Intranasal administration of human MSC for ischemic brain injury in the mouse: in vitro and in vivo neuroregenerative functions

Abstract

Intranasal treatment with C57BL/6 MSCs reduces lesion volume and improves motor and cognitive behavior in the neonatal hypoxic-ischemic (HI) mouse model. In this study, we investigated the potential of human MSCs (hMSCs) to treat HI brain injury in the neonatal mouse. Assessing the regenerative capacity of hMSCs is crucial for translation of our knowledge to the clinic. We determined the neuroregenerative potential of hMSCs in vitro and in vivo by intranasal administration 10 d post-HI in neonatal mice. HI was induced in P9 mouse pups. 1×10(6) or 2×10(6) hMSCs were administered intranasally 10 d post-HI. Motor behavior and lesion volume were measured 28 d post-HI. The in vitro capacity of hMSCs to induce differentiation of mouse neural stem cell (mNSC) was determined using a transwell co-culture differentiation assay. To determine which chemotactic factors may play a role in mediating migration of MSCs to the lesion, we performed a PCR array on 84 chemotactic factors 10 days following sham-operation, and at 10 and 17 days post-HI. Our results show that 2×10(6) hMSCs decrease lesion volume, improve motor behavior, and reduce scar formation and microglia activity. Moreover, we demonstrate that the differentiation assay reflects the neuroregenerative potential of hMSCs in vivo, as hMSCs induce mNSCs to differentiate into neurons in vitro. We also provide evidence that the chemotactic factor CXCL10 may play an important role in hMSC migration to the lesion site. This is suggested by our finding that CXCL10 is significantly upregulated at 10 days following HI, but not at 17 days after HI, a time when MSCs no longer reach the lesion when given intranasally. The results described in this work also tempt us to contemplate hMSCs not only as a potential treatment option for neonatal encephalopathy, but also for a plethora of degenerative and traumatic injuries of the nervous system.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1. In vitro proliferation of hMSCs.

Proliferating capacity of hMSCs in vitro. 1000 hMSCs were plated (T0) and proliferation was assessed at 4(T0), 24(T24), 48(T48) and 96(T96) hours after plating the MSCs by adding 3H-thymidine to the culture and measuring 3H-thymidine uptake 16 hours later. Data represent mean ± SEM. ** p<0.01; *** p<0.001 by ANOVA and Bonferroni post-hoc test. (n = 10 wells for each condition).

Figure 2. hMSCs induce differentiation of mouse NSCs in vitro.

In vitro mNSC transwell differentiation assay in co-culture with hMSCs. mNSCs were fixed at 4(T0) and 96(T96) hours after co-culture with hMSCs and stained for (A) nestin (green), (B) Olig2 (red), (C) GFAP (green) and (D) βIII-Tubulin (red). Data represent mean ± SEM. *** p<0.001 by Unpaired two-tailed T-test. Scale bar  = 100 µm. (n = 4 wells per condition).

Figure 3. PKH-26 labeled hMSCs migrate to the lesion site.

1×106 hMSCs were labeled with PKH-26 and administered intranasally at 10 days after HI. (A+B) Mice were terminated 24 hours after hMSC treatment. (A) PKH-26+ hMSCs in the ipsilateral damaged cortex. (B) Contralateral cortex shows no PKH-26+ signal. (C) qPCR validation of PCR array confirmed the upregulation of five genes 10 days after HI. At 17 days after HI 2 genes were down-regulated in comparison to 10 days following HI. (HI n = 10; sham n = 6). (D) hMSCs express CXCR3, which increases after co-culture with HI brain extract (n = 2). Data represent mean ± S.E.M. *p<0.05; **p<0.01; ***p<0.001 by ANOVA and Bonferroni post-hoc test. Dashed line  =  lesion border; Asterisk  =  lesion site. Blue  =  Dapi staining. Scale bar  = 50 µm.

Figure 4. Dose effect of hMSC on motor performance and lesion volume.

Mice were treated intranasally with either 1×106 or 2×106 hMSCs or vehicle at 10 days after HI. (A) Preference to use the unimpaired forepaw in the cylinder rearing test (CRT) was assessed at 28 days after HI. Sham-operated littermates (Sham) were used as controls. (B–C) Quantification of ipsilateral MAP2 (B) and MBP (C) area loss measured as 1- (ipsi-/contralateral MAP2- or MBP-positive area) at 28 days after HI. Representative sections of MAP2 (D) and MBP (E) staining. Data represent mean ± SEM. **p<0.01; ***p<0.001 by ANOVA and Bonferroni post-hoc test. Sham n = 13; Vehicle n = 21; 1×106 hMSC n = 11; 2×106 hMSC n = 12. Data presented in this figure are results from pups pooled out of 11 different litters. Treatment groups were randomly distributed between litters.

Figure 5. hMSCs reduce the activation of glial cells at 28 days after HI.

Mice were treated with either 1×106 or 2×106 hMSCs or vehicle intranasally at 10 days following HI. Mice were sacrificed 28 days after HI. (A) Schematic overview of fields quantified. (B) Quantification of Iba-1+ signal/mm2 or (C) GFAP+ signal/mm2. (D–G) Representative sections of Iba-1 (red) and GFAP (green) expression after sham-operation (D), vehicle (E), 1×106 hMSCs (F) or 2×106 hMSCs (G). Sections are counterstained with DAPI (blue). Scale bar  = 100 µm. Data represent mean ± SEM. * p<0.05; **p<0.01; ***p<0.001 by ANOVA and Bonferroni post-hoc test (Sham and Vehicle n = 4; 1×106 and 2×106 MSC n = 3).