Therapeutic Effect of BDNF-Overexpressing Human Neural Stem Cells (F3.BDNF) in a Contusion Model of Spinal Cord Injury in Rats.

The most common type of spinal cord injury is the contusion of the spinal cord, which causes progressive secondary tissue degeneration. In this study, we applied genetically modified human neural stem cells overexpressing BDNF (brain-derived neurotrophic factor) (F3.BDNF) to determine whether they can promote functional recovery in the spinal cord injury (SCI) model in rats. We transplanted F3.BDNF cells via intrathecal catheter delivery after a contusion of the thoracic spinal cord and found that they were migrated toward the injured spinal cord area by MR imaging. Transplanted F3.BDNF cells expressed neural lineage markers, such as NeuN, MBP, and GFAP and were functionally connected to the host neurons. The F3.BDNF-transplanted rats exhibited significantly improved locomotor functions compared with the sham group. This functional recovery was accompanied by an increased volume of spared myelination and decreased area of cystic cavity in the F3.BDNF group. We also observed that the F3.BDNF-transplanted rats showed reduced numbers of Iba1- and iNOS-positive inflammatory cells as well as GFAP-positive astrocytes. These results strongly suggest the transplantation of F3.BDNF cells can modulate inflammatory cells and glia activation and also improve the hyperalgesia following SCI.

The combination of mannitol and temozolomide increases the effectiveness of stem cell treatment in a chronic stroke model.

BACKGROUND: The blood-brain barrier (BBB) presents a significant challenge to the therapeutic efficacy of stem cells in chronic stroke. Various methods have been developed to increase BBB permeability, but these are associated with adverse effects and are, therefore, not clinically applicable. We recently identified that combination drug treatment of mannitol and temozolomide improved BBB permeability in vitro. Here, we investigated whether this combination could increase the effectiveness of stem cell treatment in an animal model of chronic ischemic stroke. METHODS: Chronic stroke was induced in rats by middle cerebral artery occlusion (MCAo). After then, rats were administered human umbilical cord-derived mesenchymal stromal cells (hUC-MSCs) by intravenous injection with or without combination drug treatment of mannitol and temozolomide. To evaluate the therapeutic efficacy, behavioral and immunohistochemical tests were performed, and the differences among control, stem cell only, combination drug only and stem cell with combination drug treatment were analyzed. RESULTS: Although no hUC-MSCs were detected in any group, treatment with stem cells and combination drug of mannitol and temozolomide increased the intracerebral delivery of hCD63-positive microvesicles compared with stem cell only treatment. Furthermore, treatment with stem cells and drug combination ameliorated behavioral deficits and increased bromodeoxyuridine-, doublecortin- and Reca-1-positive cells in the perilesional area as compared with other groups. DISCUSSION: The combination drug treatment of mannitol and temozolomide allowed for the efficient delivery of hUC-MSC-derived microvesicles into the brain in a chronic stroke rat model. This attenuated behavioral deficits, likely by improving neural regeneration and angiogenesis. Thus, combination drug treatment of mannitol and temozolomide could be a novel therapeutic option for patients with chronic ischemic stroke.

Interleukin-1 receptor antagonist-mediated neuroprotection by umbilical cord-derived mesenchymal stromal cells following transplantation into a rodent stroke model.

The human umbilical cord is a promising source of mesenchymal stromal cells (MSCs). Intravenous administration of human umbilical cord-derived MSCs (IV-hUMSCs) showed a favorable effect in a rodent stroke model by a paracrine mechanism. However, its underlying therapeutic mechanisms must be determined for clinical application. We investigated the therapeutic effects and mechanisms of our good manufacturing practice (GMP)-manufactured hUMSCs using various cell doses and delivery time points in a rodent model of stroke. IV-hUMSCs at a dose of 1 × 106 cells at 24 h after stroke improved functional deficits and reduced neuronal damage by attenuation of post-ischemic inflammation. Transcriptome and immunohistochemical analyses showed that interleukin-1 receptor antagonist (IL-1ra) was highly upregulated in ED-1-positive inflammatory cells in rats treated with IV-hUMSCs. Treatment with conditioned medium of hUMSCs increased the expression of IL-1ra in a macrophage cell line via activation of cAMP-response element-binding protein (CREB). These results strongly suggest that the attenuation of neuroinflammation mediated by endogenous IL-1ra is an important therapeutic mechanism of IV-hUMSCs for the treatment of stroke.

Additional increased effects of mannitol-temozolomide combined treatment on blood-brain barrier permeability.

The blood-brain barrier (BBB) is major obstacle in drug or stem cell treatment in chronic stroke. We hypothesized that adding mannitol to temozolomide (TMZ) is a practically applicable method for resolving the low efficacy of intravenous mannitol therapy. In this study, we investigated whether BBB permeability could be increased by this combined treatment. First, we established a chronic ischemic stroke rat model and examined changes in leakage of Evans blue dye within a lesion site, and in expression of tight junction proteins (TJPs), by this combined treatment. Additionally, in an in vitro BBB model using trans-wells, we analyzed changes in diffusion of a fluorescent tracer and in expression of TJPs. Mannitol-TMZ combined treatment not only increased the amount of Evans blue dye within the stroke lesion site, but also reduced occludin expression in rat brain microvessels. The in vitro study also showed that combined treatment increased the permeability for two different-sized fluorescent tracers, especially large size, and decreased expression of TJPs, such as occludin and ZO-1. Increased BBB permeability effects were more prominent with combined than with single treatments. Mannitol-TMZ combined treatment induced a decrease of TJPs with a consequent increase in BBB permeability. This combined treatment is clinically useful and might provide new therapeutic options by enabling efficient intracerebral delivery of various drugs that could not otherwise be used to treat many CNS diseases due to their inability to penetrate the BBB.

Attenuation of Postischemic Genomic Alteration by Mesenchymal Stem Cells: a Microarray Study.

Intravenous administration of mesenchymal stem cells (IV-MSC) protects the ischemic rat brain in a stroke model, but the molecular mechanism underlying its therapeutic effect is unclear. We compared genomic profiles using the mRNA microarray technique in a rodent stroke model. Rats were treated with 1 × 10(6) IV-MSC or saline (sham group) 2 h after transient middle cerebral artery occlusion (MCAo). mRNA microarray was conducted 72 h after MCAo using brain tissue from normal rats (normal group) and the sham and MSC groups. Predicted pathway analysis was performed in differentially expressed genes (DEGs), and functional tests and immunohistochemistry for inflammation-related proteins were performed. We identified 857 DEGs between the sham and normal groups, with the majority of them (88.7%) upregulated in sham group. Predicted pathway analysis revealed that cerebral ischemia activated 10 signaling pathways mainly related to inflammation and cell cycle. IV-MSC attenuated the numbers of dysregulated genes in cerebral ischemia (118 DEGs between the MSC and normal groups). In addition, a total of 218 transcripts were differentially expressed between the MSC and sham groups, and most of them (175/218 DEGs, 80.2%) were downregulated in the MSC group. IV-MSC reduced the number of Iba-1(+) cells in the peri-infarct area, reduced the overall infarct size, and improved functional deficits in MCAo rats. In conclusion, transcriptome analysis revealed that IV-MSC attenuated postischemic genomic alterations in the ischemic brain. Amelioration of dysregulated inflammation- and cell cycle-related gene expression in the host brain is one of the molecular mechanisms of IV-MSC therapy for cerebral ischemia.

Early neuroprotective effect with lack of long-term cell replacement effect on experimental stroke after intra-arterial transplantation of adipose-derived mesenchymal stromal cells.

BACKGROUND AIMS: Adipose-derived mesenchymal stromal cells (AD-MSCs) have high proliferative capacity and ability to secrete trophic factors. Although intra-arterial (IA) transplantation of stem cells induces efficient engraftment to the host brain, it is unclear whether engrafted cells exert their long-term therapeutic effects through a bystander mechanism or a cell replacement mechanism. METHODS: After induction of ischemia in rats by middle cerebral artery occlusion, we transplanted human AD-MSCs into their carotid arteries with the use of a micro-needle, and we then investigated the therapeutic effects during the early and late phases of ischemia by means of in vivo magnetic resonance imaging, functional and histological analyses. RESULTS: During the early phase of cerebral ischemia, IA transplantation of AD-MSCs attenuated inflammation and enhanced endogenous neurogenesis. Transplanted animals showed a marked improvement in functional tests during the early phase of cerebral ischemia that was less prominent but still significant during the late phase of cerebral ischemia. Although the transplanted cells effectively migrated to the infarct area, only a small number of engrafted cells survived at 8 weeks after transplantation and differentiated into neuronal, glial and endothelial cells. CONCLUSIONS: IA transplantation of human AD-MSCs provides an effective therapeutic modality in a rodent model of stroke, of which the main effects are mediated by a bystander mechanism at the early phase of ischemia.

In Vivo Roles of a Patient-Derived Induced Pluripotent Stem Cell Line (HD72-iPSC) in the YAC128 Model of Huntington's Disease.

Induced pluripotent stem cells (iPSCs) generated from somatic cells of patients can provide immense opportunities to model human diseases, which may lead to develop novel therapeutics. Huntington's disease (HD) is a devastating neurodegenerative genetic disease, with no available therapeutic options at the moment. We recently reported the characteristics of a HD patient-derived iPSC carrying 72 CAG repeats (HD72-iPSC). In this study, we investigated the in vivo roles of HD72-iPSC in the YAC128 transgenic mice, a commonly used HD mouse model carrying 128 CAG repeats. To do this, we transplanted HD72-iPSC-derived neural precursors into the striatum of YAC128 mice bilaterally and observed a significant behavioral improvement in the grafted mice. Interestingly, the transplanted HD72-iPSC-derived neural precursors formed GABAeric neurons efficiently, but no EM48-positive protein aggregates were detected at 12 weeks after transplantation. Taken together, these results indicate no HD pathology was developed from the grafted cells, or no transmission of HD pathology from the host to the graft occurred at 12 weeks post-transplantation.

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

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.

Therapeutic potential of human induced pluripotent stem cells in experimental stroke.

Ischemic stroke mainly caused by middle cerebral artery occlusion (MCAo) is a major type of stroke, but there are currently very limited therapeutic options for its cure. Neural stem cells (NSCs) or neural precursor cells (NPCs) derived from various sources are known to survive and improve neurological functions when they are engrafted in animal models of stroke. Induced pluripotent stem cells (iPSCs) generated from somatic cells of patients are novel cells that promise the autologous cell therapy for stroke. In this study, we successfully differentiated iPSCs derived from human fibroblasts into NPCs and found their robust therapeutic potential in a rodent MCAo stroke model. We observed the significant graft-induced behavioral recovery, as well as extensive neural tissue formation. Animal MRI results indicated that the majority of contralaterally transplanted iPSC-derived NPCs migrated to the peri-infarct area, showing a pathotropism critical for tissue recovery. The transplanted animals exhibited the significant reduction of stroke-induced inflammatory response, gliosis and apoptosis, and the contribution to the endogenous neurogenesis. Our results demonstrate that iPSC-derived NPCs are effective cells for the treatment of stroke.

Therapeutic effect of BDNF-overexpressing human neural stem cells (HB1.F3.BDNF) in a rodent model of middle cerebral artery occlusion.

Ischemic stroke mainly caused by middle cerebral artery occlusion (MCAo) represents the major type of stroke; however, there are still very limited therapeutic options for the stroke-damaged patients. In this study, we evaluated the neurogenic and therapeutic potentials of human neural stem cells (NSCs) overexpressing brain-derived neurotrophic factor (HB1.F3.BDNF) following transplantation into a rodent model of MCAo. F3.BDNF human NSCs (F3.BDNF) were transplanted into the contralateral side of striatum at 7 days after MCAo, and the transplanted animals were monitored up to 8 weeks using animal MRI and various behavioral tests before they were sacrificed for immunohistochemical analysis. Interestingly, animal MRI results indicate that the majority of contralaterally transplanted neural stem cells were migrated to the peri-infarct area, showing a pathotropism. Transplanted animals exhibited significant behavioral improvements in stepping, rotarod, and modified neurological severity score (mNSS) tests. We also found that the transplanted human cells were colocalized with nestin, DCX, MAP2, DARPP-32, TH, GAD65/67-positive cells, of which results can be correlated with neural regeneration and behavioral recovery in the transplanted animals. More importantly, we were able to detect high levels of human BDNF protein expression, presumably derived from the transplanted F3.BDNF. Taken together, these results provide strong evidence that human neural stem cells (F3.BDNF) are effective in treating stroke animal models.

Neuronal properties, in vivo effects, and pathology of a Huntington's disease patient-derived induced pluripotent stem cells.

Induced pluripotent stem cells (iPSCs) generated from somatic cells of patients can be used to model different human diseases. They may also serve as sources of transplantable cells that can be used in novel cell therapies. Here, we analyzed neuronal properties of an iPSC line derived from a patient with a juvenile form of Huntington's disease (HD) carrying 72 CAG repeats (HD-iPSC). Although its initial neural inducing activity was lower than that of human embryonic stem cells, we found that HD-iPSC can give rise to GABAergic striatal neurons, the neuronal cell type that is most susceptible to degeneration in HD. We then transplanted HD-iPSC-derived neural precursors into a rat model of HD with a unilateral excitotoxic striatal lesion and observed a significant behavioral recovery in the grafted rats. Interestingly, during our in vitro culture and when the grafts were examined at 12 weeks after transplantation, no aggregate formation was detected. However, when the culture was treated with a proteasome inhibitor (MG132) or when the cells engrafted into neonatal brains were analyzed at 33 weeks, there were clear signs of HD pathology. Taken together, these results indicate that, although HD-iPSC carrying 72 CAG repeats can form GABAergic neurons and give rise to functional effects in vivo, without showing an overt HD phenotype, it is highly susceptible to proteasome inhibition and develops HD pathology at later stages of transplantation. These unique features of HD-iPSC will serve as useful tools to study HD pathology and develop novel therapeutics.

Sprouty1 regulates neural and endothelial differentiation of mouse embryonic stem cells.

Fibroblast growth factor (FGF) signaling is implicated in the control of pluripotency and lineage differentiation of both human and mouse embryonic stem cells (mESCs). FGF4 dependent stimulation of ERK1/2 signaling triggers transition of pluripotent ESCs from self-renewal and lineage commitment. In this study, Sprouty 1 (Spry1) expression was observed in undifferentiated mESCs, where it modulated ERK1/2 activity. Spry1 was confirmed as dispensable for the maintenance of self-renewal. However, suppression of Spry1 expression and subsequent activation of ERK1/2 signaling promoted neural differentiation and inhibited endothelial differentiation of mESCs. Moreover, evidence is presented which indicates that SHP2, a major determinant of balance between mESC self-renewal and differentiation, directly regulates Spry1 activity to modulate ERK1/2 signaling and lineage-specific differentiation in mESCs. Our results show that Spry1 has an essential role in the lineage specific differentiation of mESCs.