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Analysis of host-mediated repair mechanisms after human CNS-stem cell transplantation for spinal cord injury: correlation of engraftment with recovery.

Hooshmand MJ, Sontag CJ, Uchida N, Tamaki S, Anderson AJ, Cummings BJ - PLoS ONE (2009)

Bottom Line: Stereological quantification of human cells using a human-specific cytoplasmic marker demonstrated successful cell engraftment, survival, migration and limited proliferation in all hCNS-SCns transplanted animals.Biochemical analyses supplemented stereological data supporting the absence of neural stem-cell mediated host repair.However, linear regression analysis of the number of engrafted hCNS-SCns vs. the number of errors on a horizontal ladder beam task revealed a strong correlation between these variables (r = -0.78, p<0.05), suggesting that survival and engraftment were directly related to a quantitative measure of recovery.

View Article: PubMed Central - PubMed

Affiliation: Department of Anatomy and Neurobiology, University of California Irvine, Irvine, CA, USA.

ABSTRACT

Background: Human central nervous system-stem cells grown as neurospheres (hCNS-SCns) self-renew, are multipotent, and have potential therapeutic applications following trauma to the spinal cord. We have previously shown locomotor recovery in immunodeficient mice that received a moderate contusion spinal cord injury (SCI) and hCNS-SCns transplantation 9 days post-injury (dpi). Engrafted hCNS-SCns exhibited terminal differentiation to myelinating oligodendrocytes and synapse-forming neurons. Further, selective ablation of human cells using Diphtheria toxin (DT) abolished locomotor recovery in this paradigm, suggesting integration of human cells within the mouse host as a possible mechanism for the locomotor improvement. However, the hypothesis that hCNS-SCns could alter the host microenvironment as an additional or alternative mechanism of recovery remained unexplored; we tested that hypothesis in the present study.

Methods and findings: Stereological quantification of human cells using a human-specific cytoplasmic marker demonstrated successful cell engraftment, survival, migration and limited proliferation in all hCNS-SCns transplanted animals. DT administration at 16 weeks post-transplant ablated 80.5% of hCNS-SCns. Stereological quantification for lesion volume, tissue sparing, descending serotonergic host fiber sprouting, chondroitin sulfate proteoglycan deposition, glial scarring, and angiogenesis demonstrated no evidence of host modification within the mouse spinal cord as a result of hCNS-SCns transplantation. Biochemical analyses supplemented stereological data supporting the absence of neural stem-cell mediated host repair. However, linear regression analysis of the number of engrafted hCNS-SCns vs. the number of errors on a horizontal ladder beam task revealed a strong correlation between these variables (r = -0.78, p<0.05), suggesting that survival and engraftment were directly related to a quantitative measure of recovery.

Conclusions: Altogether, the data suggest that the locomotor improvements associated with hCNS-SCns transplantation were not due to modifications within the host microenvironment, supporting the hypothesis that human cell integration within the host circuitry mediates functional recovery following a 9 day delayed transplant.

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Related in: MedlinePlus

Human cell transplantation does not alter the areas of NG2 deposition or the GFAP astroglial scar.A: Estimated area occupied by the NG2 proteoglycan was analyzed using the Cavalieri estimator probe of StereoInvestigator. B: Quantification revealed no significant differences between any of the groups in the area occupied by NG2 (One-way ANOVA: F = 0.005, p = 0.99). C: Estimated area occupied by the GFAP scar was determined in the same manner as NG2. The lesion epicenter was not included in the estimated GFAP scar area. D: Stereological quantification exhibited no significant differences between any of the groups in the area of the GFAP astroglial scar (One-way ANOVA: F = 1.50, p = 0.24). Scale Bar = 250 µm for A and C.
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pone-0005871-g008: Human cell transplantation does not alter the areas of NG2 deposition or the GFAP astroglial scar.A: Estimated area occupied by the NG2 proteoglycan was analyzed using the Cavalieri estimator probe of StereoInvestigator. B: Quantification revealed no significant differences between any of the groups in the area occupied by NG2 (One-way ANOVA: F = 0.005, p = 0.99). C: Estimated area occupied by the GFAP scar was determined in the same manner as NG2. The lesion epicenter was not included in the estimated GFAP scar area. D: Stereological quantification exhibited no significant differences between any of the groups in the area of the GFAP astroglial scar (One-way ANOVA: F = 1.50, p = 0.24). Scale Bar = 250 µm for A and C.

Mentions: NG2 is a major CSPG component that is elevated starting 24 hours after injury, remaining significantly above control levels for up to 6 months post-SCI, and is inhibitory to neurite outgrowth in vitro [58]–[60] and in vivo [58], [59], [61]. Previous transplantation studies have demonstrated the capacity of embryonic radial glia to reduce NG2 deposition, thus promoting axonal regeneration [54]. In order to investigate the effect of cell transplantation at 9dpi on NG2 deposition, stereological quantification of the area occupied by the NG2 proteoglycan (Fig. 8A) was performed using the Cavalieri estimator. No significant differences (One-way ANOVA, F = 0.005, p = 0.99) were found in the area occupied by the NG2 proteogylcan between any of the groups (Fig. 8B). This data suggests that neither hCNS-SCns nor hFb alter deposition of the NG2 proteoglycan following injury.


Analysis of host-mediated repair mechanisms after human CNS-stem cell transplantation for spinal cord injury: correlation of engraftment with recovery.

Hooshmand MJ, Sontag CJ, Uchida N, Tamaki S, Anderson AJ, Cummings BJ - PLoS ONE (2009)

Human cell transplantation does not alter the areas of NG2 deposition or the GFAP astroglial scar.A: Estimated area occupied by the NG2 proteoglycan was analyzed using the Cavalieri estimator probe of StereoInvestigator. B: Quantification revealed no significant differences between any of the groups in the area occupied by NG2 (One-way ANOVA: F = 0.005, p = 0.99). C: Estimated area occupied by the GFAP scar was determined in the same manner as NG2. The lesion epicenter was not included in the estimated GFAP scar area. D: Stereological quantification exhibited no significant differences between any of the groups in the area of the GFAP astroglial scar (One-way ANOVA: F = 1.50, p = 0.24). Scale Bar = 250 µm for A and C.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2690693&req=5

pone-0005871-g008: Human cell transplantation does not alter the areas of NG2 deposition or the GFAP astroglial scar.A: Estimated area occupied by the NG2 proteoglycan was analyzed using the Cavalieri estimator probe of StereoInvestigator. B: Quantification revealed no significant differences between any of the groups in the area occupied by NG2 (One-way ANOVA: F = 0.005, p = 0.99). C: Estimated area occupied by the GFAP scar was determined in the same manner as NG2. The lesion epicenter was not included in the estimated GFAP scar area. D: Stereological quantification exhibited no significant differences between any of the groups in the area of the GFAP astroglial scar (One-way ANOVA: F = 1.50, p = 0.24). Scale Bar = 250 µm for A and C.
Mentions: NG2 is a major CSPG component that is elevated starting 24 hours after injury, remaining significantly above control levels for up to 6 months post-SCI, and is inhibitory to neurite outgrowth in vitro [58]–[60] and in vivo [58], [59], [61]. Previous transplantation studies have demonstrated the capacity of embryonic radial glia to reduce NG2 deposition, thus promoting axonal regeneration [54]. In order to investigate the effect of cell transplantation at 9dpi on NG2 deposition, stereological quantification of the area occupied by the NG2 proteoglycan (Fig. 8A) was performed using the Cavalieri estimator. No significant differences (One-way ANOVA, F = 0.005, p = 0.99) were found in the area occupied by the NG2 proteogylcan between any of the groups (Fig. 8B). This data suggests that neither hCNS-SCns nor hFb alter deposition of the NG2 proteoglycan following injury.

Bottom Line: Stereological quantification of human cells using a human-specific cytoplasmic marker demonstrated successful cell engraftment, survival, migration and limited proliferation in all hCNS-SCns transplanted animals.Biochemical analyses supplemented stereological data supporting the absence of neural stem-cell mediated host repair.However, linear regression analysis of the number of engrafted hCNS-SCns vs. the number of errors on a horizontal ladder beam task revealed a strong correlation between these variables (r = -0.78, p<0.05), suggesting that survival and engraftment were directly related to a quantitative measure of recovery.

View Article: PubMed Central - PubMed

Affiliation: Department of Anatomy and Neurobiology, University of California Irvine, Irvine, CA, USA.

ABSTRACT

Background: Human central nervous system-stem cells grown as neurospheres (hCNS-SCns) self-renew, are multipotent, and have potential therapeutic applications following trauma to the spinal cord. We have previously shown locomotor recovery in immunodeficient mice that received a moderate contusion spinal cord injury (SCI) and hCNS-SCns transplantation 9 days post-injury (dpi). Engrafted hCNS-SCns exhibited terminal differentiation to myelinating oligodendrocytes and synapse-forming neurons. Further, selective ablation of human cells using Diphtheria toxin (DT) abolished locomotor recovery in this paradigm, suggesting integration of human cells within the mouse host as a possible mechanism for the locomotor improvement. However, the hypothesis that hCNS-SCns could alter the host microenvironment as an additional or alternative mechanism of recovery remained unexplored; we tested that hypothesis in the present study.

Methods and findings: Stereological quantification of human cells using a human-specific cytoplasmic marker demonstrated successful cell engraftment, survival, migration and limited proliferation in all hCNS-SCns transplanted animals. DT administration at 16 weeks post-transplant ablated 80.5% of hCNS-SCns. Stereological quantification for lesion volume, tissue sparing, descending serotonergic host fiber sprouting, chondroitin sulfate proteoglycan deposition, glial scarring, and angiogenesis demonstrated no evidence of host modification within the mouse spinal cord as a result of hCNS-SCns transplantation. Biochemical analyses supplemented stereological data supporting the absence of neural stem-cell mediated host repair. However, linear regression analysis of the number of engrafted hCNS-SCns vs. the number of errors on a horizontal ladder beam task revealed a strong correlation between these variables (r = -0.78, p<0.05), suggesting that survival and engraftment were directly related to a quantitative measure of recovery.

Conclusions: Altogether, the data suggest that the locomotor improvements associated with hCNS-SCns transplantation were not due to modifications within the host microenvironment, supporting the hypothesis that human cell integration within the host circuitry mediates functional recovery following a 9 day delayed transplant.

Show MeSH
Related in: MedlinePlus