Contralaterally transplanted human embryonic stem cell-derived neural precursor cells (ENStem-A) migrate and improve brain functions in stroke-damaged rats

Abstract

The transplantation of neural precursor cells (NPCs) is known to be a promising approach to ameliorating behavioral deficits after stroke in a rodent model of middle cerebral artery occlusion (MCAo). Previous studies have shown that transplanted NPCs migrate toward the infarct region, survive and differentiate into mature neurons to some extent. However, the spatiotemporal dynamics of NPC migration following transplantation into stroke animals have yet to be elucidated. In this study, we investigated the fates of human embryonic stem cell (hESC)-derived NPCs (ENStem-A) for 8 weeks following transplantation into the side contralateral to the infarct region using 7.0T animal magnetic resonance imaging (MRI). T2- and T2*-weighted MRI analyses indicated that the migrating cells were clearly detectable at the infarct boundary zone by 1 week, and the intensity of the MRI signals robustly increased within 4 weeks after transplantation. Afterwards, the signals were slightly increased or unchanged. At 8 weeks, we performed Prussian blue staining and immunohistochemical staining using human-specific markers, and found that high percentages of transplanted cells migrated to the infarct boundary. Most of these cells were CXCR4-positive. We also observed that the migrating cells expressed markers for various stages of neural differentiation, including Nestin, Tuj1, NeuN, TH, DARPP-32 and SV38, indicating that the transplanted cells may partially contribute to the reconstruction of the damaged neural tissues after stroke. Interestingly, we found that the extent of gliosis (glial fibrillary acidic protein-positive cells) and apoptosis (TUNEL-positive cells) were significantly decreased in the cell-transplanted group, suggesting that hESC-NPCs have a positive role in reducing glia scar formation and cell death after stroke. No tumors formed in our study. We also performed various behavioral tests, including rotarod, stepping and modified neurological severity score tests, and found that the transplanted animals exhibited significant improvements in sensorimotor functions during the 8 weeks after transplantation. Taken together, these results strongly suggest that hESC-NPCs have the capacity to migrate to the infarct region, form neural tissues efficiently and contribute to behavioral recovery in a rodent model of ischemic stroke.

Figure 1

Characteristics of the human embryonic stem cell-derived neural precursor cells (hESC-NPCs, ENStem-A) used in this study. Immunocytochemical staining showing the expression of neural stem/progenitor makers: (a) Sox2, (b) neural cell adhesion molecule (NCAM), (c) Nestin and (d) Musashi. The nuclei were counterstained with 4′,6′-diamidino-2-phenylindole (DAPI; blue). Scale bar, 20 μm. (e) Reverse transcription-PCR (RT-PCR) analysis showing that ENStem-A cells express high levels of Sox2 and Nestin and low levels of type III-β tubulin (IIIβ-tub). The expression of microtubule-associated protein 2 (Map2) and glial fibrillary acidic protein (GFAP) was not detectable.

Figure 2

Detection and visualization of Feridex-labeled human embryonic stem cell-derived neural precursor cells (hESC-NPCs, ENStem-A) using 7.0T animal magnetic resonance imaging (MRI) and histological methods. (a) Serial T2*-weighted MR images showing the migration and distribution of the contralaterally transplanted cells. MR images were taken from 5 days to 53 days following transplantation. The red arrows indicate the cells migrating toward the ischemic peri-infarct region. (b) Hyperintense region in the T2*-weighted MR image indicating the location of transplanted cells (left) and the corresponding Prussian blue staining, confirming the presence of transplanted Feridex-labeled cells (shown in blue) (right). (c) Confocal analysis showing that human-specific mitochondria (hMito)-positive migrating human cells express the chemokine receptor CXCR4. DAPI (4′,6′-diamidino-2-phenylindole) was used to counterstain the cells. Scale bar, 20 μm.

Figure 3

Immunohistochemical analyses showing the extent of neuronal differentiation 8 weeks following the transplantation of ENStem-A cells into middle cerebral artery occlusion (MCAo)-induced rats. Transplanted human cells were identified using antibodies against human-specific nuclei (hNu) or human-specific mitochondria (hMito), which are shown in red. Transplanted cells were shown to express markers of neural stem/progenitor cells (Nestin; a) and early and general neurons (type III-β tubulin (Tuj1; b), as well as a neuron-specific nuclear protein, NeuN (c). They also expressed markers of dopaminergic neurons (tyrosine hydroxylase (TH; d), and medium spiny neurons (dopamine- and cAMP-regulated neuronal phosphoprotein-32 (DARPP-32; e). Some of the transplanted cells were positive for a synaptic vesicle protein, SVP38 (f). Scale bar, 50 μm.

Figure 4

Behavioral improvement following the transplantation of ENStem-A cells into middle cerebral artery occlusion (MCAo)-induced rats. Scores from the rotarod test (a), stepping test (b) and modified neurological severity score (mNSS) test (c) are shown for the 8-week study period. MCAo was induced at week 0. The data are presented as the mean values±s.e.m. The numbers of animals used in the ENStem-A group and the sham group were 8 and 6, respectively. *P<0.05 and **P<0.001.

Figure 5

Histological analyses on the extents of gliosis and apoptosis following cell transplantation into middle cerebral artery occlusion (MCAo)-induced rats. The transplanted group (ENStem-A) exhibited fewer numbers of glial fibrillary acidic protein (GFAP)-positive and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL)-positive cells in the peri-infarct region 8 weeks after MCAo compared with the sham group (a–f). Scale bar, 20 μm. The data are presented as the mean values±s.e.m. **P<0.001.

Figure 6

A proposed mode of action of the transplanted human embryonic stem cell-derived neural precursor cells (hESC-NPCs, ENStem-A) in a rodent model of ischemic stroke.