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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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Diphtheria toxin (DT) administration at 16 weeks post-transplant ablates hCNS-SCns.A: Immunostaining using SC121 revealed cell survival in animals that received hCNS-SCns at 9dpi and saline injection at 16 weeks post-transplant. B: High power image of (A) demonstrated the presence of healthy cells with staining of cytoplasm and processes. C: Animals receiving hCNS-SCns at 9dpi and DT at 16 weeks post-transplant demonstrated a reduction in SC121 immunostaining. D: High power image of (C) demonstrated the presence of hCNS-SCns with foamy appearance and/or morphological characteristics representative of unhealthy or apoptotic/necrotic cells (arrowheads). E: Quantification for the estimated number of hCNS-SCns 17 weeks post-transplant revealed limited proliferation of hCNS-SCns in animals receiving saline at 16 weeks post-transplant. Dashed line indicates the original transplanted dose of 75,000 hCNS-SCns. DT administration resulted in a significant reduction (80.5%) in the number of hCNS-SCns. ** denotes p<0.0001, 1-tailed t-test. Means±standard errors are shown. Scale bars = 250 µm for A and C and 25 µm for B and D.
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pone-0005871-g004: Diphtheria toxin (DT) administration at 16 weeks post-transplant ablates hCNS-SCns.A: Immunostaining using SC121 revealed cell survival in animals that received hCNS-SCns at 9dpi and saline injection at 16 weeks post-transplant. B: High power image of (A) demonstrated the presence of healthy cells with staining of cytoplasm and processes. C: Animals receiving hCNS-SCns at 9dpi and DT at 16 weeks post-transplant demonstrated a reduction in SC121 immunostaining. D: High power image of (C) demonstrated the presence of hCNS-SCns with foamy appearance and/or morphological characteristics representative of unhealthy or apoptotic/necrotic cells (arrowheads). E: Quantification for the estimated number of hCNS-SCns 17 weeks post-transplant revealed limited proliferation of hCNS-SCns in animals receiving saline at 16 weeks post-transplant. Dashed line indicates the original transplanted dose of 75,000 hCNS-SCns. DT administration resulted in a significant reduction (80.5%) in the number of hCNS-SCns. ** denotes p<0.0001, 1-tailed t-test. Means±standard errors are shown. Scale bars = 250 µm for A and C and 25 µm for B and D.

Mentions: Histological analysis in animals receiving human cells at 9dpi and 2 injections of DT at 16 weeks post-transplant demonstrated an 80.5% reduction in the number of hCNS-SCns (Fig. 4C and D) and 97.8% reduction of hFb (Fig. 5C and D) in comparison to animals that received cells and were treated with saline at 16 weeks post-transplant (Fig. 4A–B and Fig. 5A–B). Stereological quantification of the total number of engrafted human cells within the defined region of 3 mm rostral and 3 mm caudal to the injury epicenter revealed the presence of an average of 145,553±12,940 hCNS-SCns (Fig. 4E) and an average of 3,752±790 hFb (Fig. 5E) in animals receiving saline injection at 16 weeks post-transplant. In comparison to the saline-treated animals, mice receiving DT exhibited significantly less hCNS-SCns (an average of 28,454±4,007) (Fig. 4E) and hFb (an average of 82±45) (Fig. 5E), demonstrating that DT ablated the human cells (1-tailed t-tests: p<0.0001 for hCNS-SCns and p<0.0007 for hFb). It is important to note that the estimated number of human cells in animals treated with DT reported here is likely to be an overestimation of intact, surviving cells. As shown in Fig. 4D, apoptotic/necrotic appearing SC121-positive cells with nuclei and a “foamy” appearance, but without processes, were detected in animals receiving DT (arrowheads). However, in order to ensure stereological consistency and reduce bias in quantification, all SC121-positive human cells that were associated with methyl green-positive nuclei were counted, whether or not they were morphologically “unhealthy.” Accordingly, a conservative but accurate estimate for the number of hCNS-SCns in the DT-treated animals is reported. Collectively, these findings extended and confirmed our previous observations that human cells are ablated by administration of DT.


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)

Diphtheria toxin (DT) administration at 16 weeks post-transplant ablates hCNS-SCns.A: Immunostaining using SC121 revealed cell survival in animals that received hCNS-SCns at 9dpi and saline injection at 16 weeks post-transplant. B: High power image of (A) demonstrated the presence of healthy cells with staining of cytoplasm and processes. C: Animals receiving hCNS-SCns at 9dpi and DT at 16 weeks post-transplant demonstrated a reduction in SC121 immunostaining. D: High power image of (C) demonstrated the presence of hCNS-SCns with foamy appearance and/or morphological characteristics representative of unhealthy or apoptotic/necrotic cells (arrowheads). E: Quantification for the estimated number of hCNS-SCns 17 weeks post-transplant revealed limited proliferation of hCNS-SCns in animals receiving saline at 16 weeks post-transplant. Dashed line indicates the original transplanted dose of 75,000 hCNS-SCns. DT administration resulted in a significant reduction (80.5%) in the number of hCNS-SCns. ** denotes p<0.0001, 1-tailed t-test. Means±standard errors are shown. Scale bars = 250 µm for A and C and 25 µm for B and D.
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getmorefigures.php?uid=PMC2690693&req=5

pone-0005871-g004: Diphtheria toxin (DT) administration at 16 weeks post-transplant ablates hCNS-SCns.A: Immunostaining using SC121 revealed cell survival in animals that received hCNS-SCns at 9dpi and saline injection at 16 weeks post-transplant. B: High power image of (A) demonstrated the presence of healthy cells with staining of cytoplasm and processes. C: Animals receiving hCNS-SCns at 9dpi and DT at 16 weeks post-transplant demonstrated a reduction in SC121 immunostaining. D: High power image of (C) demonstrated the presence of hCNS-SCns with foamy appearance and/or morphological characteristics representative of unhealthy or apoptotic/necrotic cells (arrowheads). E: Quantification for the estimated number of hCNS-SCns 17 weeks post-transplant revealed limited proliferation of hCNS-SCns in animals receiving saline at 16 weeks post-transplant. Dashed line indicates the original transplanted dose of 75,000 hCNS-SCns. DT administration resulted in a significant reduction (80.5%) in the number of hCNS-SCns. ** denotes p<0.0001, 1-tailed t-test. Means±standard errors are shown. Scale bars = 250 µm for A and C and 25 µm for B and D.
Mentions: Histological analysis in animals receiving human cells at 9dpi and 2 injections of DT at 16 weeks post-transplant demonstrated an 80.5% reduction in the number of hCNS-SCns (Fig. 4C and D) and 97.8% reduction of hFb (Fig. 5C and D) in comparison to animals that received cells and were treated with saline at 16 weeks post-transplant (Fig. 4A–B and Fig. 5A–B). Stereological quantification of the total number of engrafted human cells within the defined region of 3 mm rostral and 3 mm caudal to the injury epicenter revealed the presence of an average of 145,553±12,940 hCNS-SCns (Fig. 4E) and an average of 3,752±790 hFb (Fig. 5E) in animals receiving saline injection at 16 weeks post-transplant. In comparison to the saline-treated animals, mice receiving DT exhibited significantly less hCNS-SCns (an average of 28,454±4,007) (Fig. 4E) and hFb (an average of 82±45) (Fig. 5E), demonstrating that DT ablated the human cells (1-tailed t-tests: p<0.0001 for hCNS-SCns and p<0.0007 for hFb). It is important to note that the estimated number of human cells in animals treated with DT reported here is likely to be an overestimation of intact, surviving cells. As shown in Fig. 4D, apoptotic/necrotic appearing SC121-positive cells with nuclei and a “foamy” appearance, but without processes, were detected in animals receiving DT (arrowheads). However, in order to ensure stereological consistency and reduce bias in quantification, all SC121-positive human cells that were associated with methyl green-positive nuclei were counted, whether or not they were morphologically “unhealthy.” Accordingly, a conservative but accurate estimate for the number of hCNS-SCns in the DT-treated animals is reported. Collectively, these findings extended and confirmed our previous observations that human cells are ablated by administration of DT.

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