Real-Time Discrimination between Proliferation and Neuronal and Astroglial Differentiation of Human Neural Stem Cells

Neural stem cells (NSCs) are characterized by a capacity for self-renewal, differentiation into multiple neural lineages, all of which are considered to be promising components for neural regeneration. However, for cell-replacement therapies, it is essential to monitor the process of in vitro NSC differentiation and identify differentiated cell phenotypes. We report a real-time and label-free method that uses a capacitance sensor array to monitor the differentiation of human fetal brain-derived NSCs (hNSCs) and to identify the fates of differentiated cells. When hNSCs were placed under proliferation or differentiation conditions in five media, proliferating and differentiating hNSCs exhibited different frequency and time dependences of capacitance, indicating that the proliferation and differentiation status of hNSCs may be discriminated in real-time using our capacitance sensor. In addition, comparison between real-time capacitance and time-lapse optical images revealed that neuronal and astroglial differentiation of hNSCs may be identified in real-time without cell labeling.ope

Glial Cell Line-derived Neurotrophic Factor-overexpressing Human Neural Stem/Progenitor Cells Enhance Therapeutic Efficiency in Rat with Traumatic Spinal Cord Injury

Spinal cord injury (SCI) causes axonal damage and demyelination, neural cell death, and comprehensive tissue loss, resulting in devastating neurological dysfunction. Neural stem/progenitor cell (NSPCs) transplantation provides therapeutic benefits for neural repair in SCI, and glial cell linederived neurotrophic factor (GDNF) has been uncovered to have capability of stimulating axonal regeneration and remyelination after SCI. In this study, to evaluate whether GDNF would augment therapeutic effects of NSPCs for SCI, GDNF-encoding or mock adenoviral vector-transduced human NSPCs (GDNF-or Mock-hNSPCs) were transplanted into the injured thoracic spinal cords of rats at 7 days after SCI. Grafted GDNFhNSPCs showed robust engraftment, long-term survival, an extensive distribution, and increased differentiation into neurons and oligodendroglial cells. Compared with Mock-hNSPC- and vehicle-injected groups, transplantation of GDNF-hNSPCs significantly reduced lesion volume and glial scar formation, promoted neurite outgrowth, axonal regeneration and myelination, increased Schwann cell migration that contributed to the myelin repair, and improved locomotor recovery. In addition, tract tracing demonstrated that transplantation of GDNF-hNSPCs reduced significantly axonal dieback of the dorsal corticospinal tract (dCST), and increased the levels of dCST collaterals, propriospinal neurons (PSNs), and contacts between dCST collaterals and PSNs in the cervical enlargement over that of the controls. Finally grafted GDNF-hNSPCs substantially reversed the increased expression of voltage-gated sodium channels and neuropeptide Y, and elevated expression of GABA in the injured spinal cord, which are involved in the attenuation of neuropathic pain after SCI. These findings suggest that implantation of GDNF-hNSPCs enhances therapeutic efficiency of hNSPCs-based cell therapy for SCI.ope

Amyloid-β oligomers regulate the properties of human neural stem cells through GSK-3β signaling

Alzheimer's disease (AD) is the most common cause of age-related dementia. The neuropathological hallmarks of AD include extracellular deposition of amyloid-β peptides and neurofibrillary tangles that lead to intracellular hyperphosphorylated tau in the brain. Soluble amyloid-β oligomers are the primary pathogenic factor leading to cognitive impairment in AD. Neural stem cells (NSCs) are able to self-renew and give rise to multiple neural cell lineages in both developing and adult central nervous systems. To explore the relationship between AD-related pathology and the behaviors of NSCs that enable neuroregeneration, a number of studies have used animal and in vitro models to investigate the role of amyloid-β on NSCs derived from various brain regions at different developmental stages. However, the Aβ effects on NSCs remain poorly understood because of conflicting results. To investigate the effects of amyloid-β oligomers on human NSCs, we established amyloid precursor protein Swedish mutant-expressing cells and identified cell-derived amyloid-β oligomers in the culture media. Human NSCs were isolated from an aborted fetal telencephalon at 13 weeks of gestation and expanded in culture as neurospheres. Human NSCs exposure to cell-derived amyloid-β oligomers decreased dividing potential resulting from senescence through telomere attrition, impaired neurogenesis and promoted gliogenesis, and attenuated mobility. These amyloid-β oligomers modulated the proliferation, differentiation and migration patterns of human NSCs via a glycogen synthase kinase-3β-mediated signaling pathway. These findings contribute to the development of human NSC-based therapy for AD by elucidating the effects of Aβ oligomers on human NSCs.ope

Brain and spinal cord injury repair by implantation of human neural progenitor cells seeded onto polymer scaffolds

Hypoxic-ischemic (HI) brain injury and spinal cord injury (SCI) lead to extensive tissue loss and axonal degeneration. The combined application of the polymer scaffold and neural progenitor cells (NPCs) has been reported to enhance neural repair, protection and regeneration through multiple modes of action following neural injury. This study investigated the reparative ability and therapeutic potentials of biological bridges composed of human fetal brain-derived NPCs seeded upon poly(glycolic acid)-based scaffold implanted into the infarction cavity of a neonatal HI brain injury or the hemisection cavity in an adult SCI. Implantation of human NPC (hNPC)-scaffold complex reduced the lesion volume, induced survival, engraftment, and differentiation of grafted cells, increased neovascularization, inhibited glial scar formation, altered the microglial/macrophage response, promoted neurite outgrowth and axonal extension within the lesion site, and facilitated the connection of damaged neural circuits. Tract tracing demonstrated that hNPC-scaffold grafts appear to reform the connections between neurons and their targets in both cerebral hemispheres in HI brain injury and protect some injured corticospinal fibers in SCI. Finally, the hNPC-scaffold complex grafts significantly improved motosensory function and attenuated neuropathic pain over that of the controls. These findings suggest that, with further investigation, this optimized multidisciplinary approach of combining hNPCs with biomaterial scaffolds provides a more versatile treatment for brain injury and SCI.ope

Human neural stem cells alleviate Alzheimer-like pathology in a mouse model

BACKGROUND: Alzheimer's disease (AD) is an inexorable neurodegenerative disease that commonly occurs in the elderly. The cognitive impairment caused by AD is associated with abnormal accumulation of amyloid-β (Aβ) and hyperphosphorylated tau, which are accompanied by inflammation. Neural stem cells (NSCs) are self-renewing, multipotential cells that differentiate into distinct neural cells. When transplanted into a diseased brain, NSCs repair and replace injured tissues after migration toward and engraftment within lesions. We investigated the therapeutic effects in an AD mouse model of human NSCs (hNSCs) that derived from an aborted human fetal telencephalon at 13 weeks of gestation. Cells were transplanted into the cerebral lateral ventricles of neuron-specific enolase promoter-controlled APPsw-expressing (NSE/APPsw) transgenic mice at 13 months of age. RESULTS: Implanted cells extensively migrated and engrafted, and some differentiated into neuronal and glial cells, although most hNSCs remained immature. The hNSC transplantation improved spatial memory in these mice, which also showed decreased tau phosphorylation and Aβ42 levels and attenuated microgliosis and astrogliosis. The hNSC transplantation reduced tau phosphorylation via Trk-dependent Akt/GSK3β signaling, down-regulated Aβ production through an Akt/GSK3β signaling-mediated decrease in BACE1, and decreased expression of inflammatory mediators through deactivation of microglia that was mediated by cell-to-cell contact, secretion of anti-inflammatory factors generated from hNSCs, or both. The hNSC transplantation also facilitated synaptic plasticity and anti-apoptotic function via trophic supplies. Furthermore, the safety and feasibility of hNSC transplantation are supported. CONCLUSIONS: These findings demonstrate the hNSC transplantation modulates diverse AD pathologies and rescue impaired memory via multiple mechanisms in an AD model. Thus, our data provide tangible preclinical evidence that human NSC transplantation could be a safe and versatile approach for treating AD patients.ope

Human fetal brain-derived neural stem/progenitor cells grafted into the adult epileptic brain restrain seizures in rat models of temporal lobe epilepsy

Cell transplantation has been suggested as an alternative therapy for temporal lobe epilepsy (TLE) because this can suppress spontaneous recurrent seizures in animal models. To evaluate the therapeutic potential of human neural stem/progenitor cells (huNSPCs) for treating TLE, we transplanted huNSPCs, derived from an aborted fetal telencephalon at 13 weeks of gestation and expanded in culture as neurospheres over a long time period, into the epileptic hippocampus of fully kindled and pilocarpine-treated adult rats exhibiting TLE. In vitro, huNSPCs not only produced all three central nervous system neural cell types, but also differentiated into ganglionic eminences-derived γ-aminobutyric acid (GABA)-ergic interneurons and released GABA in response to the depolarization induced by a high K+ medium. NSPC grafting reduced behavioral seizure duration, afterdischarge duration on electroencephalograms, and seizure stage in the kindling model, as well as the frequency and the duration of spontaneous recurrent motor seizures in pilocarpine-induced animals. However, NSPC grafting neither improved spatial learning or memory function in pilocarpine-treated animals. Following transplantation, grafted cells showed extensive migration around the injection site, robust engraftment, and long-term survival, along with differentiation into β-tubulin III+ neurons (~34%), APC-CC1+ oligodendrocytes (~28%), and GFAP+ astrocytes (~8%). Furthermore, among donor-derived cells, ~24% produced GABA. Additionally, to explain the effect of seizure suppression after NSPC grafting, we examined the anticonvulsant glial cell-derived neurotrophic factor (GDNF) levels in host hippocampal astrocytes and mossy fiber sprouting into the supragranular layer of the dentate gyrus in the epileptic brain. Grafted cells restored the expression of GDNF in host astrocytes but did not reverse the mossy fiber sprouting, eliminating the latter as potential mechanism. These results suggest that human fetal brain-derived NSPCs possess some therapeutic effect for TLE treatments although further studies to both increase the yield of NSPC grafts-derived functionally integrated GABAergic neurons and improve cognitive deficits are still needed.ope

Therapeutic Application of Neural Stem Cells for Neonatal Hypoxic-Ischemic Brain Injury

Neural stem cells (NSCs) are characterized by a capacity for self-renewal, differentiation into multiple neural cell lineages, and migration toward damaged sites in the central nervous system (CNS). NSCs expanded in culture could be implanted into the brain where they integrate into host neural circuitry and stably express foreign genes. It hence appears that transplantation of NSCs has been proposed as a promising therapeutic strategy in neurological disorders. During hypoxic-ischemic (HI) brain injury, factors are transiently elaborated to which NSCs respond by migrating to degenerating regions and differentiating towards replacement of dying neural cells. In addition, NSCs serve as vehicles for gene delivery and appear capable of simultaneous neural cell replacement and gene therapy (e.g. with factors that might enhance neuronal differentiation, neurites outgrowth, proper connectivity, neuroprotection, and/or immunomodulatory substances). When combined with certain synthetic biomaterials, NSCs may be even more effective in 'engineering' the damaged CNS towards reconstitution. Human NSCs were isolated from the forebrain of an aborted fetus at 13 weeks of gestation and were grown as neurospheres in cultures. After the characterization of human NSCs in preclinical testing and the approval of the IRB, a clinical trial of the transplantation of human NSCs into patients with severe perinatal HI brain injury has been performed. The existing data from these clinical trials have shown to be safe, well tolerated, and of neurologically-some benefits. Therefore, long-term and large scale multicenter clinical study is required to determine its precise therapeutic effect and safety.ope

Induction of intracranial glioblastoma apoptosis by transplantation of TRAIL (Tumor necrosis factor-related apoptosis-inducing ligand) expressing human neural stem cells

의과학과/석사[한글] 다형성 교모세포종 (glioblastoma multiforme)과 같은 악성 뇌종양은 침윤성이 높아 정상 뇌 조직 사이로 광범위하게 퍼져나가며, 원발성 종양 부위에서 원거리까지 전이가 일어나므로 수술적 절제, 보조적 방사선 및 화학요법에도 불구하고 예후가 극히 불량하다. 따라서 전이된 종양병변을 표적으로 하는 치료법 개발이 사망률 감소를 위하여 필수적이다. 신경줄기세포는 신경계 이식 시 전체 신경축에 걸쳐 광범위하게 이주 및 생착이 가능하다. 특히 뇌종양 부위에 이식된 경우 종괴를 둘러싸고 내부로 침윤하며, 주위조직으로 전이되는 종양세포에 밀착하여 이주하는 것이 알려졌다. TRAIL은 tumor necrosis factor protein superfamily 중 하나로, 다양한 종양세포에서는 세포사멸을 유발하나 정상세포에 대한 독성작용이 없어 종양치료제로써 주목 받고 있다. 본 연구에서는 TRAIL 발현 재조합 아데노바이러스를 제작하였고, 이를 인간 신경줄기세포 (hNSCs)에 감염하여, 종양세포의 세포사멸을 유도하는 TRAIL 단백의 발현을 확인하였다. 또한 감염된 인간 신경줄기세포를 인간 다형성 교모세포종 누드마우스 동물모델에 이식하였을 때, 생체 내에서 교모세포종의 세포사멸이 관찰되었으며, 이식된 신경줄기세포는 신경교모세포종의 세포사멸을 유도할 뿐만 아니라, 손상된 신경계를 대체할 수 있는 다양한 신경원 및 신경교 세포로 분화하는 양상도 관찰하였다. 이러한 연구는 인간 신경줄기세포를 이용, 난치성 뇌종양에 대하여 치료효과가 높고 부작용이 적은 획기적인 줄기세포 매개성 유전자치료법 개발을 가능하게 하여 향후 임상시험에 사용될 수 있을 것이며, 뇌종양세포 파괴유도뿐만 아니라 신경줄기세포의 가소성을 이용한 손상된 뇌신경 재생도 함께 유도하는 세포치료도 가능하여 다방면의 효율적 치료적용을 시도할 수 있다. [영문]When neural stem cells (NSCs) implanted into a diseased or injured nervous system, they not only showed preferential extensive migration to and engraftment within areas of discrete as well as diffuse abnormalities, but the capability to replace diseased tissue in an appropriate manner. In addition, engraftment is often followed by integration into the local neural milieu, accompanied by stable gene expression from NSCs. These unique abilities of NSCs to home in tumor cells followed by the delivery of a desired gene product make NSCs a very promising agent for the treatment of malignant brain tumors that remain virtually untreatable and inevitably lethal despite extensive surgical excision and adjuvant therapy. In this study, we have isolated neurospheres from telencephalon of gestational age 13 weeks of human fetal CNS and engineered them to secrete TRAIL and/or GFP (green fluorescent protein) with adenoviral vectors. TRAIL secreting human neural stem cells (hNSCs) showed normal growth pattern in vitro, but induced apoptosis in human glioblastoma cells after co-cultured with U87MG or U343MG cell lines. When TRAIL-secreting hNSCs implanted into intracranial glioblastomas in adult athymic nude mice, they distributed extensively throughout the tumor bed and migrated widely to infiltrating tumor satellites, while inducing potent apoptosis in both the main tumor mass and tumor satellites that resulted in a significant reduction in tumor volume. These data suggest the adjunctive use of inherently migratory hNSCs as a delivery vehicle for targeting oncolytic molecules to refractory and invasive brain tumor.restrictio

Exchange-coupled magnetic nanoparticles for efficient heat induction.

The conversion of electromagnetic energy into heat by nanoparticles has the potential to be a powerful, non-invasive technique for biotechnology applications such as drug release, disease treatment and remote control of single cell functions, but poor conversion efficiencies have hindered practical applications so far. In this Letter, we demonstrate a significant increase in the efficiency of magnetic thermal induction by nanoparticles. We take advantage of the exchange coupling between a magnetically hard core and magnetically soft shell to tune the magnetic properties of the nanoparticle and maximize the specific loss power, which is a gauge of the conversion efficiency. The optimized core-shell magnetic nanoparticles have specific loss power values that are an order of magnitude larger than conventional iron-oxide nanoparticles. We also perform an antitumour study in mice, and find that the therapeutic efficacy of these nanoparticles is superior to that of a common anticancer drug.ope

Biodegradable Nanotopography Combined with Neurotrophic Signals Enhances Contact Guidance and Neuronal Differentiation of Human Neural Stem Cells

Biophysical cues provided by nanotopographical surfaces have been used as stimuli to guide neurite extension and regulate neural stem cell (NSC) differentiation. Here, we fabricated biodegradable polymer substrates with nanoscale topography for enhancing human NSC (hNSC) differentiation and guided neurite outgrowth. The substrate was constructed from biodegradable poly(lactic-co-glycolic acid) (PLGA) using solvent-assisted capillary force lithography. We found that precoating with 3,4-dihydroxy-l-phenylalanine (DOPA) facilitated the immobilization of poly-l-lysine and fibronectin on PLGA substrates via bio-inspired catechol chemistry. The DOPA-coated nanopatterned substrates directed cellular alignment along the patterned grooves by contact guidance, leading to enhanced focal adhesion, skeletal protein reorganization, and neuronal differentiation of hNSCs as indicated by highly extended neurites from cell bodies and increased expression of neuronal markers (Tuj1 and MAP2). The addition of nerve growth factor further enhanced neuronal differentiation of hNSCs, indicating a synergistic effect of biophysical and biochemical cues on NSC differentiation. These bio-inspired PLGA nanopatterned substrates could potentially be used as implantable biomaterials for improving the efficacy of hNSCs in treating neurodegenerative diseases.restrictio