Differentiation of embryonic stem cells into retinal neurons

Mouse embryonic stem (ES) cells are continuous cell lines derived from the inner mass of blastocysts. Neural progenitors derived from these cells serve as an excellent model for controlled neural differentiation and as such have tremendous potential to understand and treat neurodegenerative diseases. Here, we demonstrate that ES cell-derived neural progenitors express regulatory factors needed for retinal differentiation and that in response to epigenetic cues a subset of them differentiate along photoreceptor lineage. During the differentiation, they activate photoreceptor regulatory genes, suggesting that ES cell-derived neural progenitors recruit mechanisms normally used for photoreceptor differentiation in vivo. These observations suggest that ES cells can serve as an excellent model for understanding mechanisms that regulate specification of retinal neurons and as an unlimited source of neural progenitors for treating degenerative diseases of the retina by cell replacement.     

Embryonic stem cell differentiation: A chromatin perspective

Embryonic stem (ES) cells hold immense promise for the treatment of human degenerative disease. Because ES cells are pluripotent, they can be directed to differentiate into a number of alternative cell-types with potential therapeutic value. Such attempts at "rationally-directed ES cell differentiation" constitute attempts to recapitulate aspects of normal development in vitro. All differentiated cells retain identical DNA content, yet gene expression varies widely from cell-type to cell-type. Therefore, a potent epigenetic system has evolved to coordinate and maintain tissue-specific patterns of gene expression. Recent advances show that mechanisms that govern epigenetic regulation of gene expression are rooted in the details of chromatin dynamics. As embryonic cells differentiate, certain genes are activated while others are silenced. These activation and silencing events are exquisitely coordinated with the allocation of cell lineages. Remodeling of the chromatin of developmentally-regulated genes occurs in conjunction with lineage commitment. Oocytes, early embryos, and ES cells contain potent chromatin-remodeling activities, an observation that suggests that chromatin dynamics may be especially important for early lineage decisions. Chromatin dynamics are also involved in the differentiation of adult stem cells, where the assembly of specialized chromatin upon tissue-specific genes has been studied in fine detail. The next few years will likely yield striking advances in the understanding of stem cell differentiation and developmental biology from the perspective of chromatin dynamics.     

Dermal fibroblast-derived growth factors restore the ability of beta(1) integrin-deficient embryonal stem cells to differentiate into keratinocytes

Embryonal stem (ES) cells that are homozygous null for the beta(1) integrin subunit fail to differentiate into keratinocytes in vitro but do differentiate in teratomas and wild-type/beta(1)-null chimeric mice. The failure of beta(1)-null ES cells to differentiate in culture might be the result of defective extracellular matrix assembly or reduced sensitivity to soluble inducing factors. By culturing embryoid bodies on dead, deepidermized human dermis (DED) we showed that epidermal basement membrane did not induce beta(1)-null ES cells to undergo keratinocyte differentiation and did not stimulate the differentiation of wild-type ES cells. Coculture with epidermal keratinocytes also had no effect. However, when human dermal fibroblasts were incorporated into DED, the number of epidermal cysts formed by wild-type ES cells increased dramatically, and small groups of keratin 14-positive cells differentiated from beta(1)-null ES cells. Fibroblast-conditioned medium stimulated differentiation of K14-positive cells in wild-type and beta(1)-null embryoid bodies. Of a range of growth factors tested, KGF, FGF10, and TGFalpha all stimulated differentiation of keratin 14-positive beta(1)-null cells, and KGF and FGF10 were shown to be produced by the fibroblasts used in coculture experiments. The effects of the growth factors on wild-type ES cells were much less pronounced, suggesting that the concentrations of inducing factors already present in the medium were not limiting for wild-type cells. We conclude that the lack of beta(1) integrins decreases the sensitivity of ES cells to soluble factors that induce keratinocyte differentiation.     

On the formation of germ cells: The good,the bad and the ugly

Mammalian germ cells are powerful cells, the only ones that transmit information to the next generation ensuring the continuation of the species. But “with great power, comes great responsibility”, meaning that germ cells are only a few steps away from turning carcinogenic. Despite recent advances little is known about germ cell formation in mammals, predominantly because of the inaccessibility of these cells. Moreover, it is difficult to pin down what in essence is characteristic of a germ cell, as germ cells keep changing place, morphology, expression markers and epigenetic identity. Formation of (primordial) germ cells in primate ES cell cultures would therefore be helpful to identify molecular signalling pathways associated with germ cell differentiation and to study epigenetic changes in germ cells. In addition, the in vitro derivation of functional germ cells from ES cells could be used in combination with therapeutic cloning to generate patient-specific ES cell lines, and can have applications in animal breeding. In this review we present the state-of-the-art on how mouse and human germ cells are formed in vivo (the good), we discuss the link between germ cells, pluripotency and germ cell tumours (the bad) and show that despite continuous progress in trying to differentiate germ cells in vitro (the ugly) the generation of functional germ cells is still a real challenge.     

Regulation beyond genome sequences: DNA and histone methylation in embryonic stem cells

Embryonic stem (ES) cells distinct themselves from other cell type populations by their pluripotent ability. The unique features of ES cells are controlled by both genetic and epigenetic factors. Studies have shown that the methylation status of DNA and histones in ES cells is quite different from that of differentiated cells and somatic stem cells. Herein, we summarized recent advances in DNA and histone methylation studies of mammalian ES cells. The methylation status of several key pluripotent regulatory genes is also discussed.     

Neural differentiation of murine embryonic stem cells ES

Pluripotent murine embryonic stem (ES) cells can differentiate into all cell types both in vivo and in vitro. Based on their capability to proliferate and differentiate, these ES cells appear as a very promising tool for cell therapy. The understanding of the molecular mechanisms underlying the neural differentiation of the ES cells is a pre-requisite for selecting adequately the cells and conditions which will be able to correctly repair damaged brain and restore altered cognitive functions. Different methods allow obtaining neural cells from ES cells. Most of the techniques differentiate ES cells by treating embryoid bodies in order to keep an embryonic organization. More recent techniques, based on conditioned media, induce a direct differentiation of ES cells into neural cells, without going through the step of embryonic bodies. Beyond the fact that these techniques allow obtaining large numbers of neural precursors and more differentiated neural cells, these approaches also provide valuable information on the process of differentiation of ES cells into neural cells. Indeed, sequential studies of this process of differentiation have revealed that globally ES cells differentiating into neural cells in vitro recapitulate the molecular events governing the in vivo differentiation of neural cells. Altogether these data suggest that murine ES cells remain a highly valuable tool to obtain large amounts of precursor and differentiated neural cells as well as to get a better understanding of the mechanisms of neural differentiation, prior to a potential move towards the use of human ES cells in therapy.