Embryonic stem (ES) cells are derived from the early preimplantation blastocyst. These cells are immortal under defined conditions in vitro, and can be indefinitely expanded without loss of pluripotency. Proof-of-concept experiments demonstrate that they have the ability to spontaneously differentiate into insulin-producing cells, even if at a very low frequency. Here we review the most recent progress at defining conditions (chemical, genetic, or otherwise) for the directed differentiation of both mouse and human ES cells into insulinproducing beta cells.
Embryonic stem cells • iPS cells • Nestin • Embryoid bodies • Teratomas
Embryonic stem (ES) cells were first derived from the early mouse blastocyst more than two decades ago. Under appropriate culture conditions (which at the time included the use of fibroblast feeder layers), these cells could be propagated indefinitely and had the potential to differentiate into derivatives of all three embryonic layers, as evidenced by their ability to both form teratomas in immunocompromised recipients and extensively contribute to the development of the embryo when injected in recipient blastocysts. ES cells exist only transiently in the inner cell mass of the early embryo . Specific culture conditions keep them cycling rapidly ex vivo (doubling time ranges from 24 to 48 h ) without loss of pluripotency for extended periods of time.
The molecular machinery behind the “stemness” of ES cells is very well conserved across species. The genes Oct3/4 , Sox2 , and Nanog lie at the core circuitry that imparts pluripotency and self-renewability.. In fact, at least two of them ( Oct3/4 and Sox2 ) have proven indispensable in the reprogramming of somatic cells into ES-like induced pluripotent stem (iPS) cells.With some exceptions, the down-regulation of these genes is associated with the activation of specific differentiation pathways. In addition to the above gene expression signature, human ES (huES) cells are characterized on the basis of their lack of stage-specific embryonic antigen 1 (SSEA-1) and presence of SSEA-3, SSEA-4, TRA-1-60, TRA-1-81, telomerase, and alkaline phosphatase.
As mentioned in the preface of this work, for decades, ES cells were chiefly considered a tool for the creation of targeted/knockout mice. However, the contribution of ES cells from non-murine species to the germ line could never be demonstrated. The first reports on somatic cell nuclear transfer (SCNT) in 1996 put and end to this search, as it was now possible to generate targeted animals of virtually any species of interest to humans in one single step.
In the meantime, steady progress was being made in refining the culture settings that would allow for the survival of nonhuman primate blastocysts up to the point where isolation of their ES cells was possible.This seminal report was followed shortly afterwards by the first report on the generation of human ES cells, 1 which opened the door to the possibility of devising regenerative therapies by means of expanding ES cells in vitro and then differentiating them into the tissue of interest. In this chapter, we review the attempts that have been made thus far to convert them into pancreatic endocrine cell types of potential use in the treatment of type I diabetes.
human embryonic stem (ES) cell colony grown over an inactivated mouse embryonic fibroblast feeder layer . When grown on Matrigel ™ , human ES cells form a monolayer . A higher magnification picture shows the morphological characteristics of ES cells: polygonal shape, high nucleus:cytoplasm ratio and refractive nucleoli.
A Venn diagram representing the overlap of the three critical regulators of the ES cell phenotype (Oct3/4, Sox2, and Nanog) promoter-bound regions. A large number of sites are co-bound by the three genes.Autoregulatory loops formed by Oct3/4, Sox2, and Nanog.
ES Cells and Gene Targeting
During the last 25 years, the application of gene targeting techniques to ES cells has become a routine procedure to generate genetically modified mice. The availability of large populations of these immortal cells makes it feasible to target specific genes, despite the low frequency of homologous recombination in mammalian cells. Targeted clones can be easily selected in vitro and used to generate chimeric mice by aggregation or injection into blastocysts. If the host blastocyst and the donor ES cells belong to different strains of mice, chimerism can be visually assessed by the mixed coat color of the resulting animals. Typically, up to 70% of injected blastocysts are overtly chimeric. The degree of chimerism varies widely from barely detectable to complete ES coloration. Since ES cells retain the potential to contribute to all embryonic lineages, some of them may partially colonize the germ line. Because of the fine-grained nature of ES cell chimerism, the germ line is usually a mixed population of donor- and host-derived cells. In order to obtain the highest possible number of ES-derived gametes, ES cells for blastocyst injection are of male genotype. The introduction of male ES cells into female host blastocysts normally results in the generation of fertile intersex animals, which only transmit the ES genotype.This is a consequence of the expression of the Y chromosome- linked sry gene, which controls mammalian sex determination. In these cases, backcrossing chimeras with the strain from which the ES cells were originally derived renders animals with the original genetic background, with an ideal 50% of the offspring carrying the modified allele. A 25% germ line transmission (one out of the first four male chimeras tested) is not unusual, although this percentage varies from clone to clone.
Tags: cytoplasm ratio, Embryoid bodies, Embryonic Stem Cells, ES Cells and Gene Targeting, iPS cells, morphological characteristics of ES cells, Nanog, Nestin, Oct3/4, refractive nucleoli, regulators of ES cell phenotype, Sox2, Teratomas