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Use of induced pluripotent stem cell derived neurons engineered to express BDNF for modulation of stressor related pathology.

Liu G, Rustom N, Litteljohn D, Bobyn J, Rudyk C, Anisman H, Hayley S - Front Cell Neurosci (2014)

Bottom Line: Combined cell and gene-based therapeutic strategies offer potential in the treatment of neurodegenerative and psychiatric conditions that have been associated with structural brain disturbances.Specifically, intracerebroventricular transplantation of iPSC-derived neural progenitors that over-expressed BDNF reversed the impact of immune (lipopolysaccharide) and chronic stressor challenges upon subventricular zone adult neurogenesis, and the iPSC-derived neural progenitor cells alone blunted the stressor-induced corticosterone response.Moreover, our findings indicate that mature dopamine producing neurons can be generated using iPSC procedures and appear to be viable when infused in vivo.

View Article: PubMed Central - PubMed

Affiliation: Hayley Lab, Department of Neuroscience, Carleton University Ottawa, ON, Canada.

ABSTRACT
Combined cell and gene-based therapeutic strategies offer potential in the treatment of neurodegenerative and psychiatric conditions that have been associated with structural brain disturbances. In the present investigation, we used a novel virus-free re-programming method to generate induced pluripotent stem cells (iPSCs), and then subsequently transformed these cells into neural cells which over-expressed brain derived neurotrophic factor (BDNF). Importantly, the infusion of iPSC derived neural cells (as a cell replacement and gene delivery tool) and BDNF (as a protective factor) influenced neuronal outcomes. Specifically, intracerebroventricular transplantation of iPSC-derived neural progenitors that over-expressed BDNF reversed the impact of immune (lipopolysaccharide) and chronic stressor challenges upon subventricular zone adult neurogenesis, and the iPSC-derived neural progenitor cells alone blunted the stressor-induced corticosterone response. Moreover, our findings indicate that mature dopamine producing neurons can be generated using iPSC procedures and appear to be viable when infused in vivo. Taken together, these data could have important implications for using gene-plus-cell replacement methods to modulate stressor related pathology.

No MeSH data available.


Related in: MedlinePlus

(A) Immature neuronal cells that were cultured in a Dopaminergic Neuronal Progenitor Medium began to show morphological signs of neuronal maturity between Day 29–50 (a,b,c) and after 6 months these cells displayed further signs of mature network formation (d). Importantly, control tail cells that were not subject to the reprogramming procedures kept their original shape at 6 months (e). As well, during this time, expression of the early immature neuronal cell marker, DCX, was greatly diminished (f), together with increased expression of the mature general neuronal cell marker, microtubule associated protein 2 (MAP2), (g), and specific dopaminergic neuronal marker, tyrosine hydroxylase (TH), (h) was evident. Further confirmation that the reprogrammed neurons were mature dopaminergic neurons was obtained using HPLC (i); wherein tail cells that were re-programmed (Tail-Dopamine cells) displayed significantly increased dopamine levels, compared to non-reprogrammed controls (Tail cells). Furthermore, dopamine levels in non-reprogrammed controls were, as expected, quite low (Tail tissue), whereas freshly dissected adult midbrain tissue had the expected highest concentration of dopamine (Brain tissue). *p < 0.05, relative to control tails. (B) Shows the time course for dopaminergic neuron derivation. The adult mouse (aged 2 months) fibroblasts were reprogrammed by vector 20866 with c-Myc, Klf4, Oct4, and Sox2 genes and an mOrange marker was used to detect gene expression after 2-weeks in cell culture (a). Induced pluripotent stem cells (iPSCs) began to show obvious outgrowths at Days 15 and 20 (b). The undifferentiated iPSCs were transformed into neural progenitor cells following culture in the Neural Induction Medium (see Materials and Methods) during Days 21–28 (c). The neural progenitor cells were further differentiated into dopaminergic neural progenitors with the Dopamine Neuronal Progenitor Medium from Days 29–35 (d). From Days 36 to 50, the cells were subsequently differentiated into mature dopaminergic neurons by culturing in the Dopamine Neuronal Differentiation Medium (e,f,g). The mature dopaminergic neurons were grown for up to 6 months (h), and were visibly easy to distinguish from the original un-reprogrammed adult fibroblasts from parallel control cultures (i).
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Figure 5: (A) Immature neuronal cells that were cultured in a Dopaminergic Neuronal Progenitor Medium began to show morphological signs of neuronal maturity between Day 29–50 (a,b,c) and after 6 months these cells displayed further signs of mature network formation (d). Importantly, control tail cells that were not subject to the reprogramming procedures kept their original shape at 6 months (e). As well, during this time, expression of the early immature neuronal cell marker, DCX, was greatly diminished (f), together with increased expression of the mature general neuronal cell marker, microtubule associated protein 2 (MAP2), (g), and specific dopaminergic neuronal marker, tyrosine hydroxylase (TH), (h) was evident. Further confirmation that the reprogrammed neurons were mature dopaminergic neurons was obtained using HPLC (i); wherein tail cells that were re-programmed (Tail-Dopamine cells) displayed significantly increased dopamine levels, compared to non-reprogrammed controls (Tail cells). Furthermore, dopamine levels in non-reprogrammed controls were, as expected, quite low (Tail tissue), whereas freshly dissected adult midbrain tissue had the expected highest concentration of dopamine (Brain tissue). *p < 0.05, relative to control tails. (B) Shows the time course for dopaminergic neuron derivation. The adult mouse (aged 2 months) fibroblasts were reprogrammed by vector 20866 with c-Myc, Klf4, Oct4, and Sox2 genes and an mOrange marker was used to detect gene expression after 2-weeks in cell culture (a). Induced pluripotent stem cells (iPSCs) began to show obvious outgrowths at Days 15 and 20 (b). The undifferentiated iPSCs were transformed into neural progenitor cells following culture in the Neural Induction Medium (see Materials and Methods) during Days 21–28 (c). The neural progenitor cells were further differentiated into dopaminergic neural progenitors with the Dopamine Neuronal Progenitor Medium from Days 29–35 (d). From Days 36 to 50, the cells were subsequently differentiated into mature dopaminergic neurons by culturing in the Dopamine Neuronal Differentiation Medium (e,f,g). The mature dopaminergic neurons were grown for up to 6 months (h), and were visibly easy to distinguish from the original un-reprogrammed adult fibroblasts from parallel control cultures (i).

Mentions: Ensuing experiments involved mature dopaminergic neurons differentiated from neural progenitors generated from our iPSC lines. Specifically, neural progenitor cells were first produced from reprogrammed adult mouse tail-tip cells using a neuronal progenitor medium during Days 29–35 (Figure 5A-a). Then, from Day 36 to 50, these cells were further directed to differentiate into midbrain mature dopaminergic neurons using a dopamine neuronal differentiation medium (see Materials and Methods). At this point, the cells showed clear signs of forming mature neural networks (Figure 5A-b,c). Importantly, long-term observation of these cell lines until 6 months after the initial fibroblast cell reprogramming revealed that the cultured dopaminergic neurons still had a pronounced ability to form mature neuronal networks (Figure 5A-d). In contrast, at the 6-month mark, tail-tip fibroblast cells grown in control cultures (i.e., without cell reprogramming and differentiating media) maintained their original shape and failed to form any visible cellular networks (Figure 5A-e).


Use of induced pluripotent stem cell derived neurons engineered to express BDNF for modulation of stressor related pathology.

Liu G, Rustom N, Litteljohn D, Bobyn J, Rudyk C, Anisman H, Hayley S - Front Cell Neurosci (2014)

(A) Immature neuronal cells that were cultured in a Dopaminergic Neuronal Progenitor Medium began to show morphological signs of neuronal maturity between Day 29–50 (a,b,c) and after 6 months these cells displayed further signs of mature network formation (d). Importantly, control tail cells that were not subject to the reprogramming procedures kept their original shape at 6 months (e). As well, during this time, expression of the early immature neuronal cell marker, DCX, was greatly diminished (f), together with increased expression of the mature general neuronal cell marker, microtubule associated protein 2 (MAP2), (g), and specific dopaminergic neuronal marker, tyrosine hydroxylase (TH), (h) was evident. Further confirmation that the reprogrammed neurons were mature dopaminergic neurons was obtained using HPLC (i); wherein tail cells that were re-programmed (Tail-Dopamine cells) displayed significantly increased dopamine levels, compared to non-reprogrammed controls (Tail cells). Furthermore, dopamine levels in non-reprogrammed controls were, as expected, quite low (Tail tissue), whereas freshly dissected adult midbrain tissue had the expected highest concentration of dopamine (Brain tissue). *p < 0.05, relative to control tails. (B) Shows the time course for dopaminergic neuron derivation. The adult mouse (aged 2 months) fibroblasts were reprogrammed by vector 20866 with c-Myc, Klf4, Oct4, and Sox2 genes and an mOrange marker was used to detect gene expression after 2-weeks in cell culture (a). Induced pluripotent stem cells (iPSCs) began to show obvious outgrowths at Days 15 and 20 (b). The undifferentiated iPSCs were transformed into neural progenitor cells following culture in the Neural Induction Medium (see Materials and Methods) during Days 21–28 (c). The neural progenitor cells were further differentiated into dopaminergic neural progenitors with the Dopamine Neuronal Progenitor Medium from Days 29–35 (d). From Days 36 to 50, the cells were subsequently differentiated into mature dopaminergic neurons by culturing in the Dopamine Neuronal Differentiation Medium (e,f,g). The mature dopaminergic neurons were grown for up to 6 months (h), and were visibly easy to distinguish from the original un-reprogrammed adult fibroblasts from parallel control cultures (i).
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
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Figure 5: (A) Immature neuronal cells that were cultured in a Dopaminergic Neuronal Progenitor Medium began to show morphological signs of neuronal maturity between Day 29–50 (a,b,c) and after 6 months these cells displayed further signs of mature network formation (d). Importantly, control tail cells that were not subject to the reprogramming procedures kept their original shape at 6 months (e). As well, during this time, expression of the early immature neuronal cell marker, DCX, was greatly diminished (f), together with increased expression of the mature general neuronal cell marker, microtubule associated protein 2 (MAP2), (g), and specific dopaminergic neuronal marker, tyrosine hydroxylase (TH), (h) was evident. Further confirmation that the reprogrammed neurons were mature dopaminergic neurons was obtained using HPLC (i); wherein tail cells that were re-programmed (Tail-Dopamine cells) displayed significantly increased dopamine levels, compared to non-reprogrammed controls (Tail cells). Furthermore, dopamine levels in non-reprogrammed controls were, as expected, quite low (Tail tissue), whereas freshly dissected adult midbrain tissue had the expected highest concentration of dopamine (Brain tissue). *p < 0.05, relative to control tails. (B) Shows the time course for dopaminergic neuron derivation. The adult mouse (aged 2 months) fibroblasts were reprogrammed by vector 20866 with c-Myc, Klf4, Oct4, and Sox2 genes and an mOrange marker was used to detect gene expression after 2-weeks in cell culture (a). Induced pluripotent stem cells (iPSCs) began to show obvious outgrowths at Days 15 and 20 (b). The undifferentiated iPSCs were transformed into neural progenitor cells following culture in the Neural Induction Medium (see Materials and Methods) during Days 21–28 (c). The neural progenitor cells were further differentiated into dopaminergic neural progenitors with the Dopamine Neuronal Progenitor Medium from Days 29–35 (d). From Days 36 to 50, the cells were subsequently differentiated into mature dopaminergic neurons by culturing in the Dopamine Neuronal Differentiation Medium (e,f,g). The mature dopaminergic neurons were grown for up to 6 months (h), and were visibly easy to distinguish from the original un-reprogrammed adult fibroblasts from parallel control cultures (i).
Mentions: Ensuing experiments involved mature dopaminergic neurons differentiated from neural progenitors generated from our iPSC lines. Specifically, neural progenitor cells were first produced from reprogrammed adult mouse tail-tip cells using a neuronal progenitor medium during Days 29–35 (Figure 5A-a). Then, from Day 36 to 50, these cells were further directed to differentiate into midbrain mature dopaminergic neurons using a dopamine neuronal differentiation medium (see Materials and Methods). At this point, the cells showed clear signs of forming mature neural networks (Figure 5A-b,c). Importantly, long-term observation of these cell lines until 6 months after the initial fibroblast cell reprogramming revealed that the cultured dopaminergic neurons still had a pronounced ability to form mature neuronal networks (Figure 5A-d). In contrast, at the 6-month mark, tail-tip fibroblast cells grown in control cultures (i.e., without cell reprogramming and differentiating media) maintained their original shape and failed to form any visible cellular networks (Figure 5A-e).

Bottom Line: Combined cell and gene-based therapeutic strategies offer potential in the treatment of neurodegenerative and psychiatric conditions that have been associated with structural brain disturbances.Specifically, intracerebroventricular transplantation of iPSC-derived neural progenitors that over-expressed BDNF reversed the impact of immune (lipopolysaccharide) and chronic stressor challenges upon subventricular zone adult neurogenesis, and the iPSC-derived neural progenitor cells alone blunted the stressor-induced corticosterone response.Moreover, our findings indicate that mature dopamine producing neurons can be generated using iPSC procedures and appear to be viable when infused in vivo.

View Article: PubMed Central - PubMed

Affiliation: Hayley Lab, Department of Neuroscience, Carleton University Ottawa, ON, Canada.

ABSTRACT
Combined cell and gene-based therapeutic strategies offer potential in the treatment of neurodegenerative and psychiatric conditions that have been associated with structural brain disturbances. In the present investigation, we used a novel virus-free re-programming method to generate induced pluripotent stem cells (iPSCs), and then subsequently transformed these cells into neural cells which over-expressed brain derived neurotrophic factor (BDNF). Importantly, the infusion of iPSC derived neural cells (as a cell replacement and gene delivery tool) and BDNF (as a protective factor) influenced neuronal outcomes. Specifically, intracerebroventricular transplantation of iPSC-derived neural progenitors that over-expressed BDNF reversed the impact of immune (lipopolysaccharide) and chronic stressor challenges upon subventricular zone adult neurogenesis, and the iPSC-derived neural progenitor cells alone blunted the stressor-induced corticosterone response. Moreover, our findings indicate that mature dopamine producing neurons can be generated using iPSC procedures and appear to be viable when infused in vivo. Taken together, these data could have important implications for using gene-plus-cell replacement methods to modulate stressor related pathology.

No MeSH data available.


Related in: MedlinePlus