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ABSTRACT: We describe procedures for engrafting human embryonic stem (ES) cells into blastocyst stage mouse embryos. In terms of developmental time in the mouse, the blastocyst represents a closely related in vivo embryonic environment for the human ES cells. This tool can be used to study embryological properties of human ES cells and, by using genetically modified or mutant human ES cells as graft, can be extended to study the basis of disease. Protocols are presented include: (a) production of host embryos for human ES cell incorporation, (b) preparation of pseudo-pregnant foster mothers for manipulated embryos, (c) preparation of human ES cells for morula aggregation or blastocyst injection, (d) injection or aggregation of human ES cells into appropriate stage embryos, and (e) growth in vitro as differentiating outgrowths or transfer to pseudo-pregnant foster mothers for in vivo development.Protocols for traditional, though less rigorous, methods for assessing in vivo potential of human ES cells (teratoma formation in immunocomprimised mice via kidney capsule, intramuscular and subcutaneous injection) are also included.
06/2007: pages 121 - 147; , ISBN: 9780470511619
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ABSTRACT: The NOTCH signaling pathway performs a wide range of critical functions in a number of different cell types during development and differentiation. The role of NOTCH signals in human embryonic stem cells (hESCs) has not been tested. We measured the activity of canonical NOTCH signaling in undifferentiated embryonic stem (ES) cells and tested the requirement for NOTCH activity in hESC self-renewal or differentiation by growing hESCs in the presence of gamma-secretase inhibitors. Our results suggest that NOTCH signaling is not required for the propagation of undifferentiated human ES cells but instead is required for the maintenance of the differentiating cell types that accumulate in human ES cell cultures. Our studies suggest that NOTCH signaling is not required in human embryonic differentiation until the formation of extraembryonic, germ layer, or tissue-specific stem cells and progenitors.
Stem Cells 08/2006; 24(7):1646-53. · 7.78 Impact Factor
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ABSTRACT: In addition to their potential for cell-based therapies in the treatment of disease and injury, the broad developmental capacity of human embryonic stem cells (hESCs) offers potential for studying the origins of all human cell types. To date, the emergence of specialized cells from hESCs has commonly been studied in tissue culture or in teratomas, yet these methods have stopped short of demonstrating the ESC potential exhibited in the mouse (mESCs), which can give rise to every cell type when combined with blastocysts. Due to obvious barriers precluding the use of human embryos in similar cell mixing experiments with hESCs, human/non-human chimeras may need to be generated for this purpose. Our results show that hESCs can engraft into mouse blastocysts, where they proliferate and differentiate in vitro and persist in mouse/human embryonic chimeras that implant and develop in the uterus of pseudopregnant foster mice. Embryonic chimeras generated in this way offer the opportunity to study the behavior of specialized human cell types in a non-human animal model. Our data demonstrate the feasibility of this approach, using mouse embryos as a surrogate for hESC differentiation.
Developmental Biology 08/2006; 295(1):90-102. · 4.07 Impact Factor
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ABSTRACT: The vesicular inhibitory amino acid transporter, VIAAT (also known as vesicular GABA transporter VGAT) transports GABA or glycine into synaptic vesicles. To initiate an analysis of the expression and regulation of VIAAT during neurogenesis we have cloned and characterized the mouse Viaat gene. We find that the mouse Viaat coding sequence is encoded by two exons spanning 5.3 kb. A survey of expression by whole mount in situ hybridization of mouse embryos indicates that Viaat is activated early in neuron differentiation and is expressed widely within the developing CNS; however, we did not detect expression in the superficial non-neural structures that express the GABA synthase Gad1. Analysis of the Viaat promoter indicates that a minimal promoter region containing a CG rich sequence is sufficient for efficient expression in neural stem and precursor cells. Our analysis of the Viaat sequence and splicing does not support the existence of two Viaat isoforms as previously proposed [Ebihara et al., Brain Res. Mol Brain Res. 110 (2003), 126-139]. Instead, the alternative isoform Viaat-a appears to be due to PCR artifacts that have occurred independently in multiple labs.
Gene 06/2005; 351:39-49. · 2.34 Impact Factor
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ABSTRACT: Embryonic stem cells (ESCs) are able to generate a wide array of differentiated cell fates while maintaining self-renewal. Understanding the biology of these choices may be central to the use of human embryonic stem cells (HESCs), both as a model for early human development as well as a resource for cell based therapies. Efforts to dissect the molecular mechanisms that mediate stem cell identity are underway, and in this review we summarize recent progress in defining the markers and pathways involved in these decisions. We discuss recent efforts to assess the molecular signature of pluripotent HESCs and highlight work demonstrating a set of genes, including representatives from the FGF, TGFbeta, and Wnt signaling pathways, that consistently mark the undifferentiated state. In addition, we describe experiments in which signaling of HESCs is augmented by chemical probing with small molecule compounds. Using these compounds, we have demonstrated an important role for Wnt signaling in HESC pluripotency and shown a requirement for TGFbeta signaling in the maintenance of the undifferentiated state. These experiments have revealed some molecular aspects of the pluripotent state and demonstrated clear differences between mouse and human ESCs in the maintenance of this identity.
Stem Cell Reviews and Reports 02/2005; 1(2):111-8. · 3.74 Impact Factor
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ABSTRACT: The use of human embryonic stem cells (hESCs) as a source of dopaminergic neurons for Parkinson's disease cell therapy will require the development of simple and reliable cell differentiation protocols. The use of cell cocultures, added extracellular signaling factors, or transgenic approaches to drive hESC differentiation could lead to additional regulatory as well as cell production delays for these therapies. Because the neuronal cell lineage seems to require limited or no signaling for its formation, we tested the ability of hESCs to differentiate to form dopamine-producing neurons in a simple serum-free suspension culture system. BG01 and BG03 hESCs were differentiated as suspension aggregates, and neural progenitors and neurons were detectable after 2-4 weeks. Plated neurons responded appropriately to electrophysiological cues. This differentiation was inhibited by early exposure to bone morphogenic protein (BMP)-4, but a pulse of BMP-4 from days 5 to 9 caused induction of peripheral neuronal differentiation. Real-time polymerase chain reaction and whole-mount immunocytochemistry demonstrated the expression of multiple markers of the midbrain dopaminergic phenotype in serum-free differentiations. Neurons expressing tyrosine hydroxylase (TH) were killed by 6-hydroxydopamine (6-OHDA), a neurotoxic catecholamine. Upon plating, these cells released dopamine and other catecholamines in response to K+ depolarization. Surviving TH+ neurons, derived from the cells differentiated in serum-free suspension cultures, were detected 8 weeks after transplantation into 6-OHDA-lesioned rat brains. This work suggests that hESCs can differentiate in simple serum-free suspension cultures to produce the large number of cells required for transplantation studies.
Stem Cells 02/2004; 22(7):1218-38. · 7.78 Impact Factor
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ABSTRACT: We have developed a culture system for the efficient and directed differentiation of human embryonic stem cells (HESCs) to neural precursors and neurons.HESC were maintained by manual passaging and were differentiated to a morphologically distinct OCT-4+/SSEA-4- monolayer cell type prior to the derivation of embryoid bodies. Embryoid bodies were grown in suspension in serum free conditions, in the presence of 50% conditioned medium from the human hepatocarcinoma cell line HepG2 (MedII).
A neural precursor population was observed within HESC derived serum free embryoid bodies cultured in MedII conditioned medium, around 7-10 days after derivation. The neural precursors were organized into rosettes comprised of a central cavity surrounded by ring of cells, 4 to 8 cells in width. The central cells within rosettes were proliferating, as indicated by the presence of condensed mitotic chromosomes and by phosphoHistone H3 immunostaining. When plated and maintained in adherent culture, the rosettes of neural precursors were surrounded by large interwoven networks of neurites. Immunostaining demonstrated the expression of nestin in rosettes and associated non-neuronal cell types, and a radial expression of Map-2 in rosettes. Differentiated neurons expressed the markers Map-2 and Neurofilament H, and a subpopulation of the neurons expressed tyrosine hydroxylase, a marker for dopaminergic neurons.
This novel directed differentiation approach led to the efficient derivation of neuronal cultures from HESCs, including the differentiation of tyrosine hydroxylase expressing neurons. HESC were morphologically differentiated to a monolayer OCT-4+ cell type, which was used to derive embryoid bodies directly into serum free conditions. Exposure to the MedII conditioned medium enhanced the derivation of neural precursors, the first example of the effect of this conditioned medium on HESC.
BMC Neuroscience 10/2003; 4:27. · 3.04 Impact Factor
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ABSTRACT: Embryonic stem (ES) cells are pluripotent cells capable of differentiating into cell lineages derived from all primary germ layers including neural cells. In this study we describe an efficient method for differentiating rhesus monkey ES cells to neural lineages and the subsequent isolation of an enriched population of Nestin and Musashi positive neural progenitor (NP) cells. Upon differentiation, these cells exhibit electrophysiological characteristics resembling cultured primary neurons. Embryoid bodies (EBs) were formed in ES growth medium supplemented with 50% MEDII. After 7 days in suspension culture, EBs were transferred to adherent culture and either differentiated in serum containing medium or expanded in serum free medium. Immunocytochemistry on differentiating cells derived from EBs revealed large networks of MAP-2 and NF200 positive neurons. DAPI staining showed that the center of the MEDII-treated EBs was filled with rosettes. NPs isolated from adherent EB cultures expanded in serum free medium were passaged and maintained in an undifferentiated state by culture in serum free N2 with 50% MEDII and bFGF. Differentiating neurons derived from NPs fired action potentials in response to depolarizing current injection and expressed functional ionotropic receptors for the neurotransmitters glutamate and gamma-aminobutyric acid (GABA). NPs derived in this way could serve as models for cellular replacement therapy in primate models of neurodegenerative disease, a source of neural cells for toxicity and drug testing, and as a model of the developing primate nervous system.
Biochemical and Biophysical Research Communications 07/2003; 306(1):191-7. · 2.48 Impact Factor
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