Rat blastocyst-derived stem cells are precursors of embryonic and extraembryonic lineages

Despite recent advances in the derivation of rat embryonic stem cells, clear comprehension of the timing and mechanisms underlying rat early embryo lineage selection is lacking. We have previously shown the in vivo contribution of rat embryonic stem-like cells exclusively to developing extraembryonic tissues. To elucidate possible mechanisms governing the in vitro and in vivo behaviors of these rat blastocyst-derived stem cells, we evaluated their developmental capacity by using several approaches. Molecular marker analysis demonstrated the expression profile of genes characterizing not only pluripotency but also extraembryonic endoderm and trophoblast. In vitro differentiation through embryoid body formation showed in vitro pluripotent capacity through differentiation into derivatives of all three embryonic germ layers. Following either blastocyst injection, diploid or tetraploid aggregation, and embryo transfer, these rat blastocyst-derived stem cells also demonstrated in vivo multipotency through contribution to multiple developmentally distinct extraembryonic lineages. Features of phenotypic heterogeneity were revealed following examination of cell line morphology and culture behavior, as well as quantitative analysis of marker expression in discrete undifferentiated and differentiated populations of cells by flow cytometry. We demonstrate for the first time that stem cells derived from the rat blastocyst have the ability to contribute to the embryonic and extraembryonic lineages. Together, these results provide a valuable new model for rat stem cell biology and for the elucidation of early lineage selection in the embryo.     

Confinement and clearance of OCT4 in the porcine embryo at stereomicroscopically defined stages around gastrulation

In the areas of developmental biology and embryonic stem cell research, reliable molecular markers of pluripotency and early lineage commitment are sparse in large animal species. In this study, we present morphological and immunohistochemical findings on the porcine embryo in the period around gastrulation, days 8-17 postinsemination, introducing a stereomicroscopical staging system in this species. In embryos at the expanding hatched blastocyst stage, OCT4 is confined to the inner cell mass. Following detachment of the hypoblast, and formation of the embryonic disk, this marker of pluripotency was selectively observed in the epiblast. A prominent crescent-shaped thickening at the posterior region of the embryonic disk marked the first polarization within this structure reflecting incipient cell ingression. Following differentiation of the epiblast, clearance of OCT4 from the three germ layers was observed at defined stages, suggesting correlations to lineage specification. In the endoderm, clearance of OCT4 was apparent from early during its formation at the primitive streak stage. The endoderm harbored progenitors of the "fourth germ layer," the primordial germ cells (PGCs), the only cells maintaining expression of OCT4 at the end of gastrulation. In the ectodermal and mesodermal cell lineages, OCT4 became undetectable at the neural groove and somite stage, respectively. As in the mouse, PGCs showed onset of c-kit expression when located in extraembryonal compartments. They appeared to follow the endoderm during extraembryonal allocation and the mesoderm on return to the genital ridge.     

Pluripotency in the light of the developmental hourglass

The hourglass model of development postulates divergence in early and late embryo development bridged by a period of developmental constraint at mid‐embryogenesis. Recently, molecular support for the hourglass model of development has accumulated, with the emphasis on studies using zebrafish and Drosophila species. Across mammals, the hourglass model and specifically divergence in early development has thus far received little attention. Divergence in mammalian pre‐implantation development is particularly interesting because of its potential impact on derivation of pluripotent embryonic stem cells. Here, we review recent findings that support the hourglass model of development. We provide striking examples of variation in key events in mammalian peri‐implantation development and their potential consequences for pluripotency of embryonic stem cell lines, including mechanisms of cell signalling and differentiation, gene regulatory networks, X‐chromosome inactivation, and epigenetic regulation. The variation in these processes indicates divergence in early mammalian development as was postulated by the hourglass model of development. We discuss the naive and primed states of pluripotency in light of this developmental divergence and their implications for human pluripotent stem cell states.     

Pluripotency in the embryo and in culture

Specific cells within the early mammalian embryo have the capacity to generate all somatic lineages plus the germline. This property of pluripotency is confined to the epiblast, a transient tissue that persists for only a few days. In vitro, however, pluripotency can be maintained indefinitely through derivation of stem cell lines. Pluripotent stem cells established from the newly formed epiblast are known as embryonic stem cells (ESCs), whereas those generated from later stages are called postimplantation epiblast stem cells (EpiSCs). These different classes of pluripotent stem cell have distinct culture requirements and gene expression programs, likely reflecting the dynamic development of the epiblast in the embryo. In this chapter we review current understanding of how the epiblast forms and relate this to the properties of derivative stem cells. We discuss whether ESCs and EpiSCs are true counterparts of different phases of epiblast development or are culture-generated phenomena. We also consider the proposition that early epiblast cells and ESCs may represent a naïve ground state without any prespecification of lineage choice, whereas later epiblasts and EpiSCs may be primed in favor of particular fates.     

On the fate of primordial germ cells injected into early mouse embryos

Primordial germ cells (PGCs) are the founder cells of the germline. Via gametogenesis and fertilisation this lineage generates a new embryo in the next generation. PGCs are also the cell of origin of multilineage teratocarcinomas. In vitro, mouse PGCs can give rise to embryonic germ (EG) cells – pluripotent stem cells that can contribute to primary chimaeras when introduced into pre-implantation embryos. Thus, PGCs can give rise to pluripotent cells in the course of the developmental cycle, during teratocarcinogenesis and by in vitro culture. However, there is no evidence that PGCs can differentiate directly into somatic cell types. Furthermore, it is generally assumed that PGCs do not contribute to chimaeras following injection into the early mouse embryo. However, these data have never been formally published. Here, we present the primary data from the original PGC-injection experiments performed 40 years ago, alongside results from more recent studies in three separate laboratories. These results have informed and influenced current models of the relationship between pluripotency and the germline cycle. Current technologies allow further experiments to confirm and expand upon these findings and allow definitive conclusions as to the developmental potency of PGCs.     

干细胞命运决定的分子调控网络

来源于囊胚期胚胎内细胞团的胚胎干细胞具有独特的生物学特性,包括无限自我更新的能力以及分化为内胚层、中胚层和外胚层各种细胞的潜能.阐明胚胎干细胞全能性维持以及向各种特定细胞分化的分子机制,不仅有助于我们了解胚胎发育过程,而且将促进胚胎干细胞尽早应用于疾病治疗.本文主要就干细胞的一种命运决定过程,维持胚胎干细胞全能性或失去全能性开始分化,结合最新的研究进展讨论该过程中的分子调控网络,包括信号转导通路、表达调控网络以及表观遗传调控.     

Stem cells to tissue: molecular,cellular and anatomical heterogeneity in skeletal muscle

Multiple waves of muscle precursors are released from skeletal muscle progenitor cells throughout developmental life, and this process is initiated in precise locations in the embryo. Skeletal muscle diversifies not only after the acquisition of muscle identity, but curiously heterogeneity is observed even in the stem cell population. Recent studies on cell lineage, cell fusion and the nature of post-natal satellite cells have expanded on our fundamental knowledge of the formation of this tissue, and how this tissue is replenished by resident and circulating regenerative stem cells during adult life.     

Building an apical domain in the early mouse embryo: Lessons,challenges and perspectives

Cell polarization is critical for lineage segregation and morphogenesis during mammalian embryogenesis. However, the processes and mechanisms that establish cell polarity in the mammalian embryo are not well understood. Recent studies suggest that unique regulatory mechanisms are deployed by the mouse embryo to establish cell polarization. In this review, we discuss the current understanding of cell polarity establishment, focusing on the formation of the apical domain in the mouse embryo. We will also discuss outstanding questions and possible directions for future study.     

The first round of mouse spermatogenesis is a distinctive program that lacks the self-renewing spermatogonia stage

Mammalian spermatogenesis is maintained by a continuous supply of differentiating cells from self-renewing stem cells. The stem cell activity resides in a small subset of primitive germ cells, the undifferentiated spermatogonia. However, the relationship between the establishment of this population and the initiation of differentiation in the developing testes remains unclear. In this study, we have investigated this issue by using the unique expression of Ngn3, which is expressed specifically in the undifferentiated spermatogonia, but not in the differentiating spermatogonia or their progenitors, the gonocytes. Our lineage analyses demonstrate that the first round of mouse spermatogenesis initiates directly from gonocytes, without passing through the Ngn3-expressing stage (Ngn3- lineage). By contrast, the subsequent rounds of spermatogenesis are derived from Ngn3-positive undifferentiated spermatogonia, which are also immediate descendents of the gonocytes and represent the stem cell function (Ngn3+ lineage). Thus, in mouse spermatogenesis, the state of the undifferentiated spermatogonia is not an inevitable step but is a developmental option that ensures continuous sperm production. In addition, the segregation of gonocytes into undifferentiated spermatogonia (Ngn3+ lineage) or differentiating spermatogonia (Ngn3- lineage) is topographically related to the establishment of the seminiferous epithelial cycle, thus suggesting a role of somatic components in the establishment of stem cells.     

NANOG在哺乳动物早期胚胎发育与干细胞中的功能研究进展

哺乳动物的早期胚胎发育和干细胞多能性由转录因子构成的基因网络所调控。2003年,在胚胎干细胞中发现的重要转录因子NANOG位于基因网络调控中心,对胚胎第二次命运决定和基态多能性的建立至关重要。该文将在NANOG生物学特征的基础上,重点讨论其在早期胚胎发育、胚胎干细胞与诱导性多能干细胞中的功能。     

Lineage choice and differentiation in mouse embryos and embryonic stem cells

The use of embryonic stem (ES) cells for generating healthy tissues has the potential to revolutionize therapies for human disease or injury, for which there are currently no effective treatments. Strategies for manipulating stem cell differentiation should be based on knowledge of the mechanisms by which lineage decisions are made during early embryogenesis. Here, we review current research into the factors influencing lineage differentiation in the mouse embryo and the application of this knowledge to in vitro differentiation of ES cells. In the mouse embryo, specification of tissue lineages requires cell-cell interactions that are influenced by coordinated cell migration and cellular neighborhood mediated by the key WNT, FGF, and TGFbeta signaling pathways. Mimicking the cellular interactions of the embryo by providing appropriate signaling molecules in culture has enabled the differentiation of ES cells to be directed predominately toward particular lineages. Multistep strategies incorporating the provision of soluble factors known to influence lineage choices in the embryo, coculture with other cells or tissues, genetic modification, and selection for desirable cell types have allowed the production of ES cell derivatives that produce beneficial effects in animal models. Increasing the efficiency of this process can only result from a better understanding of the molecular control of cell lineage determination in the embryo.     

Embryonic stem cells and cell replacement therapies in the nervous system

Embryonic stem (ES) cells are pluripotential cells derived from the pre-implantation embryo. They can proliferate indefinitely in vitro while retaining pluripotency. ES cells can also be made to differentiate into a large variety of cell types in vitro. This has paved the way to research aimed at using ES-derived cells for cell replacement therapies. Hence, mouse ES cells can efficiently differentiate into neural precursors which can further generate functional neurons, astrocytes, and oligodendrocytes. Methods have also been developed to coax mouse ES-derived neural stem cells to differentiate into either dopaminergic neurons or motoneurons. Mouse ES-derived neural stem cells, or their fully differentiated progeny, have been shown to survive, integrate, and to some extent, function following transplantation within appropriate rodent host tissue. Research on human ES cells is still in its infancy. Considerable work has to be done: (1) to master growth and genetic manipulation of human ES cells; (2) to master their differentiation into specific cell types; and (3) to demonstrate that they can provide long term therapeutical benefits upon grafting into damaged tissues in humans. From the ethical point of view, the establishment of appropriate primate model will be an obligatory prerequisite to clinical trials based on ES cells derivatives grafting.