The present study investigated the effects of zinc deficiency during in vitro maturation (IVM) of porcine oocytes. Zinc deficiency was induced by administering the membrane‑permeable zinc chelator N,N,N',N'‑tetrakis‑(2‑pyridylmethyl)‑ethylendiamine (TPEN). First, the effects of zinc deficiency during IVM on a TPEN‑treated group and a TPEN+zinc-treated group compared with a control group were assessed. The oocyte maturation rates and subsequent embryonic developmental competence of the TPEN+zinc‑treated oocytes were similar to those of the control oocytes (metaphase II [MII] rate, 93.0 and 92.7%, respectively, and blastocyst [BL] formation rate, 42.0 and 40.0%, respectively). These results were significantly different from those obtained for the TPEN‑treated oocytes (MII rate, 0.61%; BL formation rate, 0%). Although the TPEN‑treated oocytes were arrested at metaphase I (MI), the distribution of microtubules was normal. However, microfilament formation was abnormal in the TPEN‑treated oocytes. Furthermore, the effect of a temporary zinc deficiency during IVM on oocyte maturation and subsequent embryonic development was assessed. TPEN (10 µM) was added to the IVM medium for 0, 7, 15 or 22 h. The 0 h‑treated oocytes showed an 83.9% MII rate, while the 7 h‑treated oocytes had a significantly lower MII rate (44.8%). Most of the 15- and 22 h‑treated oocytes were arrested at MI (MI rate: 98.0 and 97.2%, respectively; MII rate, 0% in both groups). Reductions in the BL formation were dependent on the TPEN treatment duration (29.3, 9.2, 0, and 0% after 0, 7, 15 and 22 h, respectively). In conclusion, zinc is an essential element for successful oocyte maturation and embryonic development in pigs. Zinc deficiency caused a meiotic block and had lasting effects on early embryonic development.

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Porcine embryonic stem cells (pESCs) have great potential for application in translational biomedical research, including xenotransplantation and disease models. Obtaining high-quality blastocysts is the most important factor in the isolation and colonization of primary ESCs and the establishment of ESC lines. In pigs, in vitro-derived blastocysts have a limited cell number compared to in vivo-derived blastocysts and show an indefinite inner cell mass, which may result in failure to establish pESC lines. In the present study, the effects of resveratrol (RES), granulocyte-macrophage colony stimulating factor (GM-CSF) and β-mercaptoethanol (β-ME) on the quality of blastocysts and the efficiency of colony derivation were investigated for the establishment of ESCs. A novel culturing system was developed in which 2 µM RES was added to the oocyte in vitro maturation (IVM) medium, and 10 ng/ml pGM-CSF and 10 µM β-ME were added to embryo in vitro culture (IVC) medium. This novel system showed significantly more parthenogenetic activation (PA) blastocysts (54.5 ± 1.8% vs. 43.4 ± 1.2%; P<0.05) and in vitro fertilization (IVF) blastocysts (36.9 ± 3.3% vs. 26.2 ± 2.9%; P<0.06) at day seven as compared with that in the control system. The PA and IVF blastocysts from the novel system showed a significantly greater hatching rate (P<0.05) and greater cell numbers (55.1 ± 2.0 vs. 45.6 ± 2.0; P<0.05 and 78.9 ± 6.8 vs. 58.5 ± 7.2; P<0.06, for PA and IVF, respectively) at day seven compared to that in the control system. After seeding on feeder cells, the PA blastocysts produced by the novel system showed a significantly increased rate of attachment (28.8 ± 3.9% vs. 17.2 ± 2.4%; P<0.062). Finally, two putative pESC lines from PA embryos produced by the novel system and one by the control system were established. In conclusion, the novel system improved blastocyst quality and increased the derivation efficiency of putative pESC lines from porcine PA and IVF embryos produced in vitro.

Craniofacial skeletal muscle is composed of approximately 60 muscles, which have critical functions including food uptake, eye movements and facial expressions. Although craniofacial muscles have significantly different embryonic origin, most current skeletal muscle differentiation protocols using human induced pluripotent stem cells (iPSCs) are based on somite-derived limb and trunk muscle developmental pathways. Since the lack of a protocol for craniofacial muscles is a significant gap in the iPSC-derived muscle field, we have developed an optimized protocol to generate craniofacial myogenic precursor cells (cMPCs) from human iPSCs by mimicking key signaling pathways during craniofacial embryonic myogenesis. At each different stage, human iPSC-derived cMPCs mirror the transcription factor expression profiles seen in their counterparts during embryo development. After the bi-potential cranial pharyngeal mesoderm is established, cells are committed to cranial skeletal muscle lineages with inhibition of cardiac lineages and are purified by flow cytometry. Furthermore, identities of Ipsc-derived cMPCs are verified with human primary myoblasts from craniofacial muscles using RNA sequencing. These data suggest that our new method could provide not only in vitro research tools to study muscle specificity of muscular dystrophy but also abundant and reliable cellular resources for tissue engineering to support craniofacial reconstruction surgery.

Abstract:Pigs have great potential to provide preclinical models for human disease in translational research because of their similarities with humans. In this regard, porcine pluripotent cells, which are able to differentiate into cells of all three primary germ layers, might be a suitable animal model for further development of regenerative medicine. Here, we describe the current state of knowledge on apoptosis in pluripotent cells including inner cell mass (ICM), epiblast, embryonic stem cells (ESCs), and induced pluripotent stem cells (iPSCs). Information is focused on the apoptotic phenomenon in pluripotency, maintenance, and differentiation of pluripotent stem cells and reprogramming of somatic cells in pigs. Additionally, this review examines the multiple roles of apoptosis and summarizes recent progress in porcine pluripotent cells.