Repeated mesenchymal stem cell treatment after neonatal hypoxia-ischemia has distinct effects on formation and maturation of new neurons and oligodendrocytes leading to restoration of damage, corticospinal motor tract activity, and sensorimotor function

Abstract

Birth asphyxia is a frequent cause of perinatal morbidity and mortality with limited therapeutic options. We show that a single mesenchymal stem cell treatment at 3 d (MSC-3) after neonatal hypoxia-ischemia (HI) in postnatal day 9 mice improved sensorimotor function and reduced lesion size. A second MSC treatment at 10 d after HI (MSC-3+10) further enhanced sensorimotor improvement and recovery of MAP2 and MBP (myelin basic protein) staining. Ipsilateral anterograde corticospinal tract tracing with biotinylated dextran amine (BDA) showed that HI reduced BDA labeling of the contralateral spinal cord. Only MSC-3+10 treatment partially restored contralateral spinal cord BDA staining, indicating enhanced axonal remodeling. MSC-3 enhanced formation of bromodeoxyuridine-positive neurons and oligodendrocytes. Interestingly, the second gift at day 10 did not further increase new cell formation, whereas only MSC-10 did. These findings indicate that increased positive effect of MSC-3+10 compared with MSC-3 alone is mediated via distinct pathways. We hypothesize that MSCs adapt their growth and differentiation factor production to the needs of the environment at the time of intracranial injection. Comparing the response of MSCs to in vitro culture with HI brain extracts obtained at day 10 from MSC-3- or vehicle-treated animals by pathway-focused PCR array analysis revealed that 29 genes encoding secreted factors were indeed differentially regulated. We propose that the function of MSCs is dictated by adaptive specific signals provided by the damaged and regenerating brain.

Figure 1.

Experimental protocol.

Figure 2.

Effect of MSC treatment at 3 and 10 d after HI on functional outcome. Mice received MSCs or VEH at 3 and 10 d after HI. A, Paw preference in the cylinder rearing test was determined at 10, 21, and 28 d after the insult. B, Performance on the rotarod at 21 and 28 d after HI. Data represent mean ± SEM. VEH, n = 12; MSC-3 and MSC-3+10, n = 14. ^δp < 0.01 versus VEH at 10 d; ^§p < 0.01 versus MSC-3 at 10 d; *p < 0.01 versus VEH at same time point; ^#p < 0.01 versus MSC-3 at same time point.

Figure 3.

Cell proliferation after MSC treatment. To label proliferating cells after the first injection of MSCs at day 3 after HI, BrdU was injected daily from 3 to 5 d after HI. A, B, The number of BrdU⁺ cells in ipsilateral hippocampus (A) and cortex (B) were counted. Cell proliferation after the second injection of MSCs at day 10 was analyzed after daily EdU injection from 10 to 12 d after HI. C, D, EdU⁺ cells were counted in the hippocampus (C) and cortex (D). Data represent mean number of positive cells ± SEM. Sham controls, n = 8; VEH, n = 12; MSC-3 and MSC-3+10, n = 14. *p < 0.05. E and F display representative examples of BrdU (E) and Edu (F) staining in cortex at 28 d after HI of VEH, MSC-3, or MSC-3+10 treatment animals. Scale bars, 50 μm.

Figure 4.

Differentiation of recently divided cells after MSC treatment. A–D, BrdU⁺ cells were analyzed for coexpression of the neuronal marker NeuN (A, B) or the oligodendrocyte precursor marker Olig2 (C, D) in hippocampus (A, B) and cortex (C, D). Data represent mean percentage of double positive cells ± SEM. VEH, n = 12; MSC-3 and MSC-3+10, n = 14. *p < 0.05. E, F, Representative examples of BrdU-, NeuN- (E), or Olig2- (F), and double-positive cells at day 28 after MSC-3 treatment. Scale bars, 50 μm.

Figure 5.

A, B, Effect of treatment with MSCs on lesion size. Quantification of MAP2⁺ (A) and MBP⁺ (B) area loss expressed as ratio ipsilateral/contralateral area and representative examples of MAP2 or MBP staining at day 28 after HI. Data represent mean percentage area loss ± SEM. VEH, n = 12; MSC-3 and MSC-3+10, n = 14. *p < 0.05.

Figure 6.

Corticospinal tract remodeling after treatment with MSCs. Mice received VEH, MSC-3, or MSC-3+10 treatment and at 21 d after HI were injected with 10k biotinylated dextran amine in the ipsilateral motor cortex. A, Analysis of MAP2 staining of the motor cortex at 28 d after HI did not reveal any signs of damage. B displays representative examples of BDA staining in the dorsal funiculus of the spinal cord. Scale bar, 20 μm. C, Relative BDA intensity in the dorsal funiculus of the contralateral cervical spinal cord. Sham controls, n = 7; VEH, n = 7; MSC-3, n = 10; MSC-10, n = 11; MSC-3+10, n = 10. *p < 0.05. D, Relationship between BDA intensity in the dorsal funiculus of the contralateral cervical spinal cord and performance in the cylinder rearing test at 28 d after HI. VEH, n = 7; MSC-3, n = 10; MSC-10, n = 11; MSC-3+10, n = 10. r = −0.75; *p < 0.001. E, F, Quantification of ipsilateral GAP43⁺ (E) and synaptophysin⁺ (F) area expressed as ratio of ipsilateral/contralateral area and representative examples of GAP43 and synaptophysin staining at day 28 after HI. Data represent mean ipsilateral area ± SEM. Sham controls, n = 7; VEH, n = 12; MSC-3, n = 14; MSC-10, n = 11; MSC-3+10, n = 13. *p < 0.05.

Figure 7.

Changes in mRNA levels and protein of selected secreted factors in MSCs cultured with ischemic brain extracts. MSCs were cultured for 72 h with brain extracts obtained from HI mice treated with MSCs at 3 d after HI or with brain extracts from HI mice treated with vehicle (n = 4 per group). A, B, To validate PCR array results, four strongly upregulated (A) and three strongly downregulated (B) genes were quantified by real-time RT-PCR on single samples. C–E, The level of IL-10 (C), IL-1β (D), and IL-6 (E) protein were determined in the culture supernatant. Data represent mean ± SEM. *p < 0.05; **p < 0.001; ***p < 0.0001.