Human-Induced Pluripotent Stem Cell-Derived Neural Progenitor Cells Showed Neuronal Differentiation, Neurite Extension, and Formation of Synaptic Structures in Rodent Ischemic Stroke Brains.

Ischemic stroke is a major cerebrovascular disease with high morbidity and mortality rates; however, effective treatments for ischemic stroke-related neurological dysfunction have yet to be developed. In this study, we generated neural progenitor cells from human leukocyte antigen major loci gene-homozygous-induced pluripotent stem cells (hiPSC-NPCs) and evaluated their therapeutic effects against ischemic stroke. hiPSC-NPCs were intracerebrally transplanted into rat ischemic brains produced by transient middle cerebral artery occlusion at either the subacute or acute stage, and their in vivo survival, differentiation, and efficacy for functional improvement in neurological dysfunction were evaluated. hiPSC-NPCs were histologically identified in host brain tissues and showed neuronal differentiation into vGLUT-positive glutamatergic neurons, extended neurites into both the ipsilateral infarct and contralateral healthy hemispheres, and synaptic structures formed 12 weeks after both acute and subacute stage transplantation. They also improved neurological function when transplanted at the subacute stage with γ-secretase inhibitor pretreatment. However, their effects were modest and not significant and showed a possible risk of cells remaining in their undifferentiated and immature status in acute-stage transplantation. These results suggest that hiPSC-NPCs show cell replacement effects in ischemic stroke-damaged neural tissues, but their efficacy is insufficient for neurological functional improvement after acute or subacute transplantation. Further optimization of cell preparation methods and the timing of transplantation is required to balance the efficacy and safety of hiPSC-NPC transplantation.

Intraoperative Real-Time Near-Infrared Image-Guided Endoscopic Endonasal Surgery for Pituitary Tumors.

BACKGROUND: For endoscopic endonasal surgery of pituitary tumors, tissue identification and intraoperative judgment depend largely on surgeon expertise. In the present study, we assess whether the delayed-window indocyanine green (ICG) technique can identify pituitary gland tumors in real-time during surgery and analyze the mechanism of ICG fluorescence in the pituitary gland and tumor. METHODS: Twenty-five patients with a pituitary adenoma were administered 12.5 mg of ICG intravenously during surgery. Thereafter, near-infrared (NIR) visualization was performed from 0 to 180 minutes. Only 8 patients underwent dynamic contrast-enhanced perfusion magnetic resonance imaging (MRI) owing to predicaments with insurance coverage. Consequently, we analyzed these 8 patients extensively. RESULTS: The pituitary gland and pituitary adenoma were visualized in all 25 patients with NIR fluorescence. The relative ratio of the fluorescence emission of the normal gland to that of the tumor (signal/background ratio [SBR] of the normal gland vs. the tumor) had increased after 15 minutes, peaking (5.8) at 90 minutes, demonstrating that the pituitary gland was distinctly visualized during that period. The tumor/blood (SBR tumor) and normal gland/blood (SBR gland) NIR fluorescence was significantly and positively correlated with each transfer constant on dynamic contrast-enhanced MRI, indicating vascular permeability. CONCLUSIONS: The results from the present study exhibit the utility of the delayed-window ICG technique in distinguishing the normal pituitary gland from a tumor during endoscopic endonasal surgery from 15 to 90 minutes after ICG administration. Permeability can contribute to gadolinium enhancement on MRI, as well as ICG retention and NIR fluorescence in a normal pituitary gland and tumor.

Utility of intraoperative real-time near-infrared fluorescence surgery for spinal schwannoma.

The authors report the first cases of fluorescence-guided spinal surgery of schwannomas using near-infrared fluorescence imaging with the delayed window indocyanine (ICG) green (DWIG) technique for accurate real-time intraoperative tumor visualization. Patients with intradural spinal schwannomas received 0.5 mg/kg ICG at the beginning of surgery. After 1 hour, using the DWIG technique, near-infrared spectroscopy (NIRS) detected the spinal schwannomas, showing the exact tumor location and boundaries. DWIG with NIRS microscopy confirmed the exact location of spinal schwannomas before and after opening of the dura mater, thereby facilitating successful tumor dissection from the surrounding tissues, tumor resection, and confirmation of tumor removal. The video can be found here: https://stream.cadmore.media/r10.3171/2021.10.FOCVID21158.

Intraoperative Real-Time Near-Infrared Image-Guided Surgery to Identify Intracranial Meningiomas via Microscope.

Meningiomas are a common pathology in the central nervous system requiring complete surgical resection. However, in cases of recurrence and post-irradiation, accurate identification of tumor remnants and a dural tail under bright light remains challenging. We aimed to perform real-time intraoperative visualization of the meningioma and dural tail using a delayed-window indocyanine green (ICG) technique with microscopy. Fifteen patients with intracranial meningioma received 0.5 mg/kg ICG a few hours before observation during the surgery. We used near-infrared (NIR) fluorescence to identify the tumor location. NIR fluorescence could visualize meningiomas in 12 out of 15 cases. Near-infrared visualization during the surgery ranged from 1 to 4 h after the administration of ICG. The mean signal-to-background ratio (SBR) of the intracranial meningioma in delayed-window ICG (DWIG) was 3.3 ± 2.6. The ratio of gadolinium-enhanced T1 tumor signal to the brain (T1BR) (2.5 ± 0.9) was significantly correlated with the tumor SBR (p = 0.016). K trans , indicating blood-brain barrier permeability, was significantly correlated with tumor SBR (p < 0.0001) and T1BR (p = 0.013) on dynamic contrast-enhanced magnetic resonance imaging (MRI). DWIG demonstrated a sensitivity of 94%, specificity of 38%, positive predictive value (PPV) of 76%, and negative predictive value (NPV) of 75% for meningiomas. This is the first pilot study in which DWIG fluorescence-guided surgery was used to visualize meningioma and dural tail intraoperatively with microscopy. DWIG is comparable with second-window ICG in terms of mean SBR. Gadolinium-enhanced T1 tumor signal may predict NIR fluorescence of the intracranial meningioma. Blood-brain barrier permeability as shown by K trans on dynamic contrast-enhanced MRI can contribute to gadolinium enhancement on MRI and to ICG retention and tumor fluorescence by NIR.

Intraoperative real-time near-infrared optical imaging for the identification of metastatic brain tumors via microscope and exoscope.

OBJECTIVE: As chemotherapy and radiotherapy have developed, the role of a neurosurgeon in the treatment of metastatic brain tumors is gradually changing. Real-time intraoperative visualization of brain tumors by near-infrared spectroscopy (NIRS) is feasible. The authors aimed to perform real-time intraoperative visualization of the metastatic tumor in brain surgery using second-window indocyanine green (SWIG) with microscope and exoscope systems. METHODS: Ten patients with intraparenchymal brain metastatic tumors were administered 5 mg/kg indocyanine green (ICG) 1 day before the surgery. In some patients, a microscope was used to help identify the metastases, whereas in the others, an exoscope was used. RESULTS: NIRS with the exoscope and microscope revealed the tumor location from the brain surface and the tumor itself in all 10 patients. The NIR signal could be detected though the normal brain parenchyma up to 20 mm. While the mean signal-to-background ratio (SBR) from the brain surface was 1.82 ± 1.30, it was 3.35 ± 1.76 from the tumor. The SBR of the tumor (p = 0.030) and the ratio of Gd-enhanced T1 tumor signal to normal brain (T1BR) (p = 0.0040) were significantly correlated with the tumor diameter. The SBR of the tumor was also correlated with the T1BR (p = 0.0020). The tumor was completely removed in 9 of the 10 patients, as confirmed by postoperative Gd-enhanced MRI. This was concomitant with the absence of NIR fluorescence at the end of surgery. CONCLUSIONS: SWIG reveals the metastatic tumor location from the brain surface with both the microscope and exoscope systems. The Gd-enhanced T1 tumor signal may predict the NIR signal of the metastatic tumor, thus facilitating tumor resection.

Gene Therapy Using Neural Stem/Progenitor Cells Derived from Human Induced Pluripotent Stem Cells: Visualization of Migration and Bystander Killing Effect.

Glioblastoma is the most aggressive brain tumor characterized by diffuse infiltration into the normal brain parenchyma. Neural stem cells are known to possess the tumor-tropic migratory capacity and thus can be used as cellular vehicles for targeted delivery of therapeutic agents. In the present study, we evaluated the efficacy of herpes simplex virus thymidine kinase (HSV-TK) suicide gene therapy for glioblastoma using neural stem/progenitor cells (NS/PCs) derived from human induced pluripotent stem cells (hiPSCs). Although transduction of hiPSCs is preferable for a safe and stable supply in the clinical setting, high-level and/or constitutive HSV-TK expression was highly cytotoxic to hiPSCs. To overcome this problem, we used the tetracycline-inducible system to control the expression of HSV-TK. hiPSC-derived NS/PCs expressing HSV-TK were transplanted in an orthotopic xenograft mouse model of human glioblastoma. Glioblastoma cell growth in mice was dramatically inhibited following ganciclovir (GCV) administration. Survival of the mice was significantly prolonged with administration of GCV compared with control groups. Time-lapse imaging of organotypic brain slice cultures first demonstrated the directional migration of NS/PCs toward glioblastoma cells and the bystander killing effect upon GCV treatment. hiPSC-derived NS/PCs with HSV-TK/GCV suicide gene system may have considerable therapeutic potential for the treatment of glioblastoma. Color images are available online.

Visualization of spatiotemporal dynamics of human glioma stem cell invasion.

Glioblastoma exhibits phenotypic and genetic heterogeneity, aggressive invasiveness, therapeutic resistance, and tumor recurrence, which can be explained by the existence of glioma stem cells (GSCs). In this study, we visualized the spatiotemporal dynamics of invasion of human GSCs in an orthotopic xenograft mouse model using time-lapse imaging of organotypic brain slice cultures and three-dimensional imaging of optically cleared whole brains. GSCs implanted in the striatum exhibited directional migration toward axon bundles, perivascular area, and the subventricular zone around the inferior horn of the lateral ventricle. GSCs migrated in a helical pattern around axon bundles in the striatum and invaded broadly in both the rostral and caudal directions. GSCs in the corpus callosum migrated more rapidly and unidirectionally toward the contralateral side with pseudopod extension. These characteristics of GSC invasion shared histological features observed in glioblastoma patients. Spatiotemporal visualization techniques can contribute to the elucidation of the mechanisms underlying GSC invasion that may lead to the development of effective therapy for glioblastoma.

The Diffuse and Severe Traumatic Subarachnoid Hemorrhage Being Hard to Distinguish to Aneurysmal Subarachnoid Hemorrhage.

BACKGROUND: In primary intracerebral hemorrhage, several studies showed that contrast extravasation (CEV) is reported to be an indicator of delayed hematoma expansion, emergent hematoma removal and poor prognosis. On the contrary in head trauma, few researches validated the effectiveness of CEV because of other influences such as subarachnoid hemorrhage (SAH), contusion or brain swelling. The authors experienced a patient showing diffuse SAH caused by traumatic mechanism with acute subdural hematoma (ASDH). In the angiography, a notable rare image of CEV was found and emergency operation mainly to cease the bleeding points was performed. PATIENT PRESENTATION: A 70-year-old man was found being comatose and brought to emergency room. Computed tomography (CT) revealed diffuse SAH with left ASDH. Computed tomography angiography (CTA) also did not clarify any abnormal vessel structure except for slightly dilatation at the part of internal carotid artery, suggesting tiny ruptured aneurysm. The authors performed a cerebral angiography and resulted in no aneurysm or arteriovenous shunt detection but revealed the extravasation from middle cerebral artery and middle meningeal artery. Follow-up CT revealed increased left ASDH though 5 hours have already elapsed. The authors performed surgical intervention to halt the bleeding. CONCLUSION: The authors experienced a notably rare image of the extravasation from middle cerebral artery and middle meningeal artery in the cerebral angiography. When an obscure arterial abnormality is suspected on CTA for the unknown origin SAH with ASDH, the authors may have an affirmative attitude toward performing conventional cerebral angiography, which is most promising modality to detect the source of bleeding including CEV.

Rush progression and fatal result of septic shock related to central line catheter infection in cirrhosis patient with brain stroke.

BACKGROUND: Catheter-related blood stream infection (CRBSI) is one of the most common intractable healthcare-associated infections because catheters can be easily contaminated by resistant bacteria, and is associated with a high mortality. Central lines are currently used for administering medication to patients with severe stroke, and may thus cause CRBSI. CASE PRESENTATION: A 71-year-old woman with cirrhosis presented with subarachnoid hemorrhage (SAH) that was treated by clipping surgery. On postoperative day (POD) 38, sudden high fever (40.3 °C) was detected; the patient died a few hours later. Blood and central line cultures were positive for Klebsiella pneumoniae that may have caused CRBSI and endotoxin shock. In this case, the duration from fever detection to death was notably short. Additionally, inflammatory markers such as white blood cells (WBC) or C-reactive protein (CRP) were almost within normal ranges, even a few hours after fever was detected and before death. Cirrhosis was considered to be the cause of these phenomena. CONCLUSION: The timely diagnosis and complete treatment of patients with liver cirrhosis who develop CRBSI are highly challenging. We suggest that clinicians should rigorously apply preventive measures and strengthen CRBSI monitoring, especially in cirrhosis-associated cases.

Primary Solitary Intracranial Malignant Melanoma: A Systematic Review of Literature.

INTRODUCTION: Primary solitary intracranial malignant melanoma (PIMM) is extremely rare. In 1992, an extensive review of 81 patients with PIMM was undertaken. Imaging studies, microsurgery, and adjuvant therapy have developed considerably over the last 25 years, and targeted therapy recently has been proven successful for metastatic melanoma. These factors could influence current and future clinical PIMM results. METHODS: We undertook a literature search of PIMM patients since 1992. RESULTS: We reviewed 49 cases of PIMM. The mean age was 45.8 years. No significant sex difference was found. Intracranial hypertension and focal neurologic deficits were commonly observed around 70% and 40%, respectively. There were no significant differences of survival period according to tumor sites. Surgeries were performed in 42 of 49 patients with PIMM reviewed (92%). The mean survival of the gross total removal group was significantly longer than that of surgical results (>22 months vs. 12 months (interquartile range: 5-22 months; P = 0.026). For adjuvant therapy, 9 patients underwent chemotherapy and 18 patients underwent radiotherapy postoperatively There was no significant difference in survival period between with and without adjuvant therapies. Leptomeningeal enhancement diagnosed in the initial MRI, was the worst prognostic factor. CONCLUSIONS: Gross total removal of the PIMM was the most promising treatment. Currently adjuvant therapy has not been associated with the survival period. To improve clinical outcome, immunotherapy and targeted therapies are likely to become more important.

Grafted Miniature-Swine Neural Stem Cells of Early Embryonic Mesencephalic Neuroepithelial Origin can Repair the Damaged Neural Circuitry of Parkinson's Disease Model Rats.

Although recent progress in the use of human iPS cell-derived midbrain dopaminergic progenitors is remarkable, alternatives are essential in the strategies of treatment of basal-ganglia-related diseases. Attention has been focused on neural stem cells (NSCs) as one of the possible candidates of donor material for neural transplantation, because of their multipotency and self-renewal characteristics. In the present study, miniature-swine (mini-swine) mesencephalic neuroepithelial stem cells (M-NESCs) of embryonic 17 and 18 days grafted in the parkinsonian rat striatum were assessed immunohistochemically, behaviorally and electrophysiologically to confirm their feasibility for the neural xenografting as a donor material. Grafted mini-swine M-NESCs survived in parkinsonian rat striatum at 8 weeks after transplantation and many of them differentiated into tyrosine hydroxylase (TH)-positive cells. The parkinsonian model rats grafted with mini-swine M-NESCs exhibited a functional recovery from their parkinsonian behavioral defects. The majority of donor-derived TH-positive cells exhibited a matured morphology at 8 weeks. Whole-cell recordings from donor-derived neurons in the host rat brain slices incorporating the graft revealed the presence of multiple types of neurons including dopaminergic. Glutamatergic and GABAergic post-synaptic currents were evoked in the donor-derived cells by stimulation of the host site, suggesting they receive both excitatory and inhibitory synaptic inputs from host area. The present study shows that non-rodent mammalian M-NESCs can differentiate into functionally active neurons in the diseased xenogeneic environment and could improve the parkinsonian behavioral defects over the species. Neuroepithelial stem cells could be an attractive candidate as a source of donor material for neural transplantation.

Successful Use of Covered Stent for Carotid Artery Injury with Active Medial Projecting Extravasation.

BACKGROUND: Nontraumatic carotid artery injury with active extravasation, or carotid blowout syndrome (CBS), is relatively rare and highly difficult to treat because it is difficult to approach the lesions owing to anatomic factors. It also involves quick progression and a risk of cerebral embolization caused by thrombi and carotid artery occlusion. Recently, covered stents were revealed to be effective for CBS. However, they have several disadvantages, such as their costs, rebleeding complications, or cerebral embolic risks. A firm selection method of CBS types that are appropriate for covered-stent therapy is expected. CASE DESCRIPTION: A 38-year-old man with esophageal cancer presented with massive hematemesis. Computed tomography revealed active extravasation from the left common carotid artery with medial projection. Initially, the open direct approach failed, which resulted in further bleeding and transient cardiopulmonary arrest. With tentative hemostasis using manual finger compression, emergency angiography was performed, and a covered stent, Fluency 8 mm × 60 mm, was placed at the rupture point. He was transferred to the rehabilitation hospital 36 days after admission with a modified Rankin score of 2 without major complications. CONCLUSION: CBS cases having rupture points around the clavicle and having medial projection extravasation should be treated by covered stent placement under tentative hemostasis using manual finger pressure rather than conventional open surgical treatment.

Safe Burr Hole Surgery for Chronic Subdural Hematoma Using Dabigatran with Idarucizumab.

BACKGROUND: Chronic subdural hematoma (CSDH) is a common intracranial hematoma. The number of patients who undergo anticoagulant therapy including a direct oral anticoagulant (DOAC) is expected to increase. Recently, idarucizumab, the antidote for dabigatran, which is a DOAC, has been developed. We successfully treated CSDH with dabigatran using emergency burr hole surgery and idarucizumab. CASE DESCRIPTION: A 79-year-old Japanese man severely hit his head and visited the emergency department. Computed tomography (CT) showed tiny traumatic acute subdural hematoma, for which he was admitted. At that time, atrial fibrillation was newly detected, for which dabigatran, having a specific antidote (idarucizumab), was chosen and started 2 weeks after the discharge. Two months after the trauma episode, he revisited the emergency department because of acute left upper and lower limb motor weakness. CT revealed a midline shifted CSDH. Considering rush course of motor weakness and shifted brain, we performed emergency surgery using an antidote for dabigatran, idarucizumab. He was discharged 5 days after surgery without any complications or excessive perioperative hemorrhage. CONCLUSION: Dabigatran should be used for atrial fibrillation detected after head trauma. Emergency surgery can be safely performed for CSDH with dabigatran using idarucizumab.

A case report of adult cerebellar high-grade glioma with H3.1 K27M mutation: a rare example of an H3 K27M mutant cerebellar tumor.

Diffuse midline glioma, H3 K27M mutant, is newly recognized as a distinct category, which usually arises in the brain stem, thalamus or spinal cord of children, and young adults. The oncogenic H3 K27M mutation involves H3.3 (encoded by H3F3A) or H3.1 (encoded by HIST1H3B/HIST1H3C), and the incidence of each mutation differs among the primary sites. Recently, several papers have reported that cerebellar high-grade gliomas in both children and adults also harbor H3 K27 mutation. With the exception of one pediatric case, all of the cases carried the mutation in H3.3. We herein present the case of an adult cerebellar high-grade astrocytic tumor with H3.1 K27M mutation in a 45-year-old man, which also involvedTP53 mutation and was immunonegative for ATRX. Some groups have reported that H3.3 and H3.1 K27M mutations define subgroups of diffuse intrinsic pontine gliomas (DIPGs) with different phenotypes as well as genetic alterations. On comparing the findings of the present case, particularly TP53 mutation status and ATRX expression, to the findings of the previous studies on DIPGs, our case seems unusual among the H3.1 K27M mutant subgroup. Further studies are needed to clarify the exact frequency, clinicopathological characteristics, and genomic alterations of cerebellar gliomas harboring H3 K27M mutation.

Thyrotropin-producing pituitary adenoma simultaneously existing with Graves' disease: a case report.

BACKGROUND: Thyrotropin-producing pituitary tumor is relatively rare. In particular, concurrent cases associated with Graves' disease are extremely rare and only nine cases have been reported so far. We describe a case of a thyrotropin-producing pituitary adenoma concomitant with Graves' disease, which was successfully treated. CASE PRESENTATION: A 40-year-old Japanese woman presented with mild signs of hyperthyroidism. She had positive anti-thyroid-stimulating hormone receptor antibody, anti-thyroglobulin antibody, and anti-thyroid peroxidase antibody. Her levels of serum thyroid-stimulating hormone, which ranged from low to normal in the presence of high levels of serum free thyroid hormones, were considered to be close to a state of syndrome of inappropriate secretion of thyroid-stimulating hormone. Magnetic resonance imaging showed a macropituitary tumor. The coexistence of thyrotropin-producing pituitary adenoma and Graves' disease was suspected. Initial therapy included anti-thyroid medication, which was immediately discontinued due to worsening symptoms. Subsequently, surgical therapy for the pituitary tumor was conducted, and her levels of free thyroid hormones, including the thyroid-stimulating hormone, became normal. On postoperative examination, her anti-thyroid-stimulating hormone receptor antibody levels decreased, and the anti-thyroglobulin antibody became negative. The coexistence of thyrotropin-producing pituitary adenoma and Graves' disease is rarely reported. The diagnosis of this condition is complicated, and the appropriate treatment strategy has not been clearly established. CONCLUSIONS: This case suggests that physicians should consider the coexistence of thyrotropin-producing pituitary adenoma with Graves' disease in cases in which thyroid-stimulating hormone values range from low to normal in the presence of thyrotoxicosis, and the surgical treatment of thyrotropin-producing pituitary adenoma could be the first-line therapy in patients with both thyrotropin-producing pituitary adenoma and Graves' disease.

Human induced pluripotent stem cell-derived cortical neurons integrate in stroke-injured cortex and improve functional recovery.

Stem cell-based approaches to restore function after stroke through replacement of dead neurons require the generation of specific neuronal subtypes. Loss of neurons in the cerebral cortex is a major cause of stroke-induced neurological deficits in adult humans. Reprogramming of adult human somatic cells to induced pluripotent stem cells is a novel approach to produce patient-specific cells for autologous transplantation. Whether such cells can be converted to functional cortical neurons that survive and give rise to behavioural recovery after transplantation in the stroke-injured cerebral cortex is not known. We have generated progenitors in vitro, expressing specific cortical markers and giving rise to functional neurons, from long-term self-renewing neuroepithelial-like stem cells, produced from adult human fibroblast-derived induced pluripotent stem cells. At 2 months after transplantation into the stroke-damaged rat cortex, the cortically fated cells showed less proliferation and more efficient conversion to mature neurons with morphological and immunohistochemical characteristics of a cortical phenotype and higher axonal projection density as compared with non-fated cells. Pyramidal morphology and localization of the cells expressing the cortex-specific marker TBR1 in a certain layered pattern provided further evidence supporting the cortical phenotype of the fated, grafted cells, and electrophysiological recordings demonstrated their functionality. Both fated and non-fated cell-transplanted groups showed bilateral recovery of the impaired function in the stepping test compared with vehicle-injected animals. The behavioural improvement at this early time point was most likely not due to neuronal replacement and reconstruction of circuitry. At 5 months after stroke in immunocompromised rats, there was no tumour formation and the grafted cells exhibited electrophysiological properties of mature neurons with evidence of integration in host circuitry. Our findings show, for the first time, that human skin-derived induced pluripotent stem cells can be differentiated to cortical neuronal progenitors, which survive, differentiate to functional neurons and improve neurological outcome after intracortical implantation in a rat stroke model.

Grafted human neural stem cells enhance several steps of endogenous neurogenesis and improve behavioral recovery after middle cerebral artery occlusion in rats.

Neural stem/progenitor cells (NSPCs) in subventricular zone (SVZ) produce new striatal neurons during several months after stroke, which may contribute to recovery. Intracerebral grafts of NSPCs can exert beneficial effects after stroke through neuronal replacement, trophic actions, neuroprotection, and modulation of inflammation. Here we have explored whether human fetal striatum-derived NSPC-grafts influence striatal neurogenesis and promote recovery in stroke-damaged brain. T cell-deficient rats were subjected to 1h middle cerebral artery occlusion (MCAO). Human fetal NSPCs or vehicle were implanted into ipsilateral striatum 48 h after MCAO, animals were assessed behaviorally, and perfused at 6 or 14 weeks. Grafted human NSPCs survived in all rats, and a subpopulation had differentiated to neuroblasts or mature neurons at 6 and 14 weeks. Numbers of proliferating cells in SVZ and new migrating neuroblasts and mature neurons were higher, and numbers of activated microglia/macrophages were lower in the ischemic striatum of NSPC-grafted compared to vehicle-injected group both at 6 and 14 weeks. A fraction of grafted NSPCs projected axons from striatum to globus pallidus. The NSPC-grafted rats showed improved functional recovery in stepping and cylinder tests from 6 and 12 weeks, respectively. Our data show, for the first time, that intrastriatal implants of human fetal NSPCs exert a long-term enhancement of several steps of striatal neurogensis after stroke. The grafts also suppress striatal inflammation and ameliorate neurological deficits. Our findings support the idea that combination of NSPC transplantation and stimulation of neurogenesis from endogenous NSPCs may become a valuable strategy for functional restoration after stroke.

Human-induced pluripotent stem cells form functional neurons and improve recovery after grafting in stroke-damaged brain.

Reprogramming of adult human somatic cells to induced pluripotent stem cells (iPSCs) is a novel approach to produce patient-specific cells for autologous transplantation. Whether such cells survive long-term, differentiate to functional neurons, and induce recovery in the stroke-injured brain are unclear. We have transplanted long-term self-renewing neuroepithelial-like stem cells, generated from adult human fibroblast-derived iPSCs, into the stroke-damaged mouse and rat striatum or cortex. Recovery of forepaw movements was observed already at 1 week after transplantation. Improvement was most likely not due to neuronal replacement but was associated with increased vascular endothelial growth factor levels, probably enhancing endogenous plasticity. Transplanted cells stopped proliferating, could survive without forming tumors for at least 4 months, and differentiated to morphologically mature neurons of different subtypes. Neurons in intrastriatal grafts sent axonal projections to the globus pallidus. Grafted cells exhibited electrophysiological properties of mature neurons and received synaptic input from host neurons. Our study provides the first evidence that transplantation of human iPSC-derived cells is a safe and efficient approach to promote recovery after stroke and can be used to supply the injured brain with new neurons for replacement.

Environmental cue-dependent dopaminergic neuronal differentiation and functional effect of grafted neuroepithelial stem cells in parkinsonian brain.

OBJECTIVE: To physiologically repair damaged neural circuitry using neural transplantation, the donor cells must be able to differentiate into the optimal type and number of neurons for the host brain environment. They must also be capable of functional regulation by the host brain. These features are important to optimize functional outcome and to minimize side effects. In this study, the differentiation of grafted mesencephalic neuroepithelial stem cells in the normal and parkinsonian brain was assessed morphologically and behaviorally to confirm the influence of the host brain environment after neural transplantation regarding functional outcome and side effects of transplants. METHODS: Green fluorescent protein-positive mesencephalic neuroepithelial stem cells were dissected from early rat embryos and transplanted into normal (n = 20) and Parkinson's disease (PD) rat model striata (n = 30). The differentiation pattern of grafted cells was precisely monitored immunohistochemically, and the functional effects of grafted cells on behavior were assessed using an amphetamine-induced rotation test. RESULTS: The grafted cells survived in both normal and PD rat striata and differentiated into tyrosine hydroxylase-positive cells. In 8-week-old grafts, the number of tyrosine hydroxylase-positive cells was 3-fold higher in parkinsonian brains than in normal brains. The donor-derived tyrosine hydroxylase-positive cells exhibited a mature morphology with long, well-branched cell processes and large cell bodies, especially in parkinsonian brains. Also, the process lengths of these cells in parkinsonian brains were 3.4-fold longer than those in normal brains at 8 weeks after transplantation. The grafted PD rats exhibited a complete recovery from their behavioral defects, and no obvious contralateral rotation to the lesioned and grafted side suggestive of overdose supply from the graft was observed. However, the grafted normal rats did not exhibit any contralateral rotation to the grafted side suggestive of dopamine unbalance. CONCLUSION: Grafted mesencephalic neuroepithelial stem cells can differentiate into optimal neuron types in response to environmental cues and can affect the behavior of PD rats without any side effects.

Potential functional neural repair with grafted neural stem cells of early embryonic neuroepithelial origin.

The fate of grafted neuroepithelial stem cells in the normal mature brain environment was assessed both morphologically and electrophysiologically to confirm their feasibility in the functional repair of damaged neural circuitry. The neuroepithelial stem cells were harvested from the mesencephalic neural plate of transgenic green fluorescence protein-carrying rat embryos, and implanted into the normal adult rat striatum. The short- and long-term differentiation pattern of donor-derived cells was precisely monitored immunohistochemically. The functional abilities of the donor-derived cells and communication between them and the host were investigated using host-rat brain slices incorporating the graft with whole-cell patch-clamp recording. Vigorous differentiation of the neuroepithelial stem cells into mostly neurons was noted in the short-term with positive staining for tyrosine hydroxylase, suggesting that the donor-derived cells were exclusively following their genetically programmed fate, together with gamma-aminobutyric acid (GABA) and glutamate expression. In the long-term, the large number of donor-derived neurons was sustained, but the staining pattern showed expression of dopamine- and adenosine 3':5'-monophosphate-regulated phosphoprotein 32, suggesting that some neurons were following environmental cues, together with the appearance of some cholinergic neurons. Some donor-derived astrocytes were also seen in the graft. Many action potentials indicating the presence of both dopaminergic and non-dopaminergic patterns could be elicited and recorded in the donor-derived neurons in addition to spontaneous glutamatergic and GABAergic post-synaptic currents which were strongly shown to be of host origin. Neuroepithelial stem cells are therefore an attractive candidate as a source of donor material for intracerebral grafting in functional repair.