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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

Expression of CNS neurotransmitters in uninduced and induced MSCs.A. Whole cell extracts from uninduced (D0) and induced (D6 and D12) MSCs were prepared and analyzed by western blots with anti-tyrosine hydroxylase (TyrH), -tryptophan hydroxylase (TrypH), -glutamic acid decarboxylase (GAD) and -vesicular acetylcholine transporter (VAChT). Normalizations were performed with anti-β-actin. Human brain extract served as positive control. Representative blots are shown for three different experiments. Band densities were quantified and normalized to total protein. Results are presented as mean ± SD relative fold change, where the lowest value is arbitrarily assigned a value of 1. B. D12 induced cells were labeled with PE-anti-GAD and then counter-labeled with DAPI. Figure shows representative labelings of five different experiments. Images are shown at 20X magnification. C. Uninduced and induced MSCs were labeled at 2 day intervals, up to 12 days, with PE-anti-glutamate and then counterlabeled with DAPI. Relative fluorescence intensities were normalized to values for uninduced cells, which were arbitrarily assigned a value of 1. Figure shows representative labelings of five different experiments. Images are shown at 10X magnification.*p<0.05 vs. uninduced cells **p<0.05 vs. D12 induced cells
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Figure 02: Expression of CNS neurotransmitters in uninduced and induced MSCs.A. Whole cell extracts from uninduced (D0) and induced (D6 and D12) MSCs were prepared and analyzed by western blots with anti-tyrosine hydroxylase (TyrH), -tryptophan hydroxylase (TrypH), -glutamic acid decarboxylase (GAD) and -vesicular acetylcholine transporter (VAChT). Normalizations were performed with anti-β-actin. Human brain extract served as positive control. Representative blots are shown for three different experiments. Band densities were quantified and normalized to total protein. Results are presented as mean ± SD relative fold change, where the lowest value is arbitrarily assigned a value of 1. B. D12 induced cells were labeled with PE-anti-GAD and then counter-labeled with DAPI. Figure shows representative labelings of five different experiments. Images are shown at 20X magnification. C. Uninduced and induced MSCs were labeled at 2 day intervals, up to 12 days, with PE-anti-glutamate and then counterlabeled with DAPI. Relative fluorescence intensities were normalized to values for uninduced cells, which were arbitrarily assigned a value of 1. Figure shows representative labelings of five different experiments. Images are shown at 10X magnification.*p<0.05 vs. uninduced cells **p<0.05 vs. D12 induced cells

Mentions: Our laboratory has recently developed a separate neuronal induction protocol specific for the generation of dopaminergic neurons from human MSCs (11). We therefore examined whether the presently described protocol also produces neuronal cells capable of non-peptidergic neurotransmitter biosynthesis. To this end, expression of the rate-limiting enzymes in catecholamine (TyrH), serotonin (TrypH) and GABA (GAD) biosynthesis, as well as the vesicular acetylcholine transporter (VAChT), were examined in uninduced (D0) and induced (D6 and D12) MSCs (Fig. 2A). The expression of TyrH and TrypH was undetectable in both uninduced and induced cells (first and second rows) compared to human brain extract controls (first and second rows, far right column). In contrast, a light band was detectable in D12 extracts for GAD (third row, far right column, white bar) and D0 extracts for VAChT (fourth row, far left column, gray bar).


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)

Expression of CNS neurotransmitters in uninduced and induced MSCs.A. Whole cell extracts from uninduced (D0) and induced (D6 and D12) MSCs were prepared and analyzed by western blots with anti-tyrosine hydroxylase (TyrH), -tryptophan hydroxylase (TrypH), -glutamic acid decarboxylase (GAD) and -vesicular acetylcholine transporter (VAChT). Normalizations were performed with anti-β-actin. Human brain extract served as positive control. Representative blots are shown for three different experiments. Band densities were quantified and normalized to total protein. Results are presented as mean ± SD relative fold change, where the lowest value is arbitrarily assigned a value of 1. B. D12 induced cells were labeled with PE-anti-GAD and then counter-labeled with DAPI. Figure shows representative labelings of five different experiments. Images are shown at 20X magnification. C. Uninduced and induced MSCs were labeled at 2 day intervals, up to 12 days, with PE-anti-glutamate and then counterlabeled with DAPI. Relative fluorescence intensities were normalized to values for uninduced cells, which were arbitrarily assigned a value of 1. Figure shows representative labelings of five different experiments. Images are shown at 10X magnification.*p<0.05 vs. uninduced cells **p<0.05 vs. D12 induced cells
© Copyright Policy - open acces
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2683550&req=5

Figure 02: Expression of CNS neurotransmitters in uninduced and induced MSCs.A. Whole cell extracts from uninduced (D0) and induced (D6 and D12) MSCs were prepared and analyzed by western blots with anti-tyrosine hydroxylase (TyrH), -tryptophan hydroxylase (TrypH), -glutamic acid decarboxylase (GAD) and -vesicular acetylcholine transporter (VAChT). Normalizations were performed with anti-β-actin. Human brain extract served as positive control. Representative blots are shown for three different experiments. Band densities were quantified and normalized to total protein. Results are presented as mean ± SD relative fold change, where the lowest value is arbitrarily assigned a value of 1. B. D12 induced cells were labeled with PE-anti-GAD and then counter-labeled with DAPI. Figure shows representative labelings of five different experiments. Images are shown at 20X magnification. C. Uninduced and induced MSCs were labeled at 2 day intervals, up to 12 days, with PE-anti-glutamate and then counterlabeled with DAPI. Relative fluorescence intensities were normalized to values for uninduced cells, which were arbitrarily assigned a value of 1. Figure shows representative labelings of five different experiments. Images are shown at 10X magnification.*p<0.05 vs. uninduced cells **p<0.05 vs. D12 induced cells
Mentions: Our laboratory has recently developed a separate neuronal induction protocol specific for the generation of dopaminergic neurons from human MSCs (11). We therefore examined whether the presently described protocol also produces neuronal cells capable of non-peptidergic neurotransmitter biosynthesis. To this end, expression of the rate-limiting enzymes in catecholamine (TyrH), serotonin (TrypH) and GABA (GAD) biosynthesis, as well as the vesicular acetylcholine transporter (VAChT), were examined in uninduced (D0) and induced (D6 and D12) MSCs (Fig. 2A). The expression of TyrH and TrypH was undetectable in both uninduced and induced cells (first and second rows) compared to human brain extract controls (first and second rows, far right column). In contrast, a light band was detectable in D12 extracts for GAD (third row, far right column, white bar) and D0 extracts for VAChT (fourth row, far left column, gray bar).

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