摘要: Epigenetic modifications play critical roles during somatic cell nuclear transfer (SCNT) embryo development. Whether RNA N6-methyladenosine (m(6) A) affects the developmental competency of SCNT embryos remains unclear. Here, we showed that porcine bone marrow mesenchymal stem cells (pBMSCs) presented higher RNA m(6) A levels than those of porcine embryonic fibroblasts (pEFs). SCNT embryos derived from pBMSCs had higher RNA m(6) A levels, cleavage, and blastocyst rates than those from pEFs. Compared with pEFs, the promoter region of METTL14 presented a hypomethylation status in pBMSCs. Mechanistically, DNA methylation regulated METTL14 expression by affecting the accessibility of transcription factor SP1 binding, highlighting the role of the DNA methylation/SP1/METTL14 pathway in donor cells. Inhibiting the DNA methylation level in donor cells increased the RNA m(6) A level and improved the development efficiency of SCNT embryos. Overexpression of METTL14 significantly increased the RNA m(6) A level in donor cells and the development efficiency of SCNT embryos, whereas knockdown of METTL14 suggested the opposite result. Moreover, we revealed that RNA m(6) A-regulated TOP2B mRNA stability, translation level, and DNA damage during SCNT embryo development. Collectively, our results highlight the crosstalk between RNA m(6) A and DNA methylation, and the crucial role of RNA m(6) A during nuclear reprogramming in SCNT embryo development.

摘要: Glaucoma and other optic neuropathies lead to progressive and irreversible vision loss by damaging retinal ganglion cells (RGCs) and their axons. Cell replacement therapy is a potential promising treatment. However, current methods to obtain RGCs have inherent limitations, including time-consuming procedures, inefficient yields and complex protocols, which hinder their practical application. Here, we have developed a straightforward, rapid and efficient approach for directly inducing RGCs from mouse embryonic fibroblasts (MEFs) using a combination of triple transcription factors (TFs): ASCL1, BRN3B and PAX6 (ABP). We showed that on the 6th day following ABP induction, neurons with molecular characteristics of RGCs were observed, and more than 60% of induced neurons became iRGCs (induced retinal ganglion cells) in the end. Transplanted iRGCs had the ability to survive and appropriately integrate into the RGC layer of mouse retinal explants and N-methyl-D-aspartic acid (NMDA)-damaged retinas. Moreover, they exhibited electrophysiological properties typical of RGCs, and were able to regrow dendrites and axons and form synaptic connections with host retinal cells. Together, we have established a rapid and efficient approach to acquire functional RGCs for potential cell replacement therapy to treat glaucoma and other optic neuropathies. The transcription factor (TF) combination ASCL1 + BRN3B + PAX6 could efficiently reprogram mouse embryonic fibroblasts (MEFs) into induced retinal ganglion cells (iRGCs). These iRGCs not only expressed several molecular markers of native RGCs, but also exhibited typical RGC electrophysiological properties. Upon transplantation into mouse retinal explants or NMDA-damaged retinas, iRGCs could integrate into the ganglion cell layer, survive for a prolonged period, grow long neuron fibres, form synapses with host cells, and exhibit electrophysiological functions.image

摘要: Adipose-derived stem cells (ASCs) have shown efficacy in promoting hair growth, while DKK1 inhibits the WNT pathway, which is associated with hair loss. Our study focused on investigating the expression of DKK1 in alopecia areata (AA), a condition characterised by significant increases in the DKK1 levels in human and mouse ASCs. Treatment of interferon-gamma increased the expression of DKK1 via STAT3 phosphorylation in ASCs. Treatment with recombinant DKK1 resulted in a decrease of cell growth in outer root sheath cells, whereas the use of a DKK1 neutralising antibody promoted hair growth. These results indicate that ASCs secrete DKK1, playing a crucial role in the progression and development of AA. Consequently, we generated DKK1 knockout (KO) ASCs using the Crispr/Cas9 system and evaluated their hair growth-promoting effects in an AA model. The DKK1 KO in ASCs led to enhanced cell motility and reduced cellular senescence by activating the WNT signalling pathway, while it reduced the expression of inflammatory cytokines by inactivating the NF-kB pathway. As expected, the intravenous injection of DKK1-KO-ASCs in AA mice, and the treatment with a conditioned medium derived from DKK1-KO-ASCs in hair organ culture proved to be more effective compared with the use of naive ASCs and their conditioned medium. Overall, these findings suggest that DKK1 represents a novel therapeutic target for treating AA, and cell therapy using DKK1-KO-ASCs demonstrates greater efficiency.