Strategies to Improve the Efficiency of Somatic Cell Nuclear Transfer

Mammalian oocytes can reprogram differentiated somatic cells into a totipotent state through somatic cell nuclear transfer (SCNT), which is known as cloning. Although many mammalian species have been successfully cloned, the majority of cloned embryos failed to develop to term, resulting in the overall cloning efficiency being still low. There are many factors contributing to the cloning success. Aberrant epigenetic reprogramming is a major cause for the developmental failure of cloned embryos and abnormalities in the cloned offspring. Numerous research groups attempted multiple strategies to technically improve each step of the SCNT procedure and rescue abnormal epigenetic reprogramming by modulating DNA methylation and histone modifications, overexpression or repression of embryonic-related genes, etc. Here, we review the recent approaches for technical SCNT improvement and ameliorating epigenetic modifications in donor cells, oocytes, and cloned embryos in order to enhance cloning efficiency.     

Signaling Pathways Impact on Induction of Corneal Epithelial-like Cells Derived from Human Wharton’s Jelly Mesenchymal Stem Cells

Corneal epithelium, the outmost layer of the cornea, comprises corneal epithelial cells (CECs) that are continuously renewed by limbal epithelial stem cells (LESCs). Loss or dysfunction of LESCs causes limbal stem cell deficiency (LSCD) which results in corneal epithelial integrity loss and visual impairment. To regenerate the ocular surface, transplantation of stem cell-derived CECs is necessary. Human Wharton’s jelly derived mesenchymal stem cells (WJ-MSCs) are a good candidate for cellular therapies in allogeneic transplantation. This study aimed to test the effects of treatments on three signaling pathways involved in CEC differentiation as well as examine the optimal protocol for inducing corneal epithelial differentiation of human WJ-MSCs. All-trans retinoic acid (RA, 5 or 10 µM) inhibited the Wnt signaling pathway via suppressing the translocation of β-catenin from the cytoplasm into the nucleus. SB505124 downregulated the TGF-β signaling pathway via reducing phosphorylation of Smad2. BMP4 did not increase phosphorylation of Smad1/5/8 that is involved in BMP signaling. The combination of RA, SB505124, BMP4, and EGF for the first 3 days of differentiation followed by supplementing hormonal epidermal medium for an additional 6 days could generate corneal epithelial-like cells that expressed a CEC specific marker CK12. This study reveals that WJ-MSCs have the potential to transdifferentiate into CECs which would be beneficial for further applications in LSCD treatment therapy.     

Development of single mouse blastomeres into blastocysts, outgrowths and the establishment of embryonic stem cells

The recently developed technique of establishing embryonic stem (ES) cell lines from single blastomeres (BTMs) of early mouse and human embryos has created significant interest in this source of ES cells. However, sister BTMs of an early embryo might not have equal competence for the development of different lineages or the derivation of ES cells. Therefore, single BTMs from two- and four-cell embryos of outbred mice were individually placed in sequential cultures to enhance the formation of the inner cell mass (ICM) and the establishment of embryonic outgrowth. The outgrowths were then used for the derivation of ES cell lines. Based on the expression of ICM (Sox2) and trophectoderm (Cdx2) markers, it was determined that ICM marker was lacking in blastocysts derived from 12% of BTMs from two-cell stage and 20% from four-cell stage. Four ES cell lines (5.6%; 4/72) were established ater culture of single BTMs from two-cell embryos, and their pluripotency was demonstrated by their differentiation into neuronal cell types. Our results demonstrate that sister BTMs of an early embryo are not equally competent for ICM marker expression. However, we demonstrated the feasibility of establishing ES cells from a single BTM of outbred mice.