Control of ground-state pluripotency by allelic regulation of Nanog

Pluripotency is established through genome-wide reprogramming during mammalian pre-implantation development, resulting in the formation of the naive epiblast. Reprogramming involves both the resetting of epigenetic marks and the activation of pluripotent-cell-specific genes such as Nanog and Oct4 (also known as Pou5f1). The tight regulation of these genes is crucial for reprogramming, but the mechanisms that regulate their expression in vivo have not been uncovered. Here we show that Nanog--but not Oct4--is monoallelically expressed in early pre-implantation embryos. Nanog then undergoes a progressive switch to biallelic expression during the transition towards ground-state pluripotency in the naive epiblast of the late blastocyst. Embryonic stem (ES) cells grown in leukaemia inhibitory factor (LIF) and serum express Nanog mainly monoallelically and show asynchronous replication of the Nanog locus, a feature of monoallelically expressed genes, but ES cells activate both alleles when cultured under 2i conditions, which mimic the pluripotent ground state in vitro. Live-cell imaging with reporter ES cells confirmed the allelic expression of Nanog and revealed allelic switching. The allelic expression of Nanog is regulated through the fibroblast growth factor-extracellular signal-regulated kinase signalling pathway, and it is accompanied by chromatin changes at the proximal promoter but occurs independently of DNA methylation. Nanog-heterozygous blastocysts have fewer inner-cell-mass derivatives and delayed primitive endoderm formation, indicating a role for the biallelic expression of Nanog in the timely maturation of the inner cell mass into a fully reprogrammed pluripotent epiblast. We suggest that the tight regulation of Nanog dose at the chromosome level is necessary for the acquisition of ground-state pluripotency during development. Our data highlight an unexpected role for allelic expression in controlling the dose of pluripotency factors in vivo, adding an extra level to the regulation of reprogramming.     

The molecular mechanism of embryonic stem cell pluripotency maintenance

In vitro cultured embryonic stem （ES） cells are derived from the inner cell mass （ICM） of pre-implantation embryos, and are capable of giving rise to all cell and tissue types of the three germ layers upon being injected back into blastocysts. These ceils are therefore said to possess pluripotency that can be maintained infinitely in culture under optimal conditions. Such pluripotency maintenance is believed to be due to the symmetrical cleavage of the cells in an undifferentiated state. The pluripotency of ES cells is the basis for their various practical and potential applications. ES cells can be used as donor cells to generate knockout or transgenic animals, as in vitro models of mammalian development, and as cell resources for cell therapy in regenerative medicine. The further success in these applications, particularly in the last two, is dependent on the establishment of a culture system with components in the medium clearly defined and the subsequent procedures for controlled differentiation of the cells into specific lineages. In turn, elucidating the molecular mechanism for pluripotency maintenance of ES cells is the prerequisite. This paper summarizes the recent progresses in this area, focusing mainly on the LIF/STAT3, BMPs/Smads, canonical Wnt, TGFβ/activin/nodal, PI3K and FGF signaling pathways and the genes such as oct4, nanog that are crucial in ES cell pluripotency maintenance. The regulatory systems of pluripotency maintenance in both mouse and human ES cells are also discussed. We believe that the cross-talkings between these signaling pathways, as well as the regulatory system underlying pluripotency maintenance will be the main focus in the area of ES cell researches in the future.     

Derivation of pluripotent epiblast stem cells from mammalian embryos

Although the first mouse embryonic stem (ES) cell lines were derived 25 years ago using feeder-layer-based blastocyst cultures, subsequent efforts to extend the approach to other mammals, including both laboratory and domestic species, have been relatively unsuccessful. The most notable exceptions were the derivation of non-human primate ES cell lines followed shortly thereafter by their derivation of human ES cells. Despite the apparent common origin and the similar pluripotency of mouse and human embryonic stem cells, recent studies have revealed that they use different signalling pathways to maintain their pluripotent status. Mouse ES cells depend on leukaemia inhibitory factor and bone morphogenetic protein, whereas their human counterparts rely on activin (INHBA)/nodal (NODAL) and fibroblast growth factor (FGF). Here we show that pluripotent stem cells can be derived from the late epiblast layer of post-implantation mouse and rat embryos using chemically defined, activin-containing culture medium that is sufficient for long-term maintenance of human embryonic stem cells. Our results demonstrate that activin/Nodal signalling has an evolutionarily conserved role in the derivation and the maintenance of pluripotency in these novel stem cells. Epiblast stem cells provide a valuable experimental system for determining whether distinctions between mouse and human embryonic stem cells reflect species differences or diverse temporal origins.     

用流式细胞仪和RAPD快速鉴定柑橘体细胞杂种

利用流式细胞仪和随机扩增多态DNA（RAPD）方法对获得的3例柑橘体细胞杂种进行鉴定,结果表明,两者相结合可快速有效地鉴定体细胞杂种．所检测的3个组合共9株再生植株,有8株是四倍体体细胞杂种,1株为二倍体叶肉亲本型杂种．体细胞杂种一般表现为具有双亲的特征带,且综合双亲的所有带,但在部分杂种中检测到了双亲都没有的新带,也发现有丢失亲本的特征带或共有带的现象,说明融合后染色体发生了重组和交换;有的引物只检测到叶肉亲本的特征带,根据柑橘叶肉细胞无论单独培养还是共培养均不能再生的事实,可推测其为体细胞杂种或二倍体叶肉亲本型胞质杂种．对这两种方法相结合用于体细胞杂种鉴定的可行性进行了讨论．     

Generation of germline-competent induced pluripotent stem cells

We have previously shown that pluripotent stem cells can be induced from mouse fibroblasts by retroviral introduction of Oct3/4 (also called Pou5f1), Sox2, c-Myc and Klf4, and subsequent selection for Fbx15 (also called Fbxo15) expression. These induced pluripotent stem (iPS) cells (hereafter called Fbx15 iPS cells) are similar to embryonic stem (ES) cells in morphology, proliferation and teratoma formation; however, they are different with regards to gene expression and DNA methylation patterns, and fail to produce adult chimaeras. Here we show that selection for Nanog expression results in germline-competent iPS cells with increased ES-cell-like gene expression and DNA methylation patterns compared with Fbx15 iPS cells. The four transgenes (Oct3/4, Sox2, c-myc and Klf4) were strongly silenced in Nanog iPS cells. We obtained adult chimaeras from seven Nanog iPS cell clones, with one clone being transmitted through the germ line to the next generation. Approximately 20% of the offspring developed tumours attributable to reactivation of the c-myc transgene. Thus, iPS cells competent for germline chimaeras can be obtained from fibroblasts, but retroviral introduction of c-Myc should be avoided for clinical application.     

Embryonic and extraembryonic stem cell lines derived from single mouse blastomeres

The most basic objection to human embryonic stem (ES) cell research is rooted in the fact that ES cell derivation deprives embryos of any further potential to develop into a complete human being. ES cell lines are conventionally isolated from the inner cell mass of blastocysts and, in a few instances, from cleavage stage embryos. So far, there have been no reports in the literature of stem cell lines derived using an approach that does not require embryo destruction. Here we report an alternative method of establishing ES cell lines-using a technique of single-cell embryo biopsy similar to that used in pre-implantation genetic diagnosis of genetic defects-that does not interfere with the developmental potential of embryos. Five putative ES and seven trophoblast stem (TS) cell lines were produced from single blastomeres, which maintained normal karyotype and markers of pluripotency or TS cells for up to more than 50 passages. The ES cells differentiated into derivatives of all three germ layers in vitro and in teratomas, and showed germ line transmission. Single-blastomere-biopsied embryos developed to term without a reduction in their developmental capacity. The ability to generate human ES cells without the destruction of ex utero embryos would reduce or eliminate the ethical concerns of many.     

Changing potency by spontaneous fusion

Recent reports have suggested that mammalian stem cells residing in one tissue may have the capacity to produce differentiated cell types for other tissues and organs 1-9. Here we define a mechanism by which progenitor cells of the central nervous system can give rise to non-neural derivatives. Cells taken from mouse brain were co-cultured with pluripotent embryonic stem cells. Following selection for a transgenic marker carried only by the brain cells, undifferentiated stem cells are recovered in which the brain cell genome has undergone epigenetic reprogramming. However, these cells also carry a transgenic marker and chromosomes derived from the embryonic stem cells. Therefore the altered phenotype does not arise by direct conversion of brain to embryonic stem cell but rather through spontaneous generation of hybrid cells. The tetraploid hybrids exhibit full pluripotent character, including multilineage contribution to chimaeras. We propose that transdetermination consequent to cell fusion 10 could underlie many observations otherwise attributed to an intrinsic plasticity of tissue stem cells 9.     

Derivation of androgenetic embryonic stem cells from m-carboxycinnamic acid bishydroxamide (CBHA) treated androgenetic embryos

The androgenetic embyronic stem (aES) cells are useful models in studying the effects of imprinted genes on pluripotency maintaining and embryo development. The expression patterns of imprinted genes are significantly different between uniparental derived aES cells and zygote-derived embryonic stem (ES) cells, therefore, the imprinting related cell pluripotency needs further exploitation. Several approaches have been applied in generation of androgenetic embryos and derivation of aES cell lines. Here, we describe a method to generate androgenetic embryos by injecting two mature sperms into one enucleated oocyte. Then these androgenetic embryos were treated with a histone deacetylase inhibitor: m-carboxycinnamic acid bishydroxamide (CBHA). Further, aES cell lines were successfully derived from these treated androgenetic embryos at blastocyst stage. The CBHA could improve not only the quality of androgenetic embryos, but also the efficiencies of aES (CaES) cells derivation and chimeric mice generation. The imprinted gene expression pattern in the CBHA treated embryo-derived aES (CaES) cells was also highly similar to that of zygote-derived ES cells.     

体细胞核移植技术和诱导多功能干细胞技术的研究比较

本文主要对体细胞核移植技术和诱导多功能干细胞技术的具体操作过程、两种技术当前面临的问题以及他们的应用前景进行了简要说明.