Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
, 4 (6), e5871

Analysis of Host-Mediated Repair Mechanisms After Human CNS-stem Cell Transplantation for Spinal Cord Injury: Correlation of Engraftment With Recovery

Affiliations

Analysis of Host-Mediated Repair Mechanisms After Human CNS-stem Cell Transplantation for Spinal Cord Injury: Correlation of Engraftment With Recovery

Mitra J Hooshmand et al. PLoS One.

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.

Conflict of interest statement

Competing Interests: MJH, CJS, and BJC declare no competing interests exist. AJA serves as a paid consultant to StemCells, Inc. NU and ST have financial interest in StemCells, Inc.

Figures

Figure 1
Figure 1. Schematic of spinal cord illustrates regions of stereological quantification for human cell numbers.
Illustration depicts the five regions (lesion epicenter, spared tissue, 1 mm rostral and caudal, 2 mm rostral and caudal, 3 mm rostral and caudal) in which stereological quantification of hCNS-SCns and hFb counts were performed. Sites of cell transplantation in the two regions rostral and caudal to the lesion epicenter are also shown.
Figure 2
Figure 2. 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.
Figure 3
Figure 3. hCNS-SCns migrate rostral and caudal to the injury; hFb migrate to a lesser extent.
A: Estimated number of hCNS-SCns at specific distances from the injury epicenter and along the spinal cord (shown in Fig. 1) was quantified. hCNS-SCns migrated at least 3 mm rostral and 3 mm caudal to the site of injury, but the majority of hCNS-SCns (average of 77,180±7,423) were found at regions 1 mm rostral and caudal to the lesion epicenter. B: Estimated number of hFb was quantified in the same manner as hCNS-SCns. While hFb were also found to migrate 3 mm rostral and 3 mm cadudal to the lesion site, less hFb were found away from the injury epicenter compared to hCNS-SCns. The majority of hFb were also found in regions 1 mm rostral and caudal to the injury site (average of 2,196±548) corresponding the original site of transplant. Bars represent group means±standard errors.
Figure 4
Figure 4. 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.
Figure 5
Figure 5. Diphtheria toxin (DT) administration at 16 weeks post-transplant ablates hFB.
A: Immunostaining using SC121 illustrated limited cell engraftment/survival (inset) in animals that received hFb at 9dpi and saline injection at 16 weeks post-transplant. B: High power image of inset in (A) revealed the presence of hFb near the site of transplantation. C: Animals receiving hFb at 9dpi and DT at 16 weeks post-transplant demonstrated a reduction in SC121 immunostaining. D: High power image of (C) demonstrated the absence of SC121 immunoreactivity in animals receiving DT at 16 weeks post-transplant. E: Quantification for the estimated number of hFb in animals receiving saline at 16 weeks post-transplant revealed limited survival. Dashed line indicates the original transplanted dose of 50,000 hFb. DT administration resulted in a significant reduction in the number of hFb (97.8%) suggesting that DT ablated the transplanted human cells. * denotes p<0.0007, 1-tailed t-test. Means±standard errors are shown. Scale bars = 250 µm for A and C and 25 µm for B and D.
Figure 6
Figure 6. Human cell transplantation does not alter the volumes of lesion epicenter or spared tisse.
A: The lesion epicenter was identified as the region devoid of GFAP immunostaining. Regions 1 mm rostral and 1 mm caudal to the border of the lesion were selected for assessment of spared tissue. B: Volume assessments were performed using the Cavalieri estimator probe of StereoInvestigator and revealed no significant differences in the estimated lesion volume between any of the groups (One-way ANOVA: F = 0.51, p = 0.60). C: No significant differences were found in the estimated volume of spared tissue between any of the groups (One-way ANOVA: F = 0.20, p = 0.82). Scale bar = 250 µm.
Figure 7
Figure 7. Human cell transplantation does not alter raphespinal sprouting/regeneration.
A: A contour was drawn around a 500 µm long region at the caudal edge of the lesion epicenter and 5-HT fiber length in this area was quantified using the Isotropic Virtual Planes probe of StereoInvestigator. B: Stereological analysis revealed no significant differences in the estimated raphespinal fiber length between any of the groups (One-way ANOVA: F = 1.67, p = 0.20). Scale bar = 250 µm.
Figure 8
Figure 8. 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.
Figure 9
Figure 9. Human cell transplantation does not alter angiogenesis.
A: Estimated length of blood vessels at the injury epicenter and at regions 1 mm rostral and 1 mm caudal to the lesion site were quantified using the Space Balls probe of StereoInvestigator. B: The injury epicenter was identified using DAPI immunofluorescent staining within the PE-CAM1-stained sections. C: While there was a trend toward an increase in blood vessel length at regions 1 mm rostral and caudal to the lesion, this difference was not statistically significant (One-way ANOVA: F = 1.87, p = 0.17). D: No significant differences in the blood vessel length were detected between the groups at the injury epicenter (One-way ANOVA: F = 0.65, p = 0.53). Scale Bar = 250 µm for A and B.
Figure 10
Figure 10. 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).
Figure 11
Figure 11. hCNS-SCns engraftment directly correlates with a quantitative measure of behavior, but not other measures of histological recovery.
A: Linear regression analysis revealed a significant negative correlation between hCNS-SCns engraftment and the number of errors made on the horizontal ladderbeam task (Pearson r = −0.78, p = 0.038, 2-tailed t-test). B-D: Linear regression analyses for the estimated number of hCNS-SCns and other measures of host recovery revealed no significant correlations between cell engraftment and lesion volume (B) (Pearson r = −0.70, p = 0.08, 2-tailed t-test), volume of spared tissue (B) (Pearson r = 0.42, p = 0.34, 2-tailed t-test), serotonergic fiber sprouting (C) (Pearson r = 0.16, p = 0.77, 2-tailed t-test), NG2 area (D) (Pearson r = 0.16, p = 0.77, 2-tailed t-test), and area of the GFAP astroglial scar (D) (Pearson r = −0.69, p = 0.08, 2-tailed t-test). * denotes p<0.05.
Figure 12
Figure 12. hFb engraftment does not correlate with behavioral or histological measures of recovery.
A: In contrast to hCNS-SCns, linear regression analysis revealed a positive, but non-significant correlation between hFb engraftment and the number of errors made on the horizontal ladderbeam task (Pearson r = 0.49, p = 0.26, 2-tailed t-test). B–D: Linear regression analyses for the estimated number of hFb and other measures of host recovery revealed no significant correlations between cell engraftment and lesion volume (B) (Pearson r: r = 0.59, p = 0.16, 2-tailed t-test), volume of spared tissue (B) (Pearson r: r = 0.55, p = 0.21, 2-tailed t-test), serotonergic fiber sprouting (C) (Pearson r: r = −0.53, p = 0.22, 2-tailed t-test), NG2 area (D) (Pearson r: r = 0.01, p = 0.98, 2-tailed t-test), and area of the GFAP astroglial scar (D) (Pearson r: r = 0.22, p = 0.63, 2-tailed t-test).

Similar articles

See all similar articles

Cited by 48 PubMed Central articles

See all "Cited by" articles

References

    1. Cummings BJ, Uchida N, Tamaki SJ, Salazar DL, Hooshmand M, et al. Human neural stem cells differentiate and promote locomotor recovery in spinal cord-injured mice. Proc Natl Acad Sci U S A. 2005;102:14069–14074. - PMC - PubMed
    1. Keirstead HS, Nistor G, Bernal G, Totoiu M, Cloutier F, et al. Human embryonic stem cell-derived oligodendrocyte progenitor cell transplants remyelinate and restore locomotion after spinal cord injury. J Neurosci. 2005;25:4694–4705. - PMC - PubMed
    1. Karimi-Abdolrezaee S, Eftekharpour E, Wang J, Morshead CM, Fehlings MG. Delayed transplantation of adult neural precursor cells promotes remyelination and functional neurological recovery after spinal cord injury. J Neurosci. 2006;26:3377–3389. - PMC - PubMed
    1. McDonald JW, Liu XZ, Qu Y, Liu S, Mickey SK, et al. Transplanted embryonic stem cells survive, differentiate and promote recovery in injured rat spinal cord. Nat Med. 1999;5:1410–1412. - PubMed
    1. Tarasenko YI, Gao J, Nie L, Johnson KM, Grady JJ, et al. Human fetal neural stem cells grafted into contusion-injured rat spinal cords improve behavior. J Neurosci Res. 2007;85:47–57. - PubMed

Publication types

Substances

Feedback