Forkhead box family transcription factors as versatile regulators for cellular reprogramming to pluripotency

Forkhead box (Fox) transcription factors play important roles in mammalian development and disease. However, their function in mouse somatic cell reprogramming remains unclear. Here, we report that FoxD subfamily and FoxG1 accelerate induced pluripotent stem cells (iPSCs) generation from mouse fibroblasts as early as day4 while FoxA and FoxO subfamily impede this process obviously. More importantly, FoxD3, FoxD4 and FoxG1 can replace Oct4 respectively and generate iPSCs with germline transmission together with Sox2 and Klf4. On the contrary, FoxO6 almost totally blocks reprogramming through inhibiting cell proliferation, suppressing the expression of pluripotent genes and hindering the process of mesenchymal to epithelial transition (MET). Thus, our study uncovers unexpected roles of Fox transcription factors in reprogramming and offers new insights into cell fate transition.

Induced Pluripotent Stem Cells

The isolation of human embryonic stem cells in 1998 has since fueled the ideology that stem cells may eventually be used for human disease therapies as well as the regeneration of tissues and organs. The transformation of somatic cells to a pluripotent state via somatic nuclear transfer and embryonic stem cell fusion brought the scientific community nearer to understanding the molecular mechanisms that govern cellular pluripotency. In 2006, the first induced pluripotent stem (iPS) cell was reported, where a mouse somatic cell was successfully converted to a pluripotent state via transduction of four essential factors. This cellular breakthrough has allowed for robust scientific investigations of human diseases that were once extremely difficult to study. Scientists and pharmaceuticals now use iPS cells as means for disease investigations, drug development and cell or tissue transplantation. There is little doubt that scientific progress on iPS cells will change many aspects of medicine in the next couple of decades.

Effect of melatonin treatment on developmental potential of somatic cell nuclear-transferred mouse oocytes in vitro

SummaryThe beneficial effect of supplementing culture medium with melatonin has been reported during in vitro embryo development of species such as mouse, bovine and porcine. However, the effect of melatonin on mouse somatic cell nuclear transfer remains unknown. In this study, we assessed the effects of various concentrations of melatonin (10−6 to 10−12 M) on the in vitro development of mouse somatic cell nuclear transfer embryos for 96 h. Embryos cultured without melatonin were used as control. There was no significant difference in cleavage rates between the groups supplemented with melatonin, dimethyl sulphoxide (DMSO) and the control. The rate of development to blastocyst stage was significantly higher in the group supplemented with 10−12 M melatonin compared with the control group (P < 0.05). Thus, our data demonstrated that adding melatonin to pre-implantation mouse nuclear-transferred embryos can accelerate blastocyst formation.

27 PSAMMAPLIN A INCREASES DEVELOPMENT AND QUALITY OF MOUSE SOMATIC CELL NUCLEAR TRANSFER EMBRYOS

Among the many biological and technical factors affecting the success rate of mouse somatic cell nuclear transfer (SCNT), faulty reprogramming of the differentiated donor nucleus to a totipotent embryonic state by the recipient oocyte seems key. Accordingly, treatment of SCNT embryos with epigenetic modifiers such as valproic acid (VPA), a histone deacetylase inhibitor (HDACi), enhances cloning efficiency. Psammaplin A (PsA) is a natural and potent DNA methyltransferase inhibitor and HDACi that has never been used in nuclear reprogramming studies. The purpose of our study was to determine the effect of PsA on the development and quality of mouse SCNT embryos, and to compare it to that of VPA. To this aim, mechanically enucleated oocytes from B6CBAF1 female mice were reconstructed with cumulus cell nuclei, activated, and cultured in the presence of the epigenetic modifier. Embryos that reached the blastocyst stage were differentially stained for counting inner cell mass (ICM) and trophectoderm cells. Alternatively, 2-cell embryos were transferred to CD1 recipient females to assess full-term development. In a first set of experiments, embryos were exposed to different concentrations of PsA (5, 10, and 20 µM) or VPA (2 and 4 mM) for 1 to 2 h after reconstruction and 6 h of activation (total 8–9 h). We found that 10 µM PsA and 2 mM VPA significantly increased blastocyst rates (37.3 and 31 v. 23.3% for the control group), although no differences were found in blastocyst quality (10.4–13.6 ICM cells). In a second set of experiments, we studied the effect of treatment duration by incubating the embryos in 10 µM PsA or 2 mM VPA for 8 to 9, 16, or 24 h after reconstruction. With PsA, all treatments showed equivalent blastocyst rates (35.2–43.3%), which were significantly higher than in the control group (20%), but only treatments for 16 and 24 h yielded blastocysts with significantly higher numbers of ICM cells (16.3 and 18.5 v. 10 for the control group). With VPA, treatments for 8 to 9 h and 16 h were equivalent in terms of blastocyst rates (34.0 and 32.5%) and significantly higher than the control group, but only VPA 16 h yielded blastocysts with a significantly higher number of ICM cells (15.6). In a third set of experiments, we studied the full-term development of embryos treated with 10 µM PsA or 2 mM VPA for 16 h and we found that both treatments, but especially the PsA treatment, resulted in higher birth rates than those obtained in the control group, although the differences were not statistically significant (1.79 and 0.86 v. 0.46%). Finally, when the actin polymerization inhibitor latrunculin A was used instead of cytochalasin B in the SCNT protocol during oocyte micromanipulation and activation, we obtained a 3-fold increase in the birth rate of embryos treated with PsA (5.29%). In conclusion, PsA enhances development and quality of mouse SCNT embryos, to a greater extent than VPA, and when combined with the use of latrunculin A instead of cytochalasin B, it results in an 11.5-fold increase in full-term development. Support from MEC AGL-2011-23784, 2009-SGR-282, and PIF-UAB Fellowships is acknowledged.