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.

2006-03-01·Stem Cells2区 · 医学

Characterization of a New NIH-Registered Variant Human Embryonic Stem Cell Line, BG01V: A Tool for Human Embryonic Stem Cell Research

2区 · 医学

Article

作者: Brimble, Sandii N. ; Plaia, Todd W. ; Zeng, Xianmin ; Ording, Carol ; Josephson, Richard ; Freed, William J. ; Toumadje, Arazdordi ; Rao, Mahendra S. ; Liu, Ying ; Sherrer, Eric S. ; Maitra, Anirban ; Auerbach, Jonathan M. ; Schulz, Thomas C. ; Uhl, Elizabeth W.

AbstractHuman embryonic stem cells (hESCs) offer a renewable source of a wide range of cell types for use in research and cell-based therapies. Characterizing these cells provides important information about their current state and affords relevant details for subsequent manipulations. For example, identifying genes expressed during culture, as well as their temporal expression order after passaging and conditions influencing the formation of all three germ layers may be helpful for the production of functional beta islet cells used in treating type I diabetes. Although several hESC lines have demonstrated karyotypic instability during extended time in culture, select variant lines exhibit characteristics similar to their normal parental lines. Such variant lines may be excellent tools and abundant sources of cells for pilot studies and in vitro differentiation research in which chromosome number is not a concern, similar to the role currently played by embryonal carcinoma cell lines. It is crucial that the cells be surveyed at a genetic and proteomic level during extensive propagation, expansion, and manipulation in vitro. Here we describe a comprehensive characterization of the variant hESC line BG01V, which was derived from the karyotypically normal, parental hESC line BG01. Our characterization process employs cytogenetic analysis, short tandem repeat and HLA typing, mitochondrial DNA sequencing, gene expression analysis using quantitative reverse transcription-polymerase chain reaction and microarray, assessment of telomerase activity, methylation analysis, and immunophenotyping and teratoma formation, in addition to screening for bacterial, fungal, mycoplasma, and human pathogen contamination.

2004-12-01·STEM CELLS2区 · 医学

Differentiation of Human Embryonic Stem Cells to Dopaminergic Neurons in Serum‐Free Suspension Culture

2区 · 医学

Article

作者: Weiler, Deb A. ; Schulz, Thomas C. ; Lambert, Nevin A. ; Pensa, Kate A. ; Davidson, Bruce P. ; Lyons, Ian G. ; Meedeniya, Adrian C.B. ; Palmarini, Gail M. ; Noggle, Scott A. ; Condie, Brian G.

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.

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