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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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hCNS-SCns engraft, survive, and show limited proliferation in all transplanted animals; hFb engraft in all transplanted animals, but demonstrate poor survival.A: Immunostaining using a human-specific cytoplasmic marker (SC121) demonstrated that hCNS-SCns survived 17 weeks post-transplant. Brown indicates human cells visualized with DAB; Green indicates mouse and human nuclei visualized with methyl green.B: Engrafted hCNS-SCns migrated away from the injury and appeared to differentiate in a site-specific manner with oligodendroglial and neuronal morphologies in the white and grey matters, respectively. C: hFb also survived 17 weeks post-transplant, but were localized near the site of transplantation (arrowhead).D: High power image of arrowhead in (C) demonstrated the presence of hFb at the site of injection.E: At 9dpi, animals received either 75,000 hCNS-SCns or 50,000 hFb. Human cells engrafted in 100% of the animals. At 17 weeks post-transplant, an average of 194% of the initial dose of hCNS-SCns and 7.5% of the initial dose of hFb were found in each animal, suggesting limited hCNS-SCns proliferation. Bars represent group means±standard errors. Scale bars = 250 µm for A and C and 25 µm for B and D.
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pone-0005871-g002: hCNS-SCns engraft, survive, and show limited proliferation in all transplanted animals; hFb engraft in all transplanted animals, but demonstrate poor survival.A: Immunostaining using a human-specific cytoplasmic marker (SC121) demonstrated that hCNS-SCns survived 17 weeks post-transplant. Brown indicates human cells visualized with DAB; Green indicates mouse and human nuclei visualized with methyl green.B: Engrafted hCNS-SCns migrated away from the injury and appeared to differentiate in a site-specific manner with oligodendroglial and neuronal morphologies in the white and grey matters, respectively. C: hFb also survived 17 weeks post-transplant, but were localized near the site of transplantation (arrowhead).D: High power image of arrowhead in (C) demonstrated the presence of hFb at the site of injection.E: At 9dpi, animals received either 75,000 hCNS-SCns or 50,000 hFb. Human cells engrafted in 100% of the animals. At 17 weeks post-transplant, an average of 194% of the initial dose of hCNS-SCns and 7.5% of the initial dose of hFb were found in each animal, suggesting limited hCNS-SCns proliferation. Bars represent group means±standard errors. Scale bars = 250 µm for A and C and 25 µm for B and D.

Mentions: One of the challenges associated with any cell transplantation strategy, especially in a xenograft paradigm, is the ability to overcome the host immune response and obtain successful cell engraftment. In the present study, we used immunodeficient NOD-scid mice to reduce confounds of xenograft rejection, avoid immunosuppressant drugs, and enhance hCNS-SCns engraftment success. Immunostaining using the human-specific cytoplasmic marker, SC121, revealed that hCNS-SCns engrafted in 100% of the animals, and that engrafted cells survived 17 weeks post-transplant (Fig. 2A). Engrafted hCNS-SCns migrated along the spinal cord and appeared to differentiate in a site-specific manner consistent with neuronal and oligodendroglial morphologies in the grey and white matter, respectively (Fig. 2B). Confocal analysis of hCNS-SCns differentiation was not repeated in the present study but was performed in our previous publication [1], where we reported evidence for β-tubulin or APC/CC1 expression in 26.4% and 64.1% of engrafted hCNS-SCns, respectively. hFb also engrafted in 100% of the animals and survived 17 weeks post-transplant (Fig. 2C). In contrast to hCNS-SCns, engrafted hFb were highly localized to the region corresponding to the site of transplantation and exhibited limited migration (Fig. 2C and D).


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)

hCNS-SCns engraft, survive, and show limited proliferation in all transplanted animals; hFb engraft in all transplanted animals, but demonstrate poor survival.A: Immunostaining using a human-specific cytoplasmic marker (SC121) demonstrated that hCNS-SCns survived 17 weeks post-transplant. Brown indicates human cells visualized with DAB; Green indicates mouse and human nuclei visualized with methyl green.B: Engrafted hCNS-SCns migrated away from the injury and appeared to differentiate in a site-specific manner with oligodendroglial and neuronal morphologies in the white and grey matters, respectively. C: hFb also survived 17 weeks post-transplant, but were localized near the site of transplantation (arrowhead).D: High power image of arrowhead in (C) demonstrated the presence of hFb at the site of injection.E: At 9dpi, animals received either 75,000 hCNS-SCns or 50,000 hFb. Human cells engrafted in 100% of the animals. At 17 weeks post-transplant, an average of 194% of the initial dose of hCNS-SCns and 7.5% of the initial dose of hFb were found in each animal, suggesting limited hCNS-SCns proliferation. Bars represent group means±standard errors. Scale bars = 250 µm for A and C and 25 µm for B and D.
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pone-0005871-g002: hCNS-SCns engraft, survive, and show limited proliferation in all transplanted animals; hFb engraft in all transplanted animals, but demonstrate poor survival.A: Immunostaining using a human-specific cytoplasmic marker (SC121) demonstrated that hCNS-SCns survived 17 weeks post-transplant. Brown indicates human cells visualized with DAB; Green indicates mouse and human nuclei visualized with methyl green.B: Engrafted hCNS-SCns migrated away from the injury and appeared to differentiate in a site-specific manner with oligodendroglial and neuronal morphologies in the white and grey matters, respectively. C: hFb also survived 17 weeks post-transplant, but were localized near the site of transplantation (arrowhead).D: High power image of arrowhead in (C) demonstrated the presence of hFb at the site of injection.E: At 9dpi, animals received either 75,000 hCNS-SCns or 50,000 hFb. Human cells engrafted in 100% of the animals. At 17 weeks post-transplant, an average of 194% of the initial dose of hCNS-SCns and 7.5% of the initial dose of hFb were found in each animal, suggesting limited hCNS-SCns proliferation. Bars represent group means±standard errors. Scale bars = 250 µm for A and C and 25 µm for B and D.
Mentions: One of the challenges associated with any cell transplantation strategy, especially in a xenograft paradigm, is the ability to overcome the host immune response and obtain successful cell engraftment. In the present study, we used immunodeficient NOD-scid mice to reduce confounds of xenograft rejection, avoid immunosuppressant drugs, and enhance hCNS-SCns engraftment success. Immunostaining using the human-specific cytoplasmic marker, SC121, revealed that hCNS-SCns engrafted in 100% of the animals, and that engrafted cells survived 17 weeks post-transplant (Fig. 2A). Engrafted hCNS-SCns migrated along the spinal cord and appeared to differentiate in a site-specific manner consistent with neuronal and oligodendroglial morphologies in the grey and white matter, respectively (Fig. 2B). Confocal analysis of hCNS-SCns differentiation was not repeated in the present study but was performed in our previous publication [1], where we reported evidence for β-tubulin or APC/CC1 expression in 26.4% and 64.1% of engrafted hCNS-SCns, respectively. hFb also engrafted in 100% of the animals and survived 17 weeks post-transplant (Fig. 2C). In contrast to hCNS-SCns, engrafted hFb were highly localized to the region corresponding to the site of transplantation and exhibited limited migration (Fig. 2C and D).

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