Glycogen synthase kinase 3ß and activin/nodal inhibition in human embryonic stem cells induces a pre-neuroepithelial state that is required for specification to a floor plate cell lineage.

The floor plate is one of the major organizers of the developing nervous system through its secretion of sonic hedgehog (Shh). Although the floor plate is located within the neural tube, the derivation of the floor plate during development is still debatable and some studies suggest that floor plate cells are specified by Shh in a temporarily restricted window different to neuroepithelial cells. Using human embryonic stem cells (hESC) as a model of neurogenesis, we sought to determine how floor plate cells may be temporarily specified by SHH signaling during human embryogenesis. We found that inhibition of both GSK3ß and activin/nodal pathways in hESC induces a cellular state of SOX2+/PAX6- expression, we describe as "pre-neuroepithelial." Exposure of SHH during this pre-neuroepithelial period causes the expression of GLI transcription factors to function as activators and consequently upregulate expression of the floor plate marker, FOXA2, while also supressing PAX6 expression to inhibit neuroepithelial fate. FOXA2+ cells were able to efficiently generate mesencephalic dopaminergic neurons, a floor plate derivative. Overall, this study demonstrates a highly efficient system for generating floor plate cells from hESC and, most importantly, reveals that specification of floor plate cells is temporally dependent, whereby it occurs prior to the onset of PAX6 expression, within a pre-neuroepithelial stage.

In vivo tissue engineering chamber supports human induced pluripotent stem cell survival and rapid differentiation.

Pluripotent stem cells are a potential source of autologous cells for cell and tissue regenerative therapies. They have the ability to renew indefinitely while retaining the capacity to differentiate into all cell types in the body. With developments in cell therapy and tissue engineering these cells may provide an option for treating tissue loss in organs which do not repair themselves. Limitations to clinical translation of pluripotent stem cells include poor cell survival and low cell engraftment in vivo and the risk of teratoma formation when the cells do survive through implantation. In this study, implantation of human induced-pluripotent stem (hiPS) cells, suspended in Matrigel, into an in vivo vascularized tissue engineering chamber in nude rats resulted in substantial engraftment of the cells into the highly vascularized rat tissues formed within the chamber. Differentiation of cells in the chamber environment was shown by teratoma formation, with all three germ lineages evident within 4 weeks. The rate of teratoma formation was higher with partially differentiated hiPS cells (as embryoid bodies) compared to undifferentiated hiPS cells (100% versus 60%). In conclusion, the in vivo vascularized tissue engineering chamber supports the survival through implantation of human iPS cells and their differentiated progeny, as well as a novel platform for rapid teratoma assay screening for pluripotency.

Derivation, propagation and differentiation of human embryonic stem cells.

Embryonic stem (ES) cells are in vitro cultivated pluripotent cells derived from the inner cell mass (ICM) of the embryonic blastocyst. Attesting to their pluripotency, ES cells can be differentiated into representative derivatives of all three embryonic germ layers (endoderm, ectoderm and mesoderm) both in vitro and in vivo. Although mouse ES cells have been studied for many years, human ES cells have only more recently been derived and successfully propagated. Many biochemical differences and culture requirements between mouse and human ES cells have been described, yet despite these differences the study of murine ES cells has provided important insights into methodologies aimed at generating a greater and more in depth understanding of human ES cell biology. One common feature of both mouse and human ES cells is their capacity to undergo controlled differentiation into spheroid structures termed embryoid bodies (EBs). EBs recapitulate several aspects of early development, displaying regional-specific differentiation programs into derivatives of all three embryonic germ layers. For this reason, EB formation has been utilised as an initial step in a wide range of studies aimed at differentiating both mouse and human ES cells into a specific and desired cell type. Recent reports utilising specific growth factor combinations and cell-cell induction systems have provided alternative strategies for the directed differentiation of cells into a desired lineage. According to each one of these strategies, however, a relatively high cell lineage heterogeneity remains, necessitating subsequent purification steps including mechanical dissection, selective media or fluorescent or magnetic activated cell sorting (FACS and MACS, respectively). In the future, the ability to specifically direct differentiation of human ES cells at 100% efficiency into a desired lineage will allow us to fully explore the potential of these cells in the analysis of early human development, drug discovery, drug testing and repair of damaged or diseased tissues via transplantation.

BMPs regulate differentiation of a putative visceral endoderm layer within human embryonic stem-cell-derived embryoid bodies.

Human embryonic stem cells (HESCs), pluripotent cells derived from the inner cell mass (ICM) of human blastocysts, represent a novel tool for the study of early human developmental events. When cultured in suspension with serum, HESCs form spherical structures resembling embryoid bodies (EBs). We show that differentiation of HESCs within EBs occurs radially, with central cells then undergoing apoptosis in association with EB cavitation. Cells within the outer layer of cavitating EBs display stage-specific immunoreactivity to pan-keratin, cytokeratin-8, GATA6, alpha-fetoprotein, and transthyretin specific antibodies, and hybridization to disabled-2, GATA4, and GATA6 specific riboprobes. Transmission electron microscopy of these cells reveals clathrin-coated micropinocytotic vesicles, microvilli, and many vacuoles, a phenotype consistent with mouse visceral endoderm (VE) rather than mouse definitive or parietal endoderm. When cultured in media supplemented with the BMP inhibitor noggin, or in the absence of serum, HESC derivatives do not develop the mouse VE-like phenotype. The addition of BMP-4 to noggin-treated HESCs cultured in serum or in serum-free conditions reconstituted development of the VE-like phenotype. These data demonstrate that human EBs undergo developmental events similar to those of mouse EBs and that in vitro BMP signalling induces derivatives of the human ICM to express a phenotype similar to mouse VE.

A murine respiratory-inducing niche displays variable efficiency across human and mouse embryonic stem cell species.

Human embryonic stemlike cells (hESCs) are pluripotent cells derived from blastocysts. Differentiating hESCs into respiratory lineages may benefit respiratory therapeutic programs. We previously demonstrated that 24% of all mouse embryonic stem cell (mESC) derivatives cocultured with embryonic day 11.5 (E11.5) mouse lung rudiments display immunoreactivity to the pneumonocyte II specific marker surfactant-associated protein C (Sftpc). Here we further investigate the effects of this inductive niche in terms of its competence to induce hESC derivative SFTPC immunoreactivity and the expression of other markers of terminal lung secretory units. When hESCs were cocultured as single cells, clumps of approximately 10 cells or embryoid bodies (EBs), hESC derivatives formed pan-keratin-positive epithelial tubules at high frequency (>30% of all hESC derivatives). However, human-specific SFTPC immunoreactivity associated with tubule formation only at low frequency (<0.1% of all hESC derivatives). Human-specific SFTPD and secretoglobin family 1A member 1 (SCGB1A1, also known as CC10) transcripts were detected by PCR after prolonged culture. Expression of other terminal lung secretory unit markers (TITF1, SFTPA, and SFTPB) was not detected at any time point analyzed. On the other hand, hESC derivatives cultured as plated EBs in media previously demonstrated to induce Sftpc expression in isolated mouse fetal tracheal epithelium expressed all terminal lung secretory unit markers examined. mESCs and hESCs thus display fundamental differences in their response to the E11.5 mouse lung inductive niche, and these data provide an important step in the delineation of signaling mechanisms capable of efficiently inducing hESC differentiation into terminal secretory units of the lung.

Stem cells: an overview.

Stem cells are specialized cells that possess a capacity to undergo self-renewal while at the same time having the ability to give rise to at least one or more differentiated or mature cell type. They therefore represent a fundamental cornerstone during the life of all vertebrates, playing central roles in the production of new and replacement cells for tissues during development and homeostasis, including repair following disease or injury. This unit is a review of stem cells, their roles in development, and their potentials as therapeutic agents.

Mouse embryonic stem cell derivation, and mouse and human embryonic stem cell culture and differentiation as embryoid bodies.

Embryonic stem (ES) cells are pluripotent cells derived from developing mouse blastocysts in vitro that maintain long-term self renewal and the capacity to give rise to all cell types in the adult body (including some extraembryonic cell types) when subjected to the appropriate conditions. It is envisaged that the development of methods enabling controlled differentiation of mouse ES cell counterparts from human blastocysts would enable the provision of an unlimited supply of tissue for cell and tissue transplantation therapies for the repair and replacement of diseased, injured, and senescent tissue. Furthermore, derivation of mouse ES cells has allowed for the generation of thousands of gene-targeted mouse mutants. Culture of mouse ES cells as embryoid bodies (EBs) has provided a convenient system for studying early mouse developmental processes, including several aspects of extraembryonic lineage and axis formation associated with the pre- and peri-gastrulating mouse embryo. Relatively little is known regarding the corresponding development of the early human embryo due to limitations associated with the acquisition of relevant tissue material for study. The transfer of methods such as EB formation to human systems should, by association, facilitate a more advanced understanding of similar processes associated with early human development. This unit describes protocols for isolating mouse embryonic stem cells and methods for propagating, freezing, and producing EBs from both mouse and human embryonic stem cells.

Human embryonic stem cells form embryoid bodies containing visceral endoderm-like derivatives.

OBJECTIVE: The objective of this study was to determine the potential of human embryonic stem (hES) cells to provide an in vitro model of human extraembryonic endoderm development. METHODS: The hES cell line HES-2 was propagated in Dulbecco's modification of Eagle's medium (DMEM) in the presence of 20% fetal calf serum (FCS) on a mouse embryonic fibroblast feeder layer. Clumps of approximately 50-100 cells were transferred to fresh DMEM and FCS and grown as embryoid bodies (EBs) in suspension culture. EBs were subjected to immunohistochemistry for endodermal, ectodermal and mesodermal specific markers and immunoreactivity analysed by confocal microscopy and on cryosections. RESULTS: HES-2 cells reproducibly formed spherical EBs after 2-3 days in suspension culture as clumps. EBs continued to expand and by 7 days had commenced cavitation in a highly reproducible and organised fashion. Culture periods longer than 10 days led to cystic structures displaying inconsistent morphological organisation. Immunolocalisation of anti-alpha-fetoprotein-, anti-neurofilament- and anti-CD31-specific antibodies at 7 days of culture provided evidence of regional differentiation of endodermal, ectodermal and mesodermal derivatives in cavitating EBs. Further, of 10 cavitating EBs analysed at 7 days of culture, all displayed immunolocalisation of anti-pan-keratin-, anti-CK8- and anti-alpha-fetoprotein-specific antibodies to a peripheral cellular layer, suggestive of yolk sac visceral endoderm (VE) formation. CONCLUSION: Cavitation, the presence of regionalised cell lineage-specific immunoreactivity and the development of a VE-like peripheral cell layer demonstrate that the HES-2 hES cell line can be induced to undergo EB formation and provide scope to study early human primitive endoderm and yolk sac VE development.