells maintained in vitro differentiation capacity. Our culture system containing a unique set of components, with animal-derived substances, maintained the self-renewal potential and pluripotency of H9 cells for eight passages. Further optimization of this system may expand the clinical application of hESCs.

During preimplantation development, embryos are exposed and have the capacity to respond to different growth factors present in the maternal environment. Among these factors, transforming growth factor β1 (TGF-β1) is a well known modulator of embryonic growth and development. However, its action during the first stages of development, when the embryo transits through the oviduct, has not been yet elucidated. The objective of the present study was to examine the effect of early exposure to exogenous TGF-β1 on embryo development and expression of pluripotency (OCT4, NANOG) and DNA methylation (DNMT1, DNMT3A, DNMT3B) genes in bovine embryos produced in vitro. First, gene expression analysis of TGF-β receptors confirmed a stage-specific expression pattern, showing greater mRNA abundance of TGFBR1 and TGFBR2 from the 2- to the 8-cell stage, before embryonic genome activation. Second, embryo culture for the first 48 h in serum-free CR1aa medium supplemented with 50 or 100 ng/ml recombinant TGF-β1 did not affect the cleavage and blastocyst rate (days 7 and 8). However, RT-qPCR analysis showed a significant increase in the relative abundance of NANOG and DNMT3A in the 8-cell stage embryos and expanded blastocysts (day 8) derived from TGF-β1 treated embryos. These results suggest an early action of exogenous TGF-β1 on the bovine embryo, highlighting the importance to provide a more comprehensive understanding of the role of TGF-β signalling during early embryogenesis.

Cyclin E1 (CCNE1) is a core component of cell cycle regulation that drives the transition into the S phase. CCNE1 plays critical roles in cell cycle, cell proliferation, and cellular functions. However, the function of CCNE1 in early embryonic development is limited. In the present study, the function and expression of Ccne1 in porcine early parthenotes were examined. Immunostaining experiments showed that CCNE1 localized in the nucleus, starting at the four-cell stage. Knockdown of Ccne1 by double-stranded RNA resulted in the failure of blastocyst formation and induced blastocyst apoptosis. Ccne1 depletion increased expression of the pro-apoptotic gene Bax, and decreased the expression of Oct4 and the rate of inner cell mass (ICM)/trophectoderm formation. The results indicated that CCNE1 affects blastocyst formation by inducing cell apoptosis and ICM formation during porcine embryonic development.

Pluripotent cells are unique because of their ability to differentiate into the cell lineages forming the entire organism. True pluripotent stem cells with germ line contribution have been reported for mice and rats. Human pluripotent cells share numerous features of pluripotentiality, but confirmation of their in vivo capacity for germ line contribution is impossible due to ethical and legal restrictions. Progress toward derivation of embryonic stem cells from domestic species has been made, but the derived cells were not able to produce germ line chimeras and thus are termed embryonic stem-like cells. However, domestic animals, in particular the domestic pig (Sus scrofa), are excellent large animals models, in which the clinical potential of stem cell therapies can be studied. Reprogramming technologies for somatic cells, including somatic cell nuclear transfer, cell fusion, in vitro culture in the presence of cell extracts, in vitro conversion of adult unipotent spermatogonial stem cells into germ line derived pluripotent stem cells, and transduction with reprogramming factors have been developed with the goal of obtaining pluripotent, germ line competent stem cells from domestic animals. This review summarizes the present state of the art in the derivation and maintenance of pluripotent stem cells in domestic animals.

In cattle, embryos elongate before implantation and after hatching. Changes in gene expression during this transition are not well studied. Especially important are variations in the expression of pluripotency-associated genes as a result of assisted reproductive biotechnologies, such as cloning and in vitro fertilization (IVF). We hypothesize that there will be a decline in the expression of key pluripotency-associated genes and an increase in the expression of IFN-τ in elongated embryos when compared with day-7 blastocysts. To test this we generated cloned and IVF bovine day-7 blastocyst and day-17 elongated embryos (day 0 = day of nucleus transfer or IVF). Gene expression in all embryos was assessed via RT-qPCR. OCT4 was overexpressed (p < 0.05) in the cloned blastocysts when compared with IVF. No differences in gene expression at this stage between cloned and IVF embryos were found for EOMES, NANOG and FGF4. At elongation EOMES, NANOG and FGF4 were upregulated in IVF embryos (p < 0.05). IFN-τ and OCT4 were expressed at similar levels. There were changes in the expression levels for all transcripts between blastogenesis and elongation. NANOG, IFN-τ and EOMES were overexpressed in all the elongated embryos (p < 0.05), FGF4 was underexpressed in both treatments. OCT4 dropped drastically in the cloned elongated embryos, but not in the IVF. Interestingly only adult donor cells (but not fetal) from which the cloned embryos originated also expressed high levels of OCT4. Our findings might help to understand the shift of gene expression during elongation and to identify key markers of embryonic development useful for embryo screening purposes.

embryonic stem cells (escs) are derived from the inner cell mass of the preimplantation stage embryo and are capable of prolonged symmetrical self-renewal (both daughter cells remain escs) as well as differentiation into derivatives of all three embryonic germ layers. escs therefore have the potential to provide an unlimited supply of transplantable cells to replace or regenerate damaged or diseased tissues. however, several barriers must be overcome before successful clinical trials are possible: for example, pure populations of the desired cell type need to be selected and expanded in clinically relevant numbers, and a method for preventing immunological rejection of the transplanted cells without long-term immunosuppressive therapy is also required. in this review, we highlight recent developments in human esc derivation and expansion, outline current understanding of the signalling pathways underlying stem cell renewal, and discuss challenging problems related to the selective differentiation and immune properties of human escs.