Neuronal differentiation of murine dental pulp stem cells in vitro

KM Ellis, David C. O'Carroll, Martin Lewis, Simon Koblar

Research output: Contribution to conferencePoster

Abstract

The potential of dental pulp stem cells (DPSCs) in regenerative medicine is becoming more widely understood. The ability of murine DPSCs to become chondrocytes, osteocytes and adipocytes has recently been demonstrated by several groups, but their neurogenic potential has not been widely shown. In this study we demonstrate neuronal differentiation of murine DPSCs in vitro. Method: mDPSCs were cultured in vitro under neuroinductive conditions (n=12) for 14 days. Cultures were subsequently assessed for neuronal and synaptic markers by immunohistochemistry (IHC) or whole cell patch clamp analysis to investigate intracellular electrophysiology. mDPSCs were also seeded onto microelectrode arrays (n=7) to assess network activity. Results: The mDPSCs showed a neuronal phenotype from 5 days in neuroinductive media and developed a neuritic network of processors. IHC revealed approximately 70% of cells expressed the pan-neuronal marker ß-III tubulin and 23% GFAP (n=8). mDPSCs also expressed markers for nestin, neurofilament-M and Schwann cell marker, S100, but not peripheral neuronal marker, periaxin. Differentiated mDPSCs stained positive for GABA and acetylcholine markers with positive expression of GAD 65/67 and ChAT, respectively, but did not contain tyrosine hydroxylase- or vGlut-2-positive cells. Intracellular electrophysiological analysis revealed the presence of L-type voltage-gated calcium channels in many distally connected neuronal-like cells. No sodium or potassium currents were found and the cells did not support spontaneous action potentials. Many cells existed in interconnected networks such that their cell capacitance was greater than that of distally connected and isolated cells. IHC revealed high expression of gap junction protein connexin 43 and the addition of 100µM gap junction blocker, 2-APB, reduced capacitance of clustered cells. Conclusion: These data suggest the differentiation of mDPSCs into a heterogeneous population of neural-like cell networks that may represent an early stage of neural development, dominated by gap junction-mediated cell-to-cell communication.
LanguageEnglish
Publication statusPublished - Oct 2012
Externally publishedYes
EventNeuroscience 2012 - New Orleans, United States
Duration: 13 Oct 201217 Oct 2012

Conference

ConferenceNeuroscience 2012
CountryUnited States
CityNew Orleans
Period13/10/1217/10/12

Cite this

Ellis, KM., O'Carroll, D. C., Lewis, M., & Koblar, S. (2012). Neuronal differentiation of murine dental pulp stem cells in vitro. Poster session presented at Neuroscience 2012, New Orleans, United States.
Ellis, KM ; O'Carroll, David C. ; Lewis, Martin ; Koblar, Simon. / Neuronal differentiation of murine dental pulp stem cells in vitro. Poster session presented at Neuroscience 2012, New Orleans, United States.
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title = "Neuronal differentiation of murine dental pulp stem cells in vitro",
abstract = "The potential of dental pulp stem cells (DPSCs) in regenerative medicine is becoming more widely understood. The ability of murine DPSCs to become chondrocytes, osteocytes and adipocytes has recently been demonstrated by several groups, but their neurogenic potential has not been widely shown. In this study we demonstrate neuronal differentiation of murine DPSCs in vitro. Method: mDPSCs were cultured in vitro under neuroinductive conditions (n=12) for 14 days. Cultures were subsequently assessed for neuronal and synaptic markers by immunohistochemistry (IHC) or whole cell patch clamp analysis to investigate intracellular electrophysiology. mDPSCs were also seeded onto microelectrode arrays (n=7) to assess network activity. Results: The mDPSCs showed a neuronal phenotype from 5 days in neuroinductive media and developed a neuritic network of processors. IHC revealed approximately 70{\%} of cells expressed the pan-neuronal marker {\ss}-III tubulin and 23{\%} GFAP (n=8). mDPSCs also expressed markers for nestin, neurofilament-M and Schwann cell marker, S100, but not peripheral neuronal marker, periaxin. Differentiated mDPSCs stained positive for GABA and acetylcholine markers with positive expression of GAD 65/67 and ChAT, respectively, but did not contain tyrosine hydroxylase- or vGlut-2-positive cells. Intracellular electrophysiological analysis revealed the presence of L-type voltage-gated calcium channels in many distally connected neuronal-like cells. No sodium or potassium currents were found and the cells did not support spontaneous action potentials. Many cells existed in interconnected networks such that their cell capacitance was greater than that of distally connected and isolated cells. IHC revealed high expression of gap junction protein connexin 43 and the addition of 100µM gap junction blocker, 2-APB, reduced capacitance of clustered cells. Conclusion: These data suggest the differentiation of mDPSCs into a heterogeneous population of neural-like cell networks that may represent an early stage of neural development, dominated by gap junction-mediated cell-to-cell communication.",
author = "KM Ellis and O'Carroll, {David C.} and Martin Lewis and Simon Koblar",
year = "2012",
month = "10",
language = "English",
note = "Neuroscience 2012 ; Conference date: 13-10-2012 Through 17-10-2012",

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Ellis, KM, O'Carroll, DC, Lewis, M & Koblar, S 2012, 'Neuronal differentiation of murine dental pulp stem cells in vitro' Neuroscience 2012, New Orleans, United States, 13/10/12 - 17/10/12, .

Neuronal differentiation of murine dental pulp stem cells in vitro. / Ellis, KM; O'Carroll, David C.; Lewis, Martin; Koblar, Simon.

2012. Poster session presented at Neuroscience 2012, New Orleans, United States.

Research output: Contribution to conferencePoster

TY - CONF

T1 - Neuronal differentiation of murine dental pulp stem cells in vitro

AU - Ellis, KM

AU - O'Carroll, David C.

AU - Lewis, Martin

AU - Koblar, Simon

PY - 2012/10

Y1 - 2012/10

N2 - The potential of dental pulp stem cells (DPSCs) in regenerative medicine is becoming more widely understood. The ability of murine DPSCs to become chondrocytes, osteocytes and adipocytes has recently been demonstrated by several groups, but their neurogenic potential has not been widely shown. In this study we demonstrate neuronal differentiation of murine DPSCs in vitro. Method: mDPSCs were cultured in vitro under neuroinductive conditions (n=12) for 14 days. Cultures were subsequently assessed for neuronal and synaptic markers by immunohistochemistry (IHC) or whole cell patch clamp analysis to investigate intracellular electrophysiology. mDPSCs were also seeded onto microelectrode arrays (n=7) to assess network activity. Results: The mDPSCs showed a neuronal phenotype from 5 days in neuroinductive media and developed a neuritic network of processors. IHC revealed approximately 70% of cells expressed the pan-neuronal marker ß-III tubulin and 23% GFAP (n=8). mDPSCs also expressed markers for nestin, neurofilament-M and Schwann cell marker, S100, but not peripheral neuronal marker, periaxin. Differentiated mDPSCs stained positive for GABA and acetylcholine markers with positive expression of GAD 65/67 and ChAT, respectively, but did not contain tyrosine hydroxylase- or vGlut-2-positive cells. Intracellular electrophysiological analysis revealed the presence of L-type voltage-gated calcium channels in many distally connected neuronal-like cells. No sodium or potassium currents were found and the cells did not support spontaneous action potentials. Many cells existed in interconnected networks such that their cell capacitance was greater than that of distally connected and isolated cells. IHC revealed high expression of gap junction protein connexin 43 and the addition of 100µM gap junction blocker, 2-APB, reduced capacitance of clustered cells. Conclusion: These data suggest the differentiation of mDPSCs into a heterogeneous population of neural-like cell networks that may represent an early stage of neural development, dominated by gap junction-mediated cell-to-cell communication.

AB - The potential of dental pulp stem cells (DPSCs) in regenerative medicine is becoming more widely understood. The ability of murine DPSCs to become chondrocytes, osteocytes and adipocytes has recently been demonstrated by several groups, but their neurogenic potential has not been widely shown. In this study we demonstrate neuronal differentiation of murine DPSCs in vitro. Method: mDPSCs were cultured in vitro under neuroinductive conditions (n=12) for 14 days. Cultures were subsequently assessed for neuronal and synaptic markers by immunohistochemistry (IHC) or whole cell patch clamp analysis to investigate intracellular electrophysiology. mDPSCs were also seeded onto microelectrode arrays (n=7) to assess network activity. Results: The mDPSCs showed a neuronal phenotype from 5 days in neuroinductive media and developed a neuritic network of processors. IHC revealed approximately 70% of cells expressed the pan-neuronal marker ß-III tubulin and 23% GFAP (n=8). mDPSCs also expressed markers for nestin, neurofilament-M and Schwann cell marker, S100, but not peripheral neuronal marker, periaxin. Differentiated mDPSCs stained positive for GABA and acetylcholine markers with positive expression of GAD 65/67 and ChAT, respectively, but did not contain tyrosine hydroxylase- or vGlut-2-positive cells. Intracellular electrophysiological analysis revealed the presence of L-type voltage-gated calcium channels in many distally connected neuronal-like cells. No sodium or potassium currents were found and the cells did not support spontaneous action potentials. Many cells existed in interconnected networks such that their cell capacitance was greater than that of distally connected and isolated cells. IHC revealed high expression of gap junction protein connexin 43 and the addition of 100µM gap junction blocker, 2-APB, reduced capacitance of clustered cells. Conclusion: These data suggest the differentiation of mDPSCs into a heterogeneous population of neural-like cell networks that may represent an early stage of neural development, dominated by gap junction-mediated cell-to-cell communication.

M3 - Poster

ER -

Ellis KM, O'Carroll DC, Lewis M, Koblar S. Neuronal differentiation of murine dental pulp stem cells in vitro. 2012. Poster session presented at Neuroscience 2012, New Orleans, United States.