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Markers and methods for cell sorting of human embryonic stem cell-derived neural cell populations.

ABSTRACT: Neural cells differentiated in vitro from human embryonic stem cells (hESC) exhibit broad cellular heterogeneity with respect to developmental stage and lineage specification. Here, we describe standard conditions for the use and discovery of markers for analysis and cell selection of hESC undergoing neuronal differentiation. To generate better-defined cell populations, we established a working protocol for sorting heterogeneous hESC-derived neural cell populations by fluorescence-activated cell sorting (FACS). Using genetically labeled synapsin-green fluorescent protein-positive hESC-derived neurons as a proof of principle, we enriched viable differentiated neurons by FACS. Cell sorting methodology using surface markers was developed, and a comprehensive profiling of surface antigens was obtained for immature embryonic stem cell types (such as stage-specific embryonic antigen [SSEA]-3, -4, TRA-1-81, TRA-1-60), neural stem and precursor cells (such as CD133, SSEA-1 [CD15], A2B5, forebrain surface embryonic antigen-1, CD29, CD146, p75 [CD271]), and differentiated neurons (such as CD24 or neural cell adhesion molecule [NCAM; CD56]). At later stages of neural differentiation, NCAM (CD56) was used to isolate hESC-derived neurons by FACS. Such FACS-sorted hESC-derived neurons survived in vivo after transplantation into rodent brain. These results and concepts provide (a) a feasible approach for experimental cell sorting of differentiated neurons, (b) an initial survey of surface antigens present during neural differentiation of hESC, and (c) a framework for developing cell selection strategies for neural cell-based therapies.

SUBMITTER: Pruszak J

PROVIDER: S-EPMC2238728 | biostudies-literature | 2007 Sep

REPOSITORIES: biostudies-literature

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Markers and methods for cell sorting of human embryonic stem cell-derived neural cell populations.

Pruszak Jan J Sonntag Kai-Christian KC Aung Moe Hein MH Sanchez-Pernaute Rosario R Isacson Ole O

Stem cells (Dayton, Ohio) 20070621 9

Neural cells differentiated in vitro from human embryonic stem cells (hESC) exhibit broad cellular heterogeneity with respect to developmental stage and lineage specification. Here, we describe standard conditions for the use and discovery of markers for analysis and cell selection of hESC undergoing neuronal differentiation. To generate better-defined cell populations, we established a working protocol for sorting heterogeneous hESC-derived neural cell populations by fluorescence-activated cell ...[more]

PMID: 17588935

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Project description:Adverse effect of alcohol on neural function has been well documented. Especially, the teratogenic effect of alcohol on neurodevelopment during embryogenesis has been demonstrated in various models, which could be a pathologic basis for fetal alcohol spectrum disorders (FASDs). While the developmental defects from alcohol abuse during gestation have been described, the specific mechanisms by which alcohol mediates these injuries have yet to be determined. Recent studies have shown that alcohol has significant effect on molecular and cellular regulatory mechanisms in embryonic stem cell (ESC) differentiation including genes involved in neural development. To test our hypothesis that alcohol induces molecular alterations during neural differentiation we have derived neural precursor cells from pluripotent human ESCs in the presence or absence of ethanol treatment. Genome-wide transcriptomic profiling identified molecular alterations induced by ethanol exposure during neural differentiation of hESCs into neural rosettes and neural precursor cell populations. The Database for Annotation, Visualization and Integrated Discovery (DAVID) functional analysis on significantly altered genes showed potential ethanol's effect on JAK-STAT signaling pathway, neuroactive ligand-receptor interaction, Toll-like receptor (TLR) signaling pathway, cytokine-cytokine receptor interaction and regulation of autophagy. We have further quantitatively verified ethanol-induced alterations of selected candidate genes. Among verified genes we further examined the expression of P2RX3, which is associated with nociception, a peripheral pain response. We found ethanol significantly reduced the level of P2RX3 in undifferentiated hESCs, but induced the level of P2RX3 mRNA and protein in hESC-derived NPCs. Our result suggests ethanol-induced dysregulation of P2RX3 along with alterations in molecules involved in neural activity such as neuroactive ligand-receptor interaction may be a molecular event associated with alcohol-related peripheral neuropathy of an enhanced nociceptive response.

Project description:BackgroundHuman embryonic stem cells (hESCs) offer a virtually unlimited source of neural cells for structural repair in neurological disorders, such as stroke. Neural cells can be derived from hESCs either by direct enrichment, or by isolating specific growth factor-responsive and expandable populations of human neural stem cells (hNSCs). Studies have indicated that the direct enrichment method generates a heterogeneous population of cells that may contain residual undifferentiated stem cells that could lead to tumor formation in vivo.Methods/principal findingsWe isolated an expandable and homogenous population of hNSCs (named SD56) from hESCs using a defined media supplemented with epidermal growth factor (EGF), basic fibroblast growth factor (bFGF) and leukemia inhibitory growth factor (LIF). These hNSCs grew as an adherent monolayer culture. They were fully neuralized and uniformly expressed molecular features of NSCs, including nestin, vimentin and radial glial markers. These hNSCs did not express the pluripotency markers Oct4 or Nanog, nor did they express markers for the mesoderm or endoderm lineages. The self-renewal property of the hNSCs was characterized by a predominant symmetrical mode of cell division. The SD56 hNSCs differentiated into neurons, astrocytes and oligodendrocytes throughout multiple passages in vitro, as well as after transplantation. Together, these criteria confirm the definitive NSC identity of the SD56 cell line. Importantly, they exhibited no chromosome abnormalities and did not form tumors after implantation into rat ischemic brains and into naïve nude rat brains and flanks. Furthermore, hNSCs isolated under these conditions migrated toward the ischemia-injured adult brain parenchyma and improved the independent use of the stroke-impaired forelimb two months post-transplantation.Conclusions/significanceThe SD56 human neural stem cells derived under the reported conditions are stable, do not form tumors in vivo and enable functional recovery after stroke. These properties indicate that this hNSC line may offer a renewable, homogenous source of neural cells that will be valuable for basic and translational research.

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Markers and methods for cell sorting of human embryonic stem cell-derived neural cell populations.

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Markers and methods for cell sorting of human embryonic stem cell-derived neural cell populations.

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