Neural stem cells: balancing self-renewal with differentiation

Stem cells are captivating because they have the potential to make multiple cell types yet maintain their undifferentiated state. Recent studies of Drosophila and mammalian neural stem cells have shed light on how stem cells regulate self-renewal versus differentiation and have revealed the proteins, processes and pathways that all converge to regulate neural progenitor self-renewal. If we can better understand how stem cells balance self-renewal versus differentiation, we will significantly advance our knowledge of embryogenesis, cancer biology and brain evolution, as well as the use of stem cells for therapeutic purposes.     

Pluripotent stem cell models of early mammalian development

Pluripotent stem cells derived from the early mammalian embryo offer a convenient model system for studying cell fate decisions in embryogenesis. The last 10 years have seen a boom in the popularity of two-dimensional micropatterns and three-dimensional stem cell culture systems as a way to recreate the architecture and interactions of particular cell populations during development. These methods enable the controlled exploration of cellular organization and patterning during development, using cell lines instead of embryos. They have established a new class of in vitro model system for pre-implantation and peri-implantation embryogenesis, ranging from models of the blastocyst stage, through gastrulation and toward early organogenesis. This review aims to set these systems in context and to highlight the strengths and suitability of each approach in modelling early mammalian development.     

Embryonic stem cell differentiation and the analysis of mammalian development

Molecular and cellular analysis of early mammalian development is compromised by the experimental inaccessibility of the embryo. Pluripotent embryonic stem (ES) cells are derived from and retain many properties of the pluripotent founder population of the embryo, the inner cell mass. Experimental manipulation of these cells and their environment in vitro provides an opportunity for the development of differentiation systems which can be used for analysis of the molecular and cellular basis of embryogenesis. In this review we discuss strengths and weaknesses of the available ES cell differentiation methodologies and their relationship to events in vivo. Exploitation of these systems is providing novel insight into embryonic processes as diverse as cell lineage establishment, cell progression during differentiation, patterning, morphogenesis and the molecular basis for cell properties in the early mammalian embryo.     

Melanocytes in development, regeneration, and cancer

The genes required for stem cell specification and lineage restriction during embryogenesis also play fundamental roles in adult tissue regeneration and cancer. This "development-regeneration-cancer" axis is exemplified by the vertebrate pigmentation system. Melanocytes exhibit almost unlimited self-renewal capacity during regenerative processes such as mammalian hair recoloration and zebrafish fin regeneration. Melanoma utilizes many regulatory signals and pathways required during ontogeny and regeneration. A discussion of these interconnections highlights how studies of stem cell function in embryonic and regenerative contexts can yield insights into melanoma biology.     

Prion potency in stem cells biology

Prion protein (PrP) can be considered a pivotal molecule because it interacts with several partners to perform a diverse range of critical biological functions that might differ in embryonic and adult cells. In recent years, there have been major advances in elucidating the putative role of PrP in the basic biology of stem cells in many different systems. Here, we review the evidence indicating that PrP is a key molecule involved in driving different aspects of the potency of embryonic and tissue-specific stem cells in self-perpetuation and differentiation in many cell types. It has been shown that PrP is involved in stem cell self-renewal, controlling pluripotency gene expression, proliferation, and neural and cardiomyocyte differentiation. PrP also has essential roles in distinct processes that regulate tissue-specific stem cell biology in nervous and hematopoietic systems and during muscle regeneration. Results from our own investigations have shown that PrP is able to modulate self-renewal and proliferation in neural stem cells, processes that are enhanced by PrP interactions with stress inducible protein 1 (STI1). Thus, the available data reveal the influence of PrP in acting upon the maintenance of pluripotent status or the differentiation of stem cells from the early embryogenesis through adulthood.     

多细胞生物干细胞不对称分裂的内在调节机制研究进展

多细胞生物的发育是从一个受精卵分化成多种类型细胞的过程。细胞多样性形成的基础是不等分裂,不等分裂是干细胞自我更新和自我维持的关键。干细胞不等分裂有细胞内和细胞外两种调节机制。果蝇神经干细胞增殖和分化、植物胚胎发育、表皮气孔形成及根内皮层的分化,是研究不等细胞分裂调节机制最多的发育背景。本综述介绍了果蝇神经干细胞和植物胚胎发育早期、表皮气孔发生及根皮层内皮层中细胞不等分裂内在调节机制的研究进展。     

The NuRD component Mbd3 is required for pluripotency of embryonic stem cells

Cells of early mammalian embryos have the potential to develop into any adult cell type, and are thus said to be pluripotent. Pluripotency is lost during embryogenesis as cells commit to specific developmental pathways. Although restriction of developmental potential is often associated with repression of inappropriate genetic programmes, the role of epigenetic silencing during early lineage commitment remains undefined. Here, we used mouse embryonic stem cells to study the function of epigenetic silencing in pluripotent cells. Embryonic stem cells lacking Mbd3 - a component of the nucleosome remodelling and histone deacetylation (NuRD) complex - were viable but failed to completely silence genes that are expressed before implantation of the embryo. Mbd3-deficient embryonic stem cells could be maintained in the absence of leukaemia inhibitory factor (LIF) and could initiate differentiation in embryoid bodies or chimeric embryos, but failed to commit to developmental lineages. Our findings define a role for epigenetic silencing in the cell-fate commitment of pluripotent cells.     

Establishment and controlled differentiation of neural crest stem cell lines using conditional transgenesis

Murine neural crest stem cells (NCSCs) are a multipotent transient population of stem cells. After being formed during early embryogenesis as a consequence of neurulation at the apical neural fold, the cells rapidly disperse throughout the embryo, migrating along specific pathways and differentiating into a wide variety of cell types. In vitro the multipotency is lost rapidly, making it difficult to study differentiation potential as well as cell fate decisions. Using a transgenic mouse line, allowing for spatio-temporal control of the transforming c-myc oncogene, we derived a cell line (JoMa1), which expressed NCSC markers in a transgene-activity dependent manner. JoMa1 cells express early NCSC markers and can be instructed to differentiate into neurons, glia, smooth muscle cells, melanocytes, and also chondrocytes. A cell-line, clonally derived from JoMa1 culture, termed JoMa1.3 showed identical behavior and was studied in more detail. This system therefore represents a powerful tool to study NCSC biology and signaling pathways. We observed that when proliferative and differentiation stimuli were given, enhanced cell death could be detected, suggesting that the two signals are incompatible in the cellular context. However, the cells regain their differentiation potential after inactivation of c-MycER(T). In summary, we have established a system, which allows for the biochemical analysis of the molecular pathways governing NCSC biology. In addition, we should be able to obtain NCSC lines from crossing the c-MycER(T) mice with mice harboring mutations affecting neural crest development enabling further insight into genetic pathways controlling neural crest differentiation.     

Mouse embryo stem cells: their identification,propagation and manipulation

The early mouse embryo contains a transient population of pluripotential stem cells which are responsible for generating both the foetal primordia and extraembryonic membranes. The characterisation of murine embryo stem cells and their isolation and propagation in culture provides the first instance in which pure populations of normal stem cells are directly accessible to the researcher. This marks a considerable advance in stem cell biology which may pave the way to the dissection of general stem cell control mechanisms and the identification of key regulatory factors. In addition, the genetic manipulation of embryo stem cells affords a unique avenue for experimental intervention in mammalian development and for controlled modification of the mouse germ line.     

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.     

Expression and knockdown of cellular prion protein (PrPC) in differentiating mouse embryonic stem cells

The mammalian cellular prion protein (PrP(C)) is a highly conserved glycoprotein that may undergo conversion into a conformationally altered isoform (scrapie prion protein or PrP(Sc)), widely believed to be the pathogenic agent of transmissible spongiform encephalopathies (TSEs). Although much is known about pathogenic PrP conversion and its role in TSEs, the normal function of PrP(C) is poorly understood. Given the abundant expression of PrP(C) in the developing mammalian CNS and the spatial association with differentiated stages of neurogenesis, recently it has been proposed that PrP(C) participates in neural cell differentiation. In the present study, we investigated the role of PrP(C) in neural development during early embryogenesis. In bovine fetuses, PrP(C) was differentially expressed in the neuroepithelium, showing higher levels at the intermediate and marginal layers where more differentiated states of neurogenesis were located. We utilized differentiating mouse embryonic stem (ES) cells to test whether PrP(C) contributed to the process of neural differentiation during early embryogenesis. PrP(C) showed increasing levels of expression starting on Day 9 until Day 18 of ES cell differentiation. PrP(C) expression was negatively correlated with pluripotency marker Oct-4 confirming that ES cells had indeed differentiated. Induction of ES cells differentiation by retinoic acid (RA) resulted in up-regulation of PrP(C) at Day 20 and nestin at Day 12. PrP(C) expression was knocked down in PrP-targeted siRNA ES cells between Days 12 and 20. PrP(C) knockdown in ES cells resulted in nestin reduction at Days 16 and 20. Analysis of bovine fetuses suggests the participation of PrP(C) in neural cell differentiation during early embryogenesis. The positive association between PrP(C) and nestin expression provide evidence for the contribution of PrP(C) to ES cell differentiation into neural progenitor cells.     

Oogenesis: Prospects and challenges for the future

Oogenesis serves a singular role in the reproductive success of plants and animals. Of their remarkable differentiation pathway what stands out is the ability of oocytes to transform from a single cell into the totipotent lineages that seed the early embryo. As our understanding that commonalities between diverse organisms at the genetic, cellular and molecular levels are conserved to achieve successful reproduction, the notion that embryogenesis presupposes oogenesis has entered the day-to-day parlance of regenerative medicine and stem cell biology. With emphasis on the mammalian oocyte, this review will cover (1) current concepts regarding the birth, survival and growth of oocytes that depends on complex patterns of cell communication between germ line and soma, (2) the notion of "maternal inheritance" from a genetic and epigenetic perspective, and (3) the relative value of model systems with reference to current clinical and biotechnology applications.     

Cell biology of stem cells: an enigma of asymmetry and self-renewal

Stem cells present a vast, new terrain of cell biology. A central question in stem cell research is how stem cells achieve asymmetric divisions to replicate themselves while producing differentiated daughter cells. This hallmark of stem cells is manifested either strictly during each mitosis or loosely among several divisions. Current research has revealed the crucial roles of niche signaling, intrinsic cell polarity, subcellular localization mechanism, asymmetric centrosomes and spindles, as well as cell cycle regulators in establishing self-renewing asymmetry during stem cell division. Much of this progress has benefited from studies in model stem cell systems such as Drosophila melanogaster neuroblasts and germline stem cells and mammalian skin stem cells. Further investigations of these questions in diverse types of stem cells will significantly advance our knowledge of cell biology and allow us to effectively harness stem cells for therapeutic applications.     

Self‐organization of human iPS cells into trophectoderm mimicking cysts induced by adhesion restriction using microstructured mesh scaffolds

Cellular dynamics leading to the formation of the trophectoderm in humans remain poorly understood owing to limited accessibility to human embryos for research into early human embryogenesis. Compared to animal models, organoids formed by self‐organization of stem cells in vitro may provide better insights into differentiation and complex morphogenetic processes occurring during early human embryogenesis. Here we demonstrate that modulating the cell culture microenvironment alone can trigger self‐organization of human induced pluripotent stem cells (hiPSCs) to yield trophectoderm‐mimicking cysts without chemical induction. To modulate the adhesion microenvironment, we used the mesh culture technique recently developed by our group, which involves culturing hiPSCs on suspended micro‐structured meshes with limited surface area for cell adhesion. We show that this adhesion‐restriction strategy can trigger a two‐stage self‐organization of hiPSCs; first into stem cell sheets, which express pluripotency signatures until around day 8–10, then into spherical cysts following differentiation and self‐organization of the sheet‐forming cells. Detailed morphological analysis using immunofluorescence microscopy with both confocal and two‐photon microscopes revealed the anatomy of the cysts as consisting of a squamous epithelial wall richly expressing E‐cadherin and CDX2. We also confirmed that the cysts exhibit a polarized morphology with basal protrusions, which show migratory behavior when anchored. Together, our results point to the formation of cysts which morphologically resemble the trophectoderm at the late‐stage blastocyst. Thus, the mesh culture microenvironment can initiate self‐organization of hiPSCs into trophectoderm‐mimicking cysts as organoids with potential application in the study of early embryogenesis and also in drug development.