Limits...
A method to generate human mesenchymal stem cell-derived neurons which express and are excited by multiple neurotransmitters.

Greco SJ, Zhou C, Ye JH, Rameshwar P - Biol Proced Online (2008)

Bottom Line: MSCs are bone marrow (BM)-derived cells which undergo lineage- specific differentiation to generate bone, fat, cartilage and muscle, but are also capable of transdifferentiating into defined ectodermal and endodermal tissues.Our neuronal protocol utilizes freshly harvested human MSCs cultured on specific surfaces and exposed to an induction cocktail consisting of low serum concentration, retinoic acid (RA), growth factors and supplements.Here we report on the types of neurotransmitters produced by the neurons, and demonstrate that the cells are electrically responsive to exogenous neurotransmitter administration.

View Article: PubMed Central - HTML - PubMed

Affiliation: Graduate School of Biomedical Sciences, UMDNJ-New Jersey Medical School, MSB, Rm. E-579 185 South Orange Ave, Newark, NJ 07103, USA.

ABSTRACT
The present study describes a protocol to generate heterogenous populations of neurotransmitter-producing neurons from human mesenchymal stem cells (MSCs). MSCs are bone marrow (BM)-derived cells which undergo lineage- specific differentiation to generate bone, fat, cartilage and muscle, but are also capable of transdifferentiating into defined ectodermal and endodermal tissues. The purpose of this study is to evaluate the potential of MSCs as an alternative source of customized neurons for experimental neurobiology or other regenerative approaches. Our neuronal protocol utilizes freshly harvested human MSCs cultured on specific surfaces and exposed to an induction cocktail consisting of low serum concentration, retinoic acid (RA), growth factors and supplements. Here we report on the types of neurotransmitters produced by the neurons, and demonstrate that the cells are electrically responsive to exogenous neurotransmitter administration.

No MeSH data available.


Related in: MedlinePlus

GABA and glutamate elicit responses in D12 induced MSCs.A. FM1-43 FX lipophilic plasma membrane dye was added at 5 µg/mL to D12 cells. Cells were cultured on glass coverslips, with dye incubation lasting for 1 min. Cells were fixed with ice-cold 3.7% formaldehyde and then examined for plasma membrane staining. Intracellular vesicle dye reuptake was studied by treating cells with 1 mM GABA (left panel) or 1 mM glutamate (middle panel) following labeling. Vehicle controls used 1% BSA (right panel). Figure represents four different experiments, each performed with cells from a different donor. Representative inward currents elicited by 1 mM GABA (B) or 1 mM glutamate (C) from cells in culture for 12 days. The peak amplitude is 49 pA for GABA-induced current, and 81 pA for glutamate-induced current. Whole-cell currents were recorded at a holding potential of -50 mV
© Copyright Policy - open acces
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2683550&req=5

Figure 05: GABA and glutamate elicit responses in D12 induced MSCs.A. FM1-43 FX lipophilic plasma membrane dye was added at 5 µg/mL to D12 cells. Cells were cultured on glass coverslips, with dye incubation lasting for 1 min. Cells were fixed with ice-cold 3.7% formaldehyde and then examined for plasma membrane staining. Intracellular vesicle dye reuptake was studied by treating cells with 1 mM GABA (left panel) or 1 mM glutamate (middle panel) following labeling. Vehicle controls used 1% BSA (right panel). Figure represents four different experiments, each performed with cells from a different donor. Representative inward currents elicited by 1 mM GABA (B) or 1 mM glutamate (C) from cells in culture for 12 days. The peak amplitude is 49 pA for GABA-induced current, and 81 pA for glutamate-induced current. Whole-cell currents were recorded at a holding potential of -50 mV

Mentions: Next, we investigated whether the D12 induced cells are capable of releasing neurotransmitters in response to glutamate or GABA treatment. For this experiment, D12 cells were incubated with the membrane dye, FM1-43 FX, as previously reported (Fig. 5A) (10). Cells undergoing exocytotic vesicular release in response to treatment will show a pattern of punctate staining along the neurites, thus indicating vesicular recycling following synaptic release. D12 cells treated for 1 min with GABA (left panel) or glutamate (middle panel) possessed cellular processes demonstrating a pattern of staining consistent with vesicular dye reuptake. Cells treated with vehicle control (BSA; right panel) did not exhibit a similar staining pattern. As shown previously, pre-conditioning with a calcium chelator such as EDTA inhibited vesicular release and dye reuptake (data not shown) (10).


A method to generate human mesenchymal stem cell-derived neurons which express and are excited by multiple neurotransmitters.

Greco SJ, Zhou C, Ye JH, Rameshwar P - Biol Proced Online (2008)

GABA and glutamate elicit responses in D12 induced MSCs.A. FM1-43 FX lipophilic plasma membrane dye was added at 5 µg/mL to D12 cells. Cells were cultured on glass coverslips, with dye incubation lasting for 1 min. Cells were fixed with ice-cold 3.7% formaldehyde and then examined for plasma membrane staining. Intracellular vesicle dye reuptake was studied by treating cells with 1 mM GABA (left panel) or 1 mM glutamate (middle panel) following labeling. Vehicle controls used 1% BSA (right panel). Figure represents four different experiments, each performed with cells from a different donor. Representative inward currents elicited by 1 mM GABA (B) or 1 mM glutamate (C) from cells in culture for 12 days. The peak amplitude is 49 pA for GABA-induced current, and 81 pA for glutamate-induced current. Whole-cell currents were recorded at a holding potential of -50 mV
© Copyright Policy - open acces
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2683550&req=5

Figure 05: GABA and glutamate elicit responses in D12 induced MSCs.A. FM1-43 FX lipophilic plasma membrane dye was added at 5 µg/mL to D12 cells. Cells were cultured on glass coverslips, with dye incubation lasting for 1 min. Cells were fixed with ice-cold 3.7% formaldehyde and then examined for plasma membrane staining. Intracellular vesicle dye reuptake was studied by treating cells with 1 mM GABA (left panel) or 1 mM glutamate (middle panel) following labeling. Vehicle controls used 1% BSA (right panel). Figure represents four different experiments, each performed with cells from a different donor. Representative inward currents elicited by 1 mM GABA (B) or 1 mM glutamate (C) from cells in culture for 12 days. The peak amplitude is 49 pA for GABA-induced current, and 81 pA for glutamate-induced current. Whole-cell currents were recorded at a holding potential of -50 mV
Mentions: Next, we investigated whether the D12 induced cells are capable of releasing neurotransmitters in response to glutamate or GABA treatment. For this experiment, D12 cells were incubated with the membrane dye, FM1-43 FX, as previously reported (Fig. 5A) (10). Cells undergoing exocytotic vesicular release in response to treatment will show a pattern of punctate staining along the neurites, thus indicating vesicular recycling following synaptic release. D12 cells treated for 1 min with GABA (left panel) or glutamate (middle panel) possessed cellular processes demonstrating a pattern of staining consistent with vesicular dye reuptake. Cells treated with vehicle control (BSA; right panel) did not exhibit a similar staining pattern. As shown previously, pre-conditioning with a calcium chelator such as EDTA inhibited vesicular release and dye reuptake (data not shown) (10).

Bottom Line: MSCs are bone marrow (BM)-derived cells which undergo lineage- specific differentiation to generate bone, fat, cartilage and muscle, but are also capable of transdifferentiating into defined ectodermal and endodermal tissues.Our neuronal protocol utilizes freshly harvested human MSCs cultured on specific surfaces and exposed to an induction cocktail consisting of low serum concentration, retinoic acid (RA), growth factors and supplements.Here we report on the types of neurotransmitters produced by the neurons, and demonstrate that the cells are electrically responsive to exogenous neurotransmitter administration.

View Article: PubMed Central - HTML - PubMed

Affiliation: Graduate School of Biomedical Sciences, UMDNJ-New Jersey Medical School, MSB, Rm. E-579 185 South Orange Ave, Newark, NJ 07103, USA.

ABSTRACT
The present study describes a protocol to generate heterogenous populations of neurotransmitter-producing neurons from human mesenchymal stem cells (MSCs). MSCs are bone marrow (BM)-derived cells which undergo lineage- specific differentiation to generate bone, fat, cartilage and muscle, but are also capable of transdifferentiating into defined ectodermal and endodermal tissues. The purpose of this study is to evaluate the potential of MSCs as an alternative source of customized neurons for experimental neurobiology or other regenerative approaches. Our neuronal protocol utilizes freshly harvested human MSCs cultured on specific surfaces and exposed to an induction cocktail consisting of low serum concentration, retinoic acid (RA), growth factors and supplements. Here we report on the types of neurotransmitters produced by the neurons, and demonstrate that the cells are electrically responsive to exogenous neurotransmitter administration.

No MeSH data available.


Related in: MedlinePlus