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Plasticity of calcium signaling cascades in human embryonic stem cell-derived neural precursors.

ABSTRACT: Human embryonic stem cell-derived neural precursors (hESC NPs) are considered to be a promising tool for cell-based therapy in central nervous system injuries and neurodegenerative diseases. The Ca(2+) ion is an important intracellular messenger essential for the regulation of various cellular functions. We investigated the role and physiology of Ca(2+) signaling to characterize the functional properties of CCTL14 hESC NPs during long-term maintenance in culture (in vitro). We analyzed changes in cytoplasmic Ca(2+) concentration ([Ca(2+)]i) evoked by high K(+), adenosine-5'-triphosphate (ATP), glutamate, ?-aminobutyric acid (GABA), and caffeine in correlation with the expression of various neuronal markers in different passages (P6 through P10) during the course of hESC differentiation. We found that only differentiated NPs from P7 exhibited significant and specific [Ca(2+)]i responses to various stimuli. About 31% of neuronal-like P7 NPs exhibited spontaneous [Ca(2+)]i oscillations. Pharmacological and immunocytochemical assays revealed that P7 NPs express L- and P/Q-type Ca(2+) channels, P2X2, P2X3, P2X7, and P2Y purinoreceptors, glutamate receptors, and ryanodine (RyR1 and RyR3) receptors. The ATP- and glutamate-induced [Ca(2+)]i responses were concentration-dependent. Higher glutamate concentrations (over 100??M) caused cell death. Responses to ATP were observed in the presence or in the absence of extracellular Ca(2+). These results emphasize the notion that with time in culture, these cells attain a transient period of operative Ca(2+) signaling that is predictive of their ability to act as stem elements.

SUBMITTER: Forostyak O

PROVIDER: S-EPMC3653370 | biostudies-other | 2013 May

REPOSITORIES: biostudies-other

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Plasticity of calcium signaling cascades in human embryonic stem cell-derived neural precursors.

Forostyak Oksana O Romanyuk Nataliya N Verkhratsky Alexei A Sykova Eva E Dayanithi Govindan G

Stem cells and development 20130219 10

Human embryonic stem cell-derived neural precursors (hESC NPs) are considered to be a promising tool for cell-based therapy in central nervous system injuries and neurodegenerative diseases. The Ca(2+) ion is an important intracellular messenger essential for the regulation of various cellular functions. We investigated the role and physiology of Ca(2+) signaling to characterize the functional properties of CCTL14 hESC NPs during long-term maintenance in culture (in vitro). We analyzed changes i ...[more]

PMID: 23294113

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Project description:Regulating the transition from lineage-restricted progenitors to terminally differentiated cells is a central aspect of nervous system development. Here, we investigated the role of the nucleoprotein Geminin in regulating neurogenesis at a mechanistic level during both Xenopus primary neurogenesis and mammalian neuronal differentiation in vitro. The latter work utilized both neural cells derived from embryonic stem and embryonal carcinoma cells in vitro and neural stem cells from mouse forebrain. In all of these contexts, Geminin antagonized the ability of neural bHLH transcription factors to activate transcriptional programs promoting neurogenesis. Furthermore, Geminin promoted a bivalent chromatin state, characterized by the presence of both activating and repressive histone modifications, at genes encoding transcription factors that promote neurogenesis. This epigenetic state restrains the expression of genes that regulate commitment of undifferentiated stem and neuronal precursor cells to neuronal lineages. Geminin is highly expressed in undifferentiated neuronal precursor cells but is downregulated prior to differentiation. Therefore, these data support a model whereby Geminin promotes the neuronal precursor cell state by modulating both the epigenetic status and expression of genes encoding neurogenesis-promoting factors. Additional developmental signals acting in these cells can then control their transition toward terminal neuronal or glial differentiation during mammalian neurogenesis. A mouse embryonic stem (ES) cell line for inducible knockdown of the small nucleoprotein Geminin was utilized. ES cells were used to generate neural precursor cells by monolayer culture in N2B27 media for 5 days, and doxycycline-inducible knockdown of Geminin was performed from day 3. Changes in gene expression resulting from Geminin knockdown were assessed by RNA sequencing. Three experimental replicates were generated for Geminin knockdown (plus Dox) with a corresponding no-Dox control. These were subjected to sequencing, and data were analyzed using TopHat and Cufflinks/Cuffdiff. Transcripts were considered as differentially expressed upon Gem knockdown if data met statistical significance cutoffs in Cuffdiff (sufficient sequence alignments were obtained for analysis and transcript had significant change in FPKM value (normalized transcript abundance; fragments per kb of exon per million fragments mapped) between the no Dox and plus Dox sample pairs) in at least two of the three replicates.

Dataset Information

Plasticity of calcium signaling cascades in human embryonic stem cell-derived neural precursors.

Publications

Plasticity of calcium signaling cascades in human embryonic stem cell-derived neural precursors.

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