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Human neural stem cells enhance structural plasticity and axonal transport in the ischaemic brain.

Andres RH, Horie N, Slikker W, Keren-Gill H, Zhan K, Sun G, Manley NC, Pereira MP, Sheikh LA, McMillan EL, Schaar BT, Svendsen CN, Bliss TM, Steinberg GK - Brain (2011)

Bottom Line: Our results show the first evidence that human neural progenitor cell treatment can significantly increase dendritic plasticity in both the ipsi- and contralesional cortex and this coincides with stem cell-induced functional recovery.Finally, we established in vitro co-culture assays in which these stem cells mimicked the effects observed in vivo.Through immunodepletion studies, we identified vascular endothelial growth factor, thrombospondins 1 and 2, and slit as mediators partially responsible for stem cell-induced effects on dendritic sprouting, axonal plasticity and axonal transport in vitro.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurosurgery, Stanford Stroke Centre, Stanford Institute for Neuro-Innovation and Translational Neurosciences, Stanford University School of Medicine, 1201 Welch Road, Stanford, CA 94305-5487, USA.

ABSTRACT
Stem cell transplantation promises new hope for the treatment of stroke although significant questions remain about how the grafted cells elicit their effects. One hypothesis is that transplanted stem cells enhance endogenous repair mechanisms activated after cerebral ischaemia. Recognizing that bilateral reorganization of surviving circuits is associated with recovery after stroke, we investigated the ability of transplanted human neural progenitor cells to enhance this structural plasticity. Our results show the first evidence that human neural progenitor cell treatment can significantly increase dendritic plasticity in both the ipsi- and contralesional cortex and this coincides with stem cell-induced functional recovery. Moreover, stem cell-grafted rats demonstrated increased corticocortical, corticostriatal, corticothalamic and corticospinal axonal rewiring from the contralesional side; with the transcallosal and corticospinal axonal sprouting correlating with functional recovery. Furthermore, we demonstrate that axonal transport, which is critical for both proper axonal function and axonal sprouting, is inhibited by stroke and that this is rescued by the stem cell treatment, thus identifying another novel potential mechanism of action of transplanted cells. Finally, we established in vitro co-culture assays in which these stem cells mimicked the effects observed in vivo. Through immunodepletion studies, we identified vascular endothelial growth factor, thrombospondins 1 and 2, and slit as mediators partially responsible for stem cell-induced effects on dendritic sprouting, axonal plasticity and axonal transport in vitro. Thus, we postulate that human neural progenitor cells aid recovery after stroke through secretion of factors that enhance brain repair and plasticity.

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Identification of secreted factors mediating neurite plasticity in vitro. (A) Representative images of cortical neurons stained for microtubule-associated protein 2 (MAP2, labels dendrites) and SMI312 (labels axons) co-cultured without (vehicle) and with human NPCs. Scale = 50 µm. (B–D) Human NPCs significantly promoted dendritic branching (B), total dendritic length (C) and axonal outgrowth (D) of the cortical neurons; *P < 0.05 ‘control human NPC’ compared with ‘control vehicle’. Neutralization of the secreted factors with antibodies or the soluble slit receptor Roundabout-Fc significantly reduced the effects of human NPCs on these parameters as indicated. *P < 0.05, **P < 0.01 compared with ‘control human NPC’; #P < 0.05 compared with ‘control vehicle’. TSP = thrombospondins.
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Figure 4: Identification of secreted factors mediating neurite plasticity in vitro. (A) Representative images of cortical neurons stained for microtubule-associated protein 2 (MAP2, labels dendrites) and SMI312 (labels axons) co-cultured without (vehicle) and with human NPCs. Scale = 50 µm. (B–D) Human NPCs significantly promoted dendritic branching (B), total dendritic length (C) and axonal outgrowth (D) of the cortical neurons; *P < 0.05 ‘control human NPC’ compared with ‘control vehicle’. Neutralization of the secreted factors with antibodies or the soluble slit receptor Roundabout-Fc significantly reduced the effects of human NPCs on these parameters as indicated. *P < 0.05, **P < 0.01 compared with ‘control human NPC’; #P < 0.05 compared with ‘control vehicle’. TSP = thrombospondins.

Mentions: In a non-contact co-culture assay (Supplementary Fig. 3), human NPCs significantly increased (per neuron) dendritic branching (Fig. 4A and B), total dendritic length (Fig. 4A and C) and axonal length (Fig. 4A and D) of co-cultured cortical neurons, thus mimicking in vitro the human NPC-mediated effects on dendritic and axonal plasticity observed in vivo. To identify potential human NPC-secreted plasticity mediators, immunodepletion studies were done for specific candidate molecules known to be involved in neuronal plasticity and shown to be expressed in our human NPCs by microarray analysis (Wright et al., 2003). Neutralization of thrombospondins 1 and 2 or human VEGF significantly reduced human NPC-induced dendritic branching and length, while neutralization of Slit only affected total dendritic length with no effect on dendritic branching (Fig. 4B and C). Human NPC-mediated axonal outgrowth was significantly reduced by neutralization of all the aforementioned factors (Fig. 4D); neutralization of thrombospondins 1 and 2 also inhibited axonal growth in vehicle-treated cortical neurons. Depletion of SPARC had no effect on either dendritic or axonal morphology (Fig. 4B–D). The effects of the neutralizing antibodies were specific as isotype control antibodies had no effect (P > 0.05). Quantitative polymerase chain reaction analysis of the stroke brains 1 week post-transplantation revealed that at least four of these factors (VEGF, thrombospondins 1, 2 and SPARC) were expressed by the transplanted human NPCs (Table 1 and Supplementary Fig. 4), indicative of a potential role in vivo.Figure 4


Human neural stem cells enhance structural plasticity and axonal transport in the ischaemic brain.

Andres RH, Horie N, Slikker W, Keren-Gill H, Zhan K, Sun G, Manley NC, Pereira MP, Sheikh LA, McMillan EL, Schaar BT, Svendsen CN, Bliss TM, Steinberg GK - Brain (2011)

Identification of secreted factors mediating neurite plasticity in vitro. (A) Representative images of cortical neurons stained for microtubule-associated protein 2 (MAP2, labels dendrites) and SMI312 (labels axons) co-cultured without (vehicle) and with human NPCs. Scale = 50 µm. (B–D) Human NPCs significantly promoted dendritic branching (B), total dendritic length (C) and axonal outgrowth (D) of the cortical neurons; *P < 0.05 ‘control human NPC’ compared with ‘control vehicle’. Neutralization of the secreted factors with antibodies or the soluble slit receptor Roundabout-Fc significantly reduced the effects of human NPCs on these parameters as indicated. *P < 0.05, **P < 0.01 compared with ‘control human NPC’; #P < 0.05 compared with ‘control vehicle’. TSP = thrombospondins.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3102243&req=5

Figure 4: Identification of secreted factors mediating neurite plasticity in vitro. (A) Representative images of cortical neurons stained for microtubule-associated protein 2 (MAP2, labels dendrites) and SMI312 (labels axons) co-cultured without (vehicle) and with human NPCs. Scale = 50 µm. (B–D) Human NPCs significantly promoted dendritic branching (B), total dendritic length (C) and axonal outgrowth (D) of the cortical neurons; *P < 0.05 ‘control human NPC’ compared with ‘control vehicle’. Neutralization of the secreted factors with antibodies or the soluble slit receptor Roundabout-Fc significantly reduced the effects of human NPCs on these parameters as indicated. *P < 0.05, **P < 0.01 compared with ‘control human NPC’; #P < 0.05 compared with ‘control vehicle’. TSP = thrombospondins.
Mentions: In a non-contact co-culture assay (Supplementary Fig. 3), human NPCs significantly increased (per neuron) dendritic branching (Fig. 4A and B), total dendritic length (Fig. 4A and C) and axonal length (Fig. 4A and D) of co-cultured cortical neurons, thus mimicking in vitro the human NPC-mediated effects on dendritic and axonal plasticity observed in vivo. To identify potential human NPC-secreted plasticity mediators, immunodepletion studies were done for specific candidate molecules known to be involved in neuronal plasticity and shown to be expressed in our human NPCs by microarray analysis (Wright et al., 2003). Neutralization of thrombospondins 1 and 2 or human VEGF significantly reduced human NPC-induced dendritic branching and length, while neutralization of Slit only affected total dendritic length with no effect on dendritic branching (Fig. 4B and C). Human NPC-mediated axonal outgrowth was significantly reduced by neutralization of all the aforementioned factors (Fig. 4D); neutralization of thrombospondins 1 and 2 also inhibited axonal growth in vehicle-treated cortical neurons. Depletion of SPARC had no effect on either dendritic or axonal morphology (Fig. 4B–D). The effects of the neutralizing antibodies were specific as isotype control antibodies had no effect (P > 0.05). Quantitative polymerase chain reaction analysis of the stroke brains 1 week post-transplantation revealed that at least four of these factors (VEGF, thrombospondins 1, 2 and SPARC) were expressed by the transplanted human NPCs (Table 1 and Supplementary Fig. 4), indicative of a potential role in vivo.Figure 4

Bottom Line: Our results show the first evidence that human neural progenitor cell treatment can significantly increase dendritic plasticity in both the ipsi- and contralesional cortex and this coincides with stem cell-induced functional recovery.Finally, we established in vitro co-culture assays in which these stem cells mimicked the effects observed in vivo.Through immunodepletion studies, we identified vascular endothelial growth factor, thrombospondins 1 and 2, and slit as mediators partially responsible for stem cell-induced effects on dendritic sprouting, axonal plasticity and axonal transport in vitro.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurosurgery, Stanford Stroke Centre, Stanford Institute for Neuro-Innovation and Translational Neurosciences, Stanford University School of Medicine, 1201 Welch Road, Stanford, CA 94305-5487, USA.

ABSTRACT
Stem cell transplantation promises new hope for the treatment of stroke although significant questions remain about how the grafted cells elicit their effects. One hypothesis is that transplanted stem cells enhance endogenous repair mechanisms activated after cerebral ischaemia. Recognizing that bilateral reorganization of surviving circuits is associated with recovery after stroke, we investigated the ability of transplanted human neural progenitor cells to enhance this structural plasticity. Our results show the first evidence that human neural progenitor cell treatment can significantly increase dendritic plasticity in both the ipsi- and contralesional cortex and this coincides with stem cell-induced functional recovery. Moreover, stem cell-grafted rats demonstrated increased corticocortical, corticostriatal, corticothalamic and corticospinal axonal rewiring from the contralesional side; with the transcallosal and corticospinal axonal sprouting correlating with functional recovery. Furthermore, we demonstrate that axonal transport, which is critical for both proper axonal function and axonal sprouting, is inhibited by stroke and that this is rescued by the stem cell treatment, thus identifying another novel potential mechanism of action of transplanted cells. Finally, we established in vitro co-culture assays in which these stem cells mimicked the effects observed in vivo. Through immunodepletion studies, we identified vascular endothelial growth factor, thrombospondins 1 and 2, and slit as mediators partially responsible for stem cell-induced effects on dendritic sprouting, axonal plasticity and axonal transport in vitro. Thus, we postulate that human neural progenitor cells aid recovery after stroke through secretion of factors that enhance brain repair and plasticity.

Show MeSH
Related in: MedlinePlus