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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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Expression levels of proteins associated with host repair remain unchanged as a result of hCNS-SCns transplantation.Biochemical analysis was performed 2 weeks post-transplant in animals that received either hCNS-SCns or vehicle control. A: No changes in Fibronectin protein expression were observed as a result of hCNS-SCns transplantation (2-tailed t-test: p = 0.234). B: Protein analysis of a CSPG protein, NG2, demonstrated no significant differences between transplanted animals and vehicle control (2-tailed t-test: p = 0.477). C: Expression levels of an additional CSPG protein, Versican, revealed no significant differences between the two groups (2-tailed t-test: p = 0.299). D: GFAP protein expression remained unchanged as a result of hCNS-SCns transplantation (2-tailed t-test: p = 0.581). E: PE-CAM1 expression levels were not altered after cell transplantation (2-tailed t-test: p = 0.496). F: β-actin was used as control to ensure equivalent protein loading (2-tailed t-test: p = 0.116).
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pone-0005871-g010: Expression levels of proteins associated with host repair remain unchanged as a result of hCNS-SCns transplantation.Biochemical analysis was performed 2 weeks post-transplant in animals that received either hCNS-SCns or vehicle control. A: No changes in Fibronectin protein expression were observed as a result of hCNS-SCns transplantation (2-tailed t-test: p = 0.234). B: Protein analysis of a CSPG protein, NG2, demonstrated no significant differences between transplanted animals and vehicle control (2-tailed t-test: p = 0.477). C: Expression levels of an additional CSPG protein, Versican, revealed no significant differences between the two groups (2-tailed t-test: p = 0.299). D: GFAP protein expression remained unchanged as a result of hCNS-SCns transplantation (2-tailed t-test: p = 0.581). E: PE-CAM1 expression levels were not altered after cell transplantation (2-tailed t-test: p = 0.496). F: β-actin was used as control to ensure equivalent protein loading (2-tailed t-test: p = 0.116).

Mentions: The histological absence of significant differences in the multiple parameters of host response after hCNS-SCns transplantation was supplemented with biochemical analyses to determine protein expression levels in spinal cord-injured animals receiving either hCNS-SCns or vehicle at 9dpi. We hypothesized that the maximum potential for trophic/neuroprotective modulation of host repair mechanisms by transplanted cells would occur within the first 2 weeks following transplantation because lesion volume, tissue sparing, astroglial scar formation and proteoglycan deposition become relatively stable within 2–3 weeks following rodent contusion SCI [69]–[72]. Hence, assessment of a supplemental set of biochemical markers for host repair at this additional time point of 2 weeks post-transplantation would have both the potential to reveal any changes missed in our analyses at 4 months post-transplantation, and the potential to reveal any changes that may have occurred acutely but transiently after transplantation. Accordingly, western blot analysis to assess possible changes in protein expression levels of fibronectin, an extracellular matrix molecule present at the lesion epicenter of injured mice, revealed no significant differences (2-tailed t-test: p = 0.234) between animals receiving hCNS-SCns and vehicle (Fig. 10A), suggesting that hCNS-SCns transplantation does not alter fibronectin deposition at 2 weeks post-transplant. The protein expression levels of two CSPG proteins, NG2 and Versican, which have been shown to increase acutely after injury and remain upregulated for prolonged periods following SCI, also did not change (2-tailed t-test: p = 0.477 for NG2 and p = 0.299 for Versican) as a result of hCNS-SCns transplantation (Fig. 10B and C). Further, GFAP protein expression (Fig. 10D), an indication of glial scarring, and PE-CAM1 expression (Fig. 10E), indicative of potential angiogenesis, were not different between transplanted animals and vehicle control (2-tailed t-test: p = 0.581 for GFAP and p = 0.496 for PE-CAM1). Protein expression level of β-actin, which was used to ensure equivalent protein loading, demonstrated no significant differences (2-tailed t-tests: p = 0.116) between transplanted animals and vehicle control (Fig. 10F). Collectively, these data supplement our stereological quantification suggesting that hCNS-SCns transplantation does not alter lesion size, CSPG deposition, GFAP glial scarring, and re-vascularization.


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)

Expression levels of proteins associated with host repair remain unchanged as a result of hCNS-SCns transplantation.Biochemical analysis was performed 2 weeks post-transplant in animals that received either hCNS-SCns or vehicle control. A: No changes in Fibronectin protein expression were observed as a result of hCNS-SCns transplantation (2-tailed t-test: p = 0.234). B: Protein analysis of a CSPG protein, NG2, demonstrated no significant differences between transplanted animals and vehicle control (2-tailed t-test: p = 0.477). C: Expression levels of an additional CSPG protein, Versican, revealed no significant differences between the two groups (2-tailed t-test: p = 0.299). D: GFAP protein expression remained unchanged as a result of hCNS-SCns transplantation (2-tailed t-test: p = 0.581). E: PE-CAM1 expression levels were not altered after cell transplantation (2-tailed t-test: p = 0.496). F: β-actin was used as control to ensure equivalent protein loading (2-tailed t-test: p = 0.116).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2690693&req=5

pone-0005871-g010: Expression levels of proteins associated with host repair remain unchanged as a result of hCNS-SCns transplantation.Biochemical analysis was performed 2 weeks post-transplant in animals that received either hCNS-SCns or vehicle control. A: No changes in Fibronectin protein expression were observed as a result of hCNS-SCns transplantation (2-tailed t-test: p = 0.234). B: Protein analysis of a CSPG protein, NG2, demonstrated no significant differences between transplanted animals and vehicle control (2-tailed t-test: p = 0.477). C: Expression levels of an additional CSPG protein, Versican, revealed no significant differences between the two groups (2-tailed t-test: p = 0.299). D: GFAP protein expression remained unchanged as a result of hCNS-SCns transplantation (2-tailed t-test: p = 0.581). E: PE-CAM1 expression levels were not altered after cell transplantation (2-tailed t-test: p = 0.496). F: β-actin was used as control to ensure equivalent protein loading (2-tailed t-test: p = 0.116).
Mentions: The histological absence of significant differences in the multiple parameters of host response after hCNS-SCns transplantation was supplemented with biochemical analyses to determine protein expression levels in spinal cord-injured animals receiving either hCNS-SCns or vehicle at 9dpi. We hypothesized that the maximum potential for trophic/neuroprotective modulation of host repair mechanisms by transplanted cells would occur within the first 2 weeks following transplantation because lesion volume, tissue sparing, astroglial scar formation and proteoglycan deposition become relatively stable within 2–3 weeks following rodent contusion SCI [69]–[72]. Hence, assessment of a supplemental set of biochemical markers for host repair at this additional time point of 2 weeks post-transplantation would have both the potential to reveal any changes missed in our analyses at 4 months post-transplantation, and the potential to reveal any changes that may have occurred acutely but transiently after transplantation. Accordingly, western blot analysis to assess possible changes in protein expression levels of fibronectin, an extracellular matrix molecule present at the lesion epicenter of injured mice, revealed no significant differences (2-tailed t-test: p = 0.234) between animals receiving hCNS-SCns and vehicle (Fig. 10A), suggesting that hCNS-SCns transplantation does not alter fibronectin deposition at 2 weeks post-transplant. The protein expression levels of two CSPG proteins, NG2 and Versican, which have been shown to increase acutely after injury and remain upregulated for prolonged periods following SCI, also did not change (2-tailed t-test: p = 0.477 for NG2 and p = 0.299 for Versican) as a result of hCNS-SCns transplantation (Fig. 10B and C). Further, GFAP protein expression (Fig. 10D), an indication of glial scarring, and PE-CAM1 expression (Fig. 10E), indicative of potential angiogenesis, were not different between transplanted animals and vehicle control (2-tailed t-test: p = 0.581 for GFAP and p = 0.496 for PE-CAM1). Protein expression level of β-actin, which was used to ensure equivalent protein loading, demonstrated no significant differences (2-tailed t-tests: p = 0.116) between transplanted animals and vehicle control (Fig. 10F). Collectively, these data supplement our stereological quantification suggesting that hCNS-SCns transplantation does not alter lesion size, CSPG deposition, GFAP glial scarring, and re-vascularization.

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