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Acquisition of RGC phenotype in human Müller glia with stem cell characteristics is accompanied by upregulation of functional nicotinic acetylcholine receptors
PURPOSE: Human Müller glia with stem cell characteristics (hMGSCs) can be induced to express genes and proteins of retinal ganglion cells (RGCs) upon in vitro inhibition of Notch-1 activity. However, it is not known whether expression of these markers is accompanied by acquisition of RGC function. T...
Autores principales: | , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Molecular Vision
2013
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3774575/ https://www.ncbi.nlm.nih.gov/pubmed/24049438 |
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author | Becker, Silke Singhal, Shweta Jones, Megan F. Eastlake, Karen Cottrill, Phillippa B. Jayaram, Hari Limb, G. Astrid |
author_facet | Becker, Silke Singhal, Shweta Jones, Megan F. Eastlake, Karen Cottrill, Phillippa B. Jayaram, Hari Limb, G. Astrid |
author_sort | Becker, Silke |
collection | PubMed |
description | PURPOSE: Human Müller glia with stem cell characteristics (hMGSCs) can be induced to express genes and proteins of retinal ganglion cells (RGCs) upon in vitro inhibition of Notch-1 activity. However, it is not known whether expression of these markers is accompanied by acquisition of RGC function. This study investigated whether hMGSCs that express RGC markers also display neural functionality, as measured by their intracellular calcium concentration ([Ca(2+)](i)) responsiveness following neurotransmitter stimulation in vitro. METHODS: Changes in mRNA expression of RGC markers and neurotransmitter receptors were assessed either by conventional or quantitative reverse transcription PCR (RT-PCR), while changes in protein levels were confirmed by immunocytochemistry. The [Ca(2+)](i) levels were estimated by fluorescence microscopy. RESULTS: We showed that while undifferentiated hMGSCs displayed a profound elevation of [Ca(2+)](i) after stimulation with N-methyl-D-aspartate (NMDA), this was lost following Notch-1 inhibition. Conversely, untreated hMGSCs did not respond to muscarinic receptor stimulation, whereas [Ca(2+)](i) was increased in differentiated hMGSCs that expressed RGC precursor markers. Differentiated hMGSC-derived RGCs, but not undifferentiated hMGSCs, responded to stimulation by nicotine with a substantial rise in [Ca(2+)](i), which was inhibited by the α4β2 and α6β2 nicotinic receptor antagonist methyllycaconitine. Notch-1 attenuation not only caused a decrease in the gene expression of the Notch effector HES1 and increased expression of RGC markers, but also an increase in the gene and protein expression of α4 and α6 nicotinic receptor subunits. CONCLUSIONS: These observations suggest that in response to Notch-1 inhibition, hMGSCs differentiate into a population of RGCs that exhibit some of the functionality observed in differentiated RGCs. |
format | Online Article Text |
id | pubmed-3774575 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2013 |
publisher | Molecular Vision |
record_format | MEDLINE/PubMed |
spelling | pubmed-37745752013-09-18 Acquisition of RGC phenotype in human Müller glia with stem cell characteristics is accompanied by upregulation of functional nicotinic acetylcholine receptors Becker, Silke Singhal, Shweta Jones, Megan F. Eastlake, Karen Cottrill, Phillippa B. Jayaram, Hari Limb, G. Astrid Mol Vis Research Article PURPOSE: Human Müller glia with stem cell characteristics (hMGSCs) can be induced to express genes and proteins of retinal ganglion cells (RGCs) upon in vitro inhibition of Notch-1 activity. However, it is not known whether expression of these markers is accompanied by acquisition of RGC function. This study investigated whether hMGSCs that express RGC markers also display neural functionality, as measured by their intracellular calcium concentration ([Ca(2+)](i)) responsiveness following neurotransmitter stimulation in vitro. METHODS: Changes in mRNA expression of RGC markers and neurotransmitter receptors were assessed either by conventional or quantitative reverse transcription PCR (RT-PCR), while changes in protein levels were confirmed by immunocytochemistry. The [Ca(2+)](i) levels were estimated by fluorescence microscopy. RESULTS: We showed that while undifferentiated hMGSCs displayed a profound elevation of [Ca(2+)](i) after stimulation with N-methyl-D-aspartate (NMDA), this was lost following Notch-1 inhibition. Conversely, untreated hMGSCs did not respond to muscarinic receptor stimulation, whereas [Ca(2+)](i) was increased in differentiated hMGSCs that expressed RGC precursor markers. Differentiated hMGSC-derived RGCs, but not undifferentiated hMGSCs, responded to stimulation by nicotine with a substantial rise in [Ca(2+)](i), which was inhibited by the α4β2 and α6β2 nicotinic receptor antagonist methyllycaconitine. Notch-1 attenuation not only caused a decrease in the gene expression of the Notch effector HES1 and increased expression of RGC markers, but also an increase in the gene and protein expression of α4 and α6 nicotinic receptor subunits. CONCLUSIONS: These observations suggest that in response to Notch-1 inhibition, hMGSCs differentiate into a population of RGCs that exhibit some of the functionality observed in differentiated RGCs. Molecular Vision 2013-09-13 /pmc/articles/PMC3774575/ /pubmed/24049438 Text en Copyright © 2013 Molecular Vision. http://creativecommons.org/licenses/by/3.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. |
spellingShingle | Research Article Becker, Silke Singhal, Shweta Jones, Megan F. Eastlake, Karen Cottrill, Phillippa B. Jayaram, Hari Limb, G. Astrid Acquisition of RGC phenotype in human Müller glia with stem cell characteristics is accompanied by upregulation of functional nicotinic acetylcholine receptors |
title | Acquisition of RGC phenotype in human Müller glia with stem cell characteristics is accompanied by upregulation of functional nicotinic acetylcholine receptors |
title_full | Acquisition of RGC phenotype in human Müller glia with stem cell characteristics is accompanied by upregulation of functional nicotinic acetylcholine receptors |
title_fullStr | Acquisition of RGC phenotype in human Müller glia with stem cell characteristics is accompanied by upregulation of functional nicotinic acetylcholine receptors |
title_full_unstemmed | Acquisition of RGC phenotype in human Müller glia with stem cell characteristics is accompanied by upregulation of functional nicotinic acetylcholine receptors |
title_short | Acquisition of RGC phenotype in human Müller glia with stem cell characteristics is accompanied by upregulation of functional nicotinic acetylcholine receptors |
title_sort | acquisition of rgc phenotype in human müller glia with stem cell characteristics is accompanied by upregulation of functional nicotinic acetylcholine receptors |
topic | Research Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3774575/ https://www.ncbi.nlm.nih.gov/pubmed/24049438 |
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