Xenograft of Human Umbilical Mesenchymal Stem Cells Promotes Recovery from Chronic Ischemic Stroke in Rats

Abstract

Stroke is a leading cause of adult disability. In our previous study, transplantation of human umbilical mesenchymal stem cells (HUMSCs) in Wharton's jelly in the acute phase of ischemic stroke promotes recovery in rats. Unfortunately, there is no cure for chronic stroke. Patients with chronic stroke can only be treated with rehabilitation or supportive interventions. This study aimed to investigate the potential of xenograft of HUMSCs for treating chronic stroke in rats. Rats were subjected to 90 min middle cerebral artery occlusion and then reperfusion to mimic ischemic cerebral stroke. On day 14 following stroke, HUMSCs were transplanted into the damaged cerebral cortex. The motor function in rats of the Stroke + HUMSCs group exhibited significant improvement compared to that of the Stroke + Saline group, and the trend persisted until day 56 post stroke. The cerebral cortex changes were tracked using magnetic resonance imaging, showing that cerebral atrophy was found starting on day 7 and was reduced significantly in rats receiving HUMSCs compared to that in the Stroke + Saline group from day 21 to day 56. HUMSCs were found to be existed in the rats' cerebral cortex on day 56, with signs of migration. The grafted HUMSCs did not differentiate into neurons or astrocytes and may release cytokines to improve neuroprotection, decrease inflammation and increase angiogenesis. Our results demonstrate that xeno-transplantation of HUMSCs has therapeutic benefits for chronic ischemic stroke. Most importantly, patients do not need to use their own HUMSCs, which is a gospel thing for clinical patients.

Keywords: MRI; Wharton’s jelly; angiogenesis; cerebral ischemia; chronic stroke; neurologic deficit; neuroprotection; transplantation; umbilical mesenchymal stem cells; xenograft.

Figure 1

HUMSCs transplantation enhanced motor function in rats with chronic stroke. (A) The diagram shows the time course for various experiments in this study. Representative MRI, TTC-stained, and cresyl violet stained images of rat brains are shown on day 1 after stroke, showing that right cerebral cortices were markedly enlarged (B). The Stroke + HUMSCs group show better functional recovery in the rotarod test (C) and cylinder test (D) than the Stroke + Saline group. Normal + Saline n = 11, Normal + HUMSCs n = 11, Stroke + Saline n = 15, Stroke + HUMSCs n = 14. ∗, vs. the Normal + Saline group at the same day, p < 0.05. #, vs. the Normal + HUMSCs group at the same day, p < 0.05. ♣, vs. the Stroke + Saline group at the same day, p < 0.05.

Figure 2

Cortical edema and atrophy examined by MRI. Brain MRI examination was conducted on day 1 (➊–➏), day 7 (➊–➏), day 14 (➊–➏), day 21 (➊–➏), day 28 (➊–➏), day 35 (➊–➏), day 42 (➊–➏), day 49 (➊–➏), and day 56 (➊–➏). Along the rostral–caudal orientation, coronal slices (➊–➎) and horizontal slices (➏) were taken for monitoring the alterations of brain damages in these rats. MRI slices of the Normal + Saline (A) and Normal + HUMSCs (B) groups did not find significant edema or atrophy areas from day 1 to day 56. MRI slices of the Stroke + Saline group (C) and the Stroke + HUMSCs group (D) were obtained on day 1 post stroke. White areas in the right cerebral cortex were seen, indicating severe inflammation and edema occurred in this region (➊–➏). On day 7 and day 14, there was a reduction in the white edema cortices, but slight atrophy had appeared in the right infarcted area when comparing to the left cerebral cortex (➊–➏). The volumes of edema (E) and atrophy (F) in the right cerebral cortex were quantified post stroke. Atrophy of the infarcted cortex becomes apparent in the Stroke + Saline group but is significantly less severe in the Stroke + HUMSCs group from day 21 to day 56. The results of brain MRI revealed that HUMSCs transplantation alleviated cerebral atrophy in rats with chronic stroke. Normal + Saline n = 4, Normal + HUMSCs n = 4, Stroke + Saline n = 4, Stroke + HUMSCs n = 4. ∗, vs. the Normal + Saline group at the same day, p < 0.05. #, vs. the Normal + HUMSCs group at the same day, p < 0.05. ♣, vs. the Stroke + Saline group at the same day, p < 0.05. indicates the first injection site.

Figure 3

HUMSCs transplantation preserved the cerebral cortex in rats with chronic stroke. On day 56 post stroke, the brains fixed with paraformaldehyde were photographed from a superior (top panel) and a lateral view (bottom panel). The brain of the Stroke + Saline group exhibited more severe atrophy in the cortex than that grafted with HUMSCs (A). Subsequently, the brains of four groups were subjected to serial coronal sections (from bregma +2.52 mm to −5.76 mm) and stained with cresyl violet to observe alterations in the cerebral cortex. The results showed that the brain grafted with HUMSCs had more preserved cortices (B). The volume of atrophy in the infarcted cortex was quantified using the sections stained by cresyl violet, the result indicated the atrophy volume of the Stroke + HUMSCs group was substantially lower than that of the Stroke + Saline group (C). Normal + Saline n = 4, Normal + HUMSCs n = 4, Stroke + Saline n = 12, Stroke + HUMSCs n = 14. ∗, vs. the Normal + Saline group, p < 0.05. #, vs. the Normal + HUMSCs group, p < 0.05. ♣, vs. the Stroke + Saline group, p < 0.05. ■ is the value of individual experiment.

Figure 4

HUMSCs transplantation increased the survival of neuronal cells in the infarcted brain in rats with chronic stroke. On day 56 post stroke, anti-NeuN immunohistochemical staining was applied to observe the number of survived neuronal cells. Brain coronal sections at bregma −1.92 mm are displayed ((A): Normal + Saline group; (B): Normal + HUMSCs group; (C): Stroke + Saline group; (D): Stroke + HUMSCs group). For high magnification, the peripheral region to the infarcted cerebral cortex (A2,B2,C2,D2) or striatum (A4,B4,C4,D4) are shown, as well as undamaged contralateral sides of the cerebral cortex (A1,B1,C1,D1) and striatum (A3,B3,C3,D3). The number of NeuN⁺ cells in the cerebral cortex (E) and striatum (F) was quantified. The results showed that the neuronal cell numbers in the cerebral cortex and striatum peripheral to the damaged brain areas were significantly higher in the Stroke + HUMSCs group than those in the Stroke + Saline group. Normal + Saline n = 4, Normal + HUMSCs n = 4, Stroke + Saline n = 9, Stroke + HUMSCs n = 10. ∗, vs. the ipsilateral region of Normal + Saline group, p < 0.05. #, vs. ipsilateral region of the Normal + HUMSCs group, p < 0.05. ♣, vs. ipsilateral region of the Stroke + Saline group, p < 0.05. ♠, contra-lateral vs. ipsi-lateral region in the Stroke + Saline group, p < 0.05. ■ is the value of individual experiment.

Figure 5

HUMSCs transplantation promoted angiogenesis in the infarcted brain of the rats with chronic stroke. On day 56 post stroke, the brains were photographed from a superior view (top panel) and a lateral view (intermediate panel and high magnification in the bottom panel) to observe the distribution of superficial blood vessels of the cerebral cortex. More blood vessels are observed on the surface of the Stroke + HUMSCs group (A). From coronal brain sections at bregma −2.4 mm, vessels in the contralateral cortex (B1,C1,D1,E1) and ipsilateral cortex (B2,C2,D2,E2) are visualized by fluorescein isothiocyanate-conjugated dextran amine. Boxed areas in the photographs are magnified in the bottom row (B1,B2,C1,C2,D1,D2,E1,E2). The total vessel length per mm² (F) and vessel density (G) in the infarcted region are significantly greater in the cortex of the Stroke + HUMSCs group than those in the Stroke + Saline group. Normal + Saline n = 4, Normal + HUMSCs n = 4, Stroke + Saline n = 4, Stroke + HUMSCs n = 4. ∗, vs. ipsilateral cortex of the Normal + Saline group, p < 0.05. #, vs. ipsilateral cortex of the Normal + HUMSCs group, p < 0.05. ♣, vs. ipsilateral cortex of the Stroke + Saline group, p < 0.05. ♠, contra-lateral vs. ipsi-lateral cortex in the Stroke + Saline group, p < 0.05. ■ is the value of individual experiment.

Figure 6

Grafted HUMSCs survived in the infarcted cortex and did not differentiate into neuronal cells or astrocytes. The nucleus of HUMSCs was labeled with bisBenzimide (blue), and the cells were transplanted into the rat’s right cerebral cortex on day 14 post stroke. On day 56, phase-contrast microscopy images of the HUMSCs group are displayed with the section at the first HUMSCs transplantation position of bregma +0.3 mm and its anterior (+0.6 mm) and posterior coronal sections (+0.1 mm). The blue clusters of cells were observed at high magnification (A). The second HUMSCs transplantation position at bregma −5.2 mm and its anterior (bregma −4.9 mm) and posterior (bregma −5.5 mm) coronal brain sections are shown. At high magnification, clusters of cells labeled with bisBenzimide were found (B). Immunostaining for human-specific nucleus antigen demonstrates the aggregation of HUMSCs around the first (C) and secondary (D) transplantation sites at 56 days post stroke. The boxed areas in the left column are magnified in the right column. On day 56 post stroke, the cerebral cortex was subjected to RT-PCR tests to examine whether human NeuN or GFAP could be detected in all the groups. The results showed that the transplanted HUMSCs did not differentiate into neuronal cells or astrocytes (E).