Mesenchymal lineage stem cells have pronounced anti-inflammatory effects in the twitcher mouse model of Krabbe's disease

Abstract

The twitcher mouse is an animal model of Krabbe's disease (KD), which is a neurodegenerative lysosomal storage disorder resulting from the absence of functional lysosomal enzyme galactocerebrosidase (GALC). This disease affects the central and peripheral nervous systems and in its most severe form results in death before the age of 2 in humans and approximately 30-40 days in mice. This study evaluates the effect of intracerebroventricular administration of mesenchymal stem cells derived from adipose tissue (ASCs) and bone marrow (BMSCs) on the pathology of KD. Subsequent to the intracerebroventricular injection of ASCs or BMSCs on postnatal day (PND) 3-4, body weight, lifespan, and neuromotor function were evaluated longitudinally beginning on PND15. At sacrifice, tissues were harvested for analysis of GALC activity, presence of myelin, infiltration of macrophages, microglial activation, inflammatory markers, and cellular persistence. Survival analysis curves indicate a statistically significant increase in lifespan in stem cell-treated twitcher mice as compared with control twitcher mice. Body weight and motor function were also improved compared with controls. The stem cells may mediate some of these benefits through an anti-inflammatory mechanism because the expression of numerous proinflammatory markers was downregulated at both transcriptional and translational levels. A marked decrease in the levels of macrophage infiltration and microglial activation was also noted. These data indicate that mesenchymal lineage stem cells are potent inhibitors of inflammation associated with KD progression and offer potential benefits as a component of a combination approach for in vivo treatment by reducing the levels of inflammation.

Figure 1. Survival and Body Weight

A. Kaplan-Meier survival curve illustrating the lifespan of twitcher mice. Cumulative survival was 36% for eGFPTgASC injected, 45% for eGFPTgBMSC injected and 0% for HBSS injected twitchers at PND40, respectively (p<0.05). B. Body weight for twitchers was measured beginning on PND15. Body weight for cell injected twitchers was significantly greater than controls (p<0.001).

Figure 2. Motor Function

A. Comparative analysis of twitching frequency for eGFPTgBMSCs compared to HBSS injected twitchers (p<0.0001). B. Twitching severity curves for both eGFPTgASC and eGFPTgBMSC injected mice are significantly different from HBSS injected controls (p<0.01). C. Mice were suspended by the tail and lowered onto a horizontal wire and then released. The wire hang maneuver curves for cell injected twitchers are significantly different from HBSS injected controls (p<0.01). D. Gait analysis was performed by averaging the hind stride length of both back paws. Cell injected twitchers had a longer hind stride length over time compared to HBSS injected controls (p<0.0001).

Figure 3

A. eGFP⁺ Cell Persistence. eGFP+ cells were tracked in injected mouse brains using real-time PCR at pre-determined time points. 1 day post injection, there was a 59% recovery of eGFPTgASCs in wild-type mouse brains and a 24% recovery in twitcher mice (p>0.05). Also, there was a 57% recovery of eGFPTgBMSCs in wild-type mice and 56% recovery in twitcher mice at this time point (p>0.05). 10 days post injection, there was a 17% recovery of the injected eGFPTgASCs in wild-type brains and a 12% recovery in twitcher mice (p>0.05). Also, there was a 35% recovery of eGFPTgBMSCs in wild-type mice and 16% recovery in twitcher mice at this time point (p>0.05). B. Lack of Transdifferentiation. eGFPTgASCs (top) and eGFPTgBMSCs (bottom) were located in cryosections of injected brains 10 days post injection and immunostained with NeuN (red) and S100 (blue) to investigate cellular differentiation along neural or glial lineages, respectively (right). No co-localization of either antibody was found with the eGFP⁺. Nuclei were stained with To-Pro-3 (blue) (left).

Figure 4

A. Western Blotting for Myelin. Protein lysates derived from twitcher brains at euthanization or wild-type mice at PND40 reveal that expression levels of myelin in normal mice is substantially greater than that of twitcher mice. The levels of myelin detected in the brains of either eGFPTgASC or eGFPTgBMSC injected mice were not increased in comparison to HBSS injected twitcher mice. B. Immunohistochemistry. Results from immunohistochemistry (IHC) on cryosections derived from twitcher brains at euthanization or wild-type mice at PND40 stained with anti-MBP (green). IHC results also suggest that the expression of myelin is not increased in stem cell injected twitcher mice compared to HBSS injected twitcher mice.

Figure 5

A. Analysis of Inflammatory Cytokines/Chemokines by Real Time RT-PCR. Total cellular RNA samples derived from twitcher brains at euthanization or wild-type mice at PND40 reveal a marked elevation in expression of inflammatory markers is present in the twitcher mouse brain at this time point. The injection with either eGFPTgASCs or eGFPTgBMSCs markedly down-regulates the expression of several inflammatory mediators. Administration of eGFPTgASCs particularly reduced expression of IL-10 and the eGFPTgBMSCs significantly reduced expression of TNFα (p<0.05). All samples were normalized to β-actin content. B. Analysis of Inflammatory Cytokines/Chemokines by Multiplex Analysis. Protein lysates derived from twitcher brains at euthanization or wild-type mice at PND40 were analyzed on a 32-plex panel multiplex plate, which confirmed that inflammatory mediators are up-regulated in the twitcher brain at the protein level. The administration of both eGFPTgASCs and eGFPTgBMSCs reduced expression of G-CSF, IL-1α, MCP-1, and LIF in a statistically significant manner (p<0.01).

Figure 6. iNOS Expression

Protein lysates derived from twitcher brains at euthanization or wild-type mice at PND40 were analyzed by Western blot and illustrate inducible nitric oxide synthase (iNOS) is substantially up-regulated in the twitcher mouse compared to wild-type mice. Injection of eGFPTgASCs and eGFPTgBMSCs substantially decreased expression of iNOS, even 4–5 weeks after administration.

Figure 7

A. Macrophage Infiltration. Protein lysates derived from twitcher brains at euthanization or wild-type mice at PND40 were analyzed by Western blot using anti-CD163 and revealed increased macrophage infiltration in the twitcher brain. Injection of eGFPTgASCs and eGFPTgBMSCs substantially decreased expression of CD163, even 4–5 weeks after administration. B. Microglial Activation. Protein lysates derived as previously mentioned were analyzed by Western blot using anti-Iba-1 for activated microglia and illustrate the increased presence of activated microglia in the twitcher brain compared to wild-type brains. These results suggest a small decrease in the numbers of activated microglia with eGFPTgASC injection and a much larger decrease with injection of eGFPTgBMSCs in the twitcher brain in comparison to HBSS injected twitchers. C. Immunohistochemistry for Iba-1. Results from IHC on cryosections derived from twitcher brains at euthanization or wild-type mice at PND40 stained with anti-Iba-1(green) support Western blotting results.