Umbilical cord blood stem cell mediated downregulation of fas improves functional recovery of rats after spinal cord injury

Abstract

Human umbilical cord blood stem cells (hUCB), due to their primitive nature and ability to develop into nonhematopoietic cells of various tissue lineages, represent a potentially useful source for cell-based therapies after spinal cord injury (SCI). To evaluate their therapeutic potential, hUCB were stereotactically transplanted into the injury epicenter, one week after SCI in rats. Our results show the presence of a substantial number of surviving hUCB in the injured spinal cord up to five weeks after transplantation. Three weeks after SCI, apoptotic cells were found especially in the dorsal white matter and gray matter, which are positive for both neuron and oligodendrocyte markers. Expression of Fas on both neurons and oligodendrocytes was efficiently downregulated by hUCB. This ultimately resulted in downregulation of caspase-3 extrinsic pathway proteins involving increased expression of FLIP, XIAP and inhibition of PARP cleavage. In hUCB-treated rats, the PI3K/Akt pathway was also involved in antiapoptotic actions. Further, structural integrity of the cytoskeletal proteins alpha-tubulin, MAP2A&2B and NF-200 has been preserved in hUCB treatments. The behavioral scores of hind limbs of hUCB-treated rats improved significantly than those of the injured group, showing functional recovery. Taken together, our results indicate that hUCB-mediated downregulation of Fas and caspases leads to functional recovery of hind limbs of rats after SCI.

Figures

Fig. 1. Trans-differentiation of hUCB into neural phenotypes

(A) Photomicrographs of hUCB in mixed culture demonstrating neural proteins expressed by the cells before transplantation. FITC-conjugated- Nestin and NF-200 (for neurons) with Texas-red conjugated- CD44 (for hUCB); Texas-red conjugated –CNPase and O1 (for oligodendrocytes) with FITC-conjugated- CD44 (for hUCB). Cells displaying neuron like morphology with long axonal projections, CNPase-immunoreactive cells displaying morphology characteristic of oligodendrocytes, with flat cell body and short or long branched projections. (B) Confocal immunohistochemistry on longitudinal sections of the injured spinal cords of rats 5 weeks after transplantation is shown. Immunofluorescence analysis of cryo-sections indicates co-localization (yellow) of FITC-conjugated –βIII tubulin and NF-200 (for neurons) with Texas-red conjugated- CD44 (for hUCB); FITC conjugated-CD44 (for hUCB) with Texas-red conjugated -APC and -MBP (for oligodendrocytes) as shown by (↑). The results are from 3 independent sections between 1 and 2mm from the injury epicenter from treated rats (n =5). Bar = 100 μm. (C) Phase-contrast image of undifferentiated hUCB. (D) Phase-contrast image of differentiating hUCB after 4 days showing mixed population of differentiated neural phenotypes. (E) Western blot showing neural proteins in differentiated population. Equal amounts of protein (40 μg) were loaded onto 10%-14% gels and transferred onto nylon membranes, which were then probed with respective antibodies. The blots were stripped and reprobed with GAPDH to assess protein levels. (F) Quantitative estimation of neural proteins in Fig. F. (U-hUCB = Undifferentiated hUCB; D- hUCB = Differentiated hUCB; Positive control = Whole brain extract of rat). Results are from 3 parallel experiments from 3 separate cord blood preparations. A subpopulation of hUCB-derived cells growing in a monolayer before differentiation was found to be negative for all investigated antigens. Merged figures include colocalized markers, shown by (↑). For panels A, B and C, Bar = 100 μm. For panel D, Bar = 200 μm. (Error bars indicate SEM. * Significant at p <0.05).

Fig. 2. Expression of apoptotic and TUNEL cells in spinal cord sections of rats

(A) Cryo-sections from injured rats after 3 weeks stained with Hoechst 33343 shows merged image showing TUNEL cells on apoptotic cells (↑). Bar = 100 μm. (B) Quantitative estimation of apoptotic and TUNEL cells. (Error bars indicate SEM. * Significant at p <0.05). (C) Quantitative estimation of TUNEL-positive cells in the tissue sections up to 2 mm rostral and caudal to the injury epicenter. (Error bars indicate SEM. * Significant at p <0.05). Results are from 3 independent sections between 1 and 2 mm from the injury epicenter (n ≥ 3).

Fig. 3. Fas immunoreactivity on TUNEL positive cells

(A) Expression of Fas (Texas-red conjugated) on TUNEL positive cells (green) from injured sections. (B) Quantitative estimation of TUNEL, Fas and TUNEL + Fas cells in injured and treated groups. (C) Cryo-sections showing Co-localization of Fas and NF-200 (specific for neurons) and (E) Fas and APC (mature marker for oligodendrocytes) established the expression of Fas on neurons and oligodendrocytes (↑) undergoing apoptosis. Fas is FITC-conjugated and NF-200 and APC are Texas-red conjugated. Note a significant decrease in the expression of Fas in hUCB-treated sections. For panels A, C and E Bar = 100 μm. (D) Quantification of NF-200 + Fas cells and (F) APC + Fas cells. (Error bars indicate SEM. * Significant at p <0.05). A total of more than 100 cells were counted per each field and more than five fields were chosen at random on each section. Results are from three sections between 1 and 2 mm caudal to the injury epicenter after 3 weeks SCI (n ≥ 3).

Fig. 4. Immunoblot analysis of Fas and other death ligands in spinal cord sections

Equal amounts of protein (40 μg) were loaded onto 10%-14% gels and transferred onto nylon membranes, which were then probed with respective antibodies. The blots were stripped and reprobed with GAPDH to assess protein levels. A shows the FasL, Fas and FADD proteins with respect to GAPDH; B shows their quantitative estimations. Inhibition of the apoptotic pathway by hUCB shows downregulation of FasL, Fas and FADD. Each blot is representative of experiments performed in duplicate with each sample (n ≥ 3). Error bars indicate SEM. * Significant at p <0.05.

Fig. 5. Activity of caspases after spinal cord injury

Equal amounts of protein (40 μg) were loaded onto 10%-14% gels and transferred onto nylon membranes, which were then probed with respective antibodies. The blots were stripped and reprobed with GAPDH to assess protein levels. (A) shows the caspase-3 mediated apoptotic pathway proteins with respect to GAPDH; (B) their quantitative estimation. Antisera for caspase-8 and caspase-3 recognize cleaved fragments also. Each blot is representative of experiments performed in duplicate with each sample (n ≥ 3). Error bars indicate SEM. * Significant at p <0.05. (C) Caspase-3 enzyme activity was assayed in tissue lysates of spinal cord. Equal amounts of protein (50 μg) were assayed for caspase-3 activity for 30 min in an ELISA plate reader, measuring absorption at 405 nm. Purified caspase-3 enzyme was used as a positive control. The injured tissues show maximum caspase-3 activity when compared to the treated tissues, which show activity similar to that of the control groups. Inh. = Ac-DEVD-CHO inhibitor. (Error bars indicate SEM. * Significant at p <0.05). The experiment is repeated twice with triplicates (n ≥ 3).

Fig. 6. Immunofluorescence analysis of caspase-3 expression in neurons and oligodendrocytes

Confocal images of cryo-sections illustrate co-localization (yellow) of activated caspase-3 (FITC-conjugated) with (A) NF-200 (Texas-red conjugated) and (B) APC (Texas-red conjugated) within the dorsal region(↑), following spinal cord contusion. The tissue sections represent regions between 1 and 2 mm caudal from the lesion epicenter after 3 weeks SCI. Bar = 100 μm. (C) Quantitation of apoptotic neurons and oligodendrocytes in tissue sections. A large number of caspase-3 positive cells are present in the injured sections, whereas the hUCB-treated sections exhibit a decreasing trend of caspase-3 positive cells. Values represent mean ± SEM values of at least 3 sections with duplicates between 1 and 2 mm from the injury epicenter. * Significant at p <0.05, (n ≥ 4).

Fig. 7. Immunoblot analysis of activated inhibitors of apoptosis in spinal cord sections

Equal amounts of protein (40 μg) were loaded onto 10%-14% gels and transferred onto nylon membranes, which were then probed with respective antibodies. The blots were stripped and reprobed with GAPDH to assess protein levels. A and C shows the inhibitors of apoptosis present in cytosol and nucleus whereas E shows the survival signaling pathway proteins with respect to GAPDH; B, D and F are their quantitative estimations, respectively. Upregulation of inhibitory proteins FLIP, XIAP, and inhibition of PARP cleavage is clearly seen hUCB treatments. Each blot is representative of experiments performed in duplicate with each sample (n ≥ 3). Error bars indicate SEM. * Significant at p <0.05.

Fig. 8. Structural integrity of the injured spinal cord maintained by hUCB after intraspinal grafting

Immunofluorescence studies of spinal cord cryo-sections showing dorsal white matter with cytoskeletal markers viz., α-tubulin (A), MAP2A & 2B (B) and NF200 (C) were performed. α-tubulin, MAP2A & 2B are Texas-red conjugated and their respective CD44 are FITC-conjugated. NF-200 is FITC-conjugated and CD44 are Texas-red conjugated. Inset shows corresponding DAPI images. Bar = 2000μm. (D) Quantitative estimation of the cross-sectional area stained. Calculation of % staining was done on only the lineage specific markers, (but not on the merged image) so that exact comparison with left panel images can be done. When compared to the injured sections, hUCB-treated sections have a high level of structural integrity of the spinal cord. Values represent mean ± SEM values of at least three sections with duplicates from each condition. * Significant at p <0.05. Results are from 3 independent sections between 1 and 2mm from the injury epicenter after 3 weeks SCI (n ≥ 3).

Fig. 9. Functional recovery of rats after hUCB-transplantation

(A) BBB scores of rats with SCI before and after hUCB transplantation. Repeated-measures of ANOVA followed by Bonferroni’s post hoc tests showed that BBB scores in hUCB-grafted animals were significantly higher than those in injured-untreated animals. Each point represents the highest locomotor score achieved each day. (B) Narrow beam scores of injured and treated rats over a period of 6 weeks and (C) Number of placing responses of injured and treated animals. Arrow (↓) indicates transplantation point. Error bars indicate ±SEM (n≥5 per group) (*p<0.01 and **p<0.05).