The cytoplasm of mouse germinal vesicle stage oocytes can enhance somatic cell nuclear reprogramming

In mammalian cloning, evidence suggests that genomic reprogramming factors are located in the nucleus rather than the cytoplasm of oocytes or zygotes. However, little is known about the mechanisms of reprogramming, and new methods using nuclear factors have not succeeded in producing cloned mice from differentiated somatic cell nuclei. We aimed to determine whether there are functional reprogramming factors present in the cytoplasm of germinal vesicle stage (GV) oocytes. We found that the GV oocyte cytoplasm could remodel somatic cell nuclei, completely demethylate histone H3 at lysine 9 and partially deacetylate histone H3 at lysines 9 and 14. Moreover, cytoplasmic lysates of GV oocytes promoted somatic cell reprogramming and cloned embryo development, when assessed by measuring histone H3-K9 hypomethylation, Oct4 and Cdx2 expression in blastocysts, and the production of cloned offspring. Thus, genomic reprogramming factors are present in the cytoplasm of the GV oocyte and could facilitate cloning technology. This finding is also useful for research on the mechanisms involved in histone deacetylation and demethylation, even though histone methylation is thought to be epigenetically stable.     

组蛋白去乙酰化酶抑制剂调控干细胞分化与体细胞重编程的研究进展

表观遗传调控,如组蛋白乙酰化修饰,是决定干细胞分化方向的重要机制。组蛋白去乙酰化酶抑制剂(HDACi)通过影响不同亚类的组蛋白去乙酰化酶(HDAC)活性,提高组蛋白乙酰化水平,调控基因表达,从而影响胚胎干细胞自我更新,以及沿神经元、心肌和造血等细胞谱系的定向分化。HDACi类小分子化合物在体细胞重编程中也有广泛的应用,可替代致癌因子c-Myc和Klf4,促进体细胞克隆。研究显示,HDACi的效应与药物剂量、细胞类型和细胞分化状态密切相关。本文主要阐述了HDACi在干细胞分化和体细胞重编程中的应用进展,并对所涉及的分子通路进行讨论,有助于揭示干细胞定向分化的关键分子机制,优化干细胞定向分化诱导策略,对干细胞诱导分化具有重要的理论和实用价值。     

The neurosteroid dehydroepiandrosterone could improve somatic cell reprogramming

Expression of four major reprogramming transgenes, including Oct4, Sox2, Klf4 and c-myc, in somatic cells enables them to have pluripotency. These cells are iPSC (induced pluripotent stem cell) that currently show the greatest potential for differentiation into cells of the three germ lineages. One of the issues facing the successful reprogramming and clinical translation of iPSC technology is the high rate of apoptosis after the reprogramming process. Reprogramming is a stressful process, and the p53 apoptotic pathway plays a negative role in cell growth and self-renewal. Apoptosis via the p53 pathway serves as a major barrier in nuclear somatic cell reprogramming during iPSC generation. DHEA (dehydroepiandrosterone) is an abundant steroid that is produced at high levels in the adrenal cells, and withdrawal of DHEA increases the levels of p53 in the epithelial and stromal cells, resulting in increased levels of apoptotic cells; meanwhile, DHEA decreases cellular apoptosis. DHEA could improve the efficacy of reprogramming yield due to a decrease in apoptosis via the p53 pathway and an increase in cell viability.     

The complex roles of histone demethylases in vivo

Comment on: Di Stefano L, et al. Genes Dev 2011; 25:17-28.     

Five classic articles in somatic cell reprogramming

Research on somatic cell reprogramming has progressed significantly over the past few decades, from nuclear transfer into frogs' eggs in 1952 to the derivation of human-induced pluripotent stem (iPS) cells in the present day. In this article, I review five landmark papers that have laid the foundation for current efforts to apply somatic cell reprogramming in the clinic.     

Role of histone acetylation in reprogramming of somatic nuclei following nuclear transfer

Before fertilization, chromatins of both mouse oocytes and spermatozoa contain very few acetylated histones. Soon after fertilization, chromatins of both gametes become highly acetylated. The same deacetylation-reacetylation changes occur with histones of somatic nuclei transferred into enucleated oocytes. The significance of these events in somatic chromatin reprogramming to the totipotent state is not known. To investigate their importance in reprogramming, we injected cumulus cell nuclei into enucleated mouse oocytes and estimated the histone deacetylation dynamics with immunocytochemistry. Other reconstructed oocytes were cultured before and/or after activation in the presence of the highly potent histone deacetylase inhibitor trychostatin A (TSA) for up to 9 h postactivation. The potential of TSA-treated and untreated oocytes to develop to the blastocyst stage and to full term was compared. Global deacetylation of histones in the cumulus nuclei occurred between 1 and 3 h after injection. TSA inhibition of histone deacetylation did not affect the blastocyst rate (37% with and 34% without TSA treatment), whereas extension of the TSA treatment beyond the activation point significantly increased the blastocyst rate (up to 81% versus 40% without TSA treatment) and quality (on average, 59 versus 45 cells in day 4 blastocysts with and without TSA treatment, respectively). TSA treatment also slightly increased full-term development (from 0.8% to 2.8%). Thus, deacetylation of somatic histones is not important for reprogramming, and hyperacetylation might actually improve reprogramming.     

Nuclear reprogramming in mammalian somatic cell nuclear cloning

Nuclear cloning is still a developing technique used to create genetically identical animals by somatic cell nuclear transfer into unfertilized eggs. Despite an intensive effort in a number of laboratories, the success rate of obtaining viable offspring from this technique remains less than 5%. In the past few years many investigators reported the reprogramming of specific nuclear activities in cloned animals, such as genome-wide gene expression patterns, DNA methylation, genetic imprinting, histone modifications and telomere length regulation. The results highlight the tremendous difficulty the clones face to reprogram the original differentiation status of the donor nuclei. Nevertheless, nuclei prepared from terminally differentiated lymphocytes can overcome this barrier and produce apparently normal mice. Study of this striking nuclear reprogramming activity should significantly contribute to our understanding of cell differentiation in more physiological settings.