Il Sun Kim

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.

Human neurospheres derived from the fetal central nervous system are regionally and temporally specified but are not committed.

Proliferating single cells were isolated from various CNS regions (telencephalon, diencephalon, midbrain, cerebellum, pons and medulla, and spinal cord) of human fetal cadavers at 13 weeks of gestation and grown as neurospheres in long-term cultures. We investigated whether neural stem cells (NSCs) or progenitors within spheres have specific regional or temporal characteristics with regard to growth, differentiation, and region-specific gene expression, and whether these molecular specifications are reversible. Regardless of regional origin, all of the neurospheres were found to contain cells of different subtypes, which suggests that multipotent NSCs, progenitors or radial glial cells co-exist with restricted neuronal or glial progenitors within the neurospheres. Neurospheres from the forebrain grew faster and gave rise to significantly more neurons than did those from either the midbrain or hindbrain, and regional differences in neuronal differentiation appeared to be sustained during long-term passage of neurospheres in culture. There was also a trend towards a reduction in neuronal emergence from the respective neurospheres over time in culture, although the percentages of neurons generated from cerebellum-derived neurospheres increased dramatically. These results suggest that differences in neuronal differentiation for the various neurospheres are spatially and temporally determined. In addition, the properties of glial fibrillary acidic protein (GFAP)-, glutamate-, and gamma-aminobutyric acid (GABA)-expressing cells derived from neurospheres of the respective CNS regions appear to be regionally and temporally different. Isolated human neurospheres from different CNS compartments expressed distinctive molecular markers of regional identity and maintained these patterns of region-specific gene expression during long-term passage in vitro. To determine the potential of human neurospheres for regional fate plasticity, single spheres from the respective regions were co-cultured with embryonic day 16.5 (E16.5 d) mouse brain slices. Specific cues from the developing mouse brain tissues induced the human neurospheres to express different marker genes of regional identity and to suppress the expression of their original marker genes. Thus, even the early regional identities of human neurospheres may not be irreversible and may be altered by local inductive cues. These findings have important implications for understanding the characteristics of growth, differentiation, and molecular specification of human neurospheres derived from the developing CNS, as well as the therapeutic potential for neural repair.

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.

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.

Clinical Trial of Human Fetal Brain-Derived Neural Stem/Progenitor Cell Transplantation in Patients with Traumatic Cervical Spinal Cord Injury

In a phase I/IIa open-label and nonrandomized controlled clinical trial, we sought to assess the safety and neurological effects of human neural stem/progenitor cells (hNSPCs) transplanted into the injured cord after traumatic cervical spinal cord injury (SCI). Of 19 treated subjects, 17 were sensorimotor complete and 2 were motor complete and sensory incomplete. hNSPCs derived from the fetal telencephalon were grown as neurospheres and transplanted into the cord. In the control group, who did not receive cell implantation but were otherwise closely matched with the transplantation group, 15 patients with traumatic cervical SCI were included. At 1 year after cell transplantation, there was no evidence of cord damage, syrinx or tumor formation, neurological deterioration, and exacerbating neuropathic pain or spasticity. The American Spinal Injury Association Impairment Scale (AIS) grade improved in 5 of 19 transplanted patients, 2 (A → C), 1 (A → B), and 2 (B → D), whereas only one patient in the control group showed improvement (A → B). Improvements included increased motor scores, recovery of motor levels, and responses to electrophysiological studies in the transplantation group. Therefore, the transplantation of hNSPCs into cervical SCI is safe and well-tolerated and is of modest neurological benefit up to 1 year after transplants. This trial is registered with Clinical Research Information Service (CRIS), Registration Number: KCT0000879.

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.

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.

Neurogenin-2-transduced human neural progenitor cells attenuate neonatal hypoxic-ischemic brain injury

&NA; Neonatal hypoxic‐ischemic (HI) brain injury leads to high mortality and neurodevelopmental disabilities. Multipotent neural progenitor cells (NPCs) with self‐renewing capacity have the potential to reduce neuronal loss and improve the compromised environment in the HI brain injury. However, the therapeutic efficacy of neuronal‐committed progenitor cells and the underlying mechanisms of recovery are not yet fully understood. Therefore, this study investigated the regenerative ability and action mechanisms of neuronally committed human NPCs (hNPCs) transduced with neurogenin‐2 (NEUROG2) in neonatal HI brain injury. NEUROG2‐ or green fluorescent protein (GFP)–encoding adenoviral vector–transduced hNPCs (NEUROG2‐ or GFP‐NPCs) were transplanted into neonatal mouse brains with HI injury. Grafted NEUROG2‐NPCs showed robust dispersion and engraftment, prolonged survival, and neuronal differentiation in HI brain injury. NEUROG2‐NPCs significantly improved neurological behaviors, decreased cellular apoptosis, and increased the neurite outgrowth and axonal sprouting in HI brain injury. In contrast, GFP‐NPC grafts moderately enhanced axonal extension with limited behavioral recovery. Notably, NEUROG2‐NPCs showed increased secretion of multiple factors, such as nerve growth factor, brain‐derived neurotrophic factor, neurotrophin‐3 (NTF3), fibroblast growth factor 9 (FGF9), ciliary neurotrophic factor (CNTF), and thrombospondins 1 and 2 (THBS 1/2), which promoted SH‐SY5Y neuroblastoma cell survival and neurite outgrowth. Thus, we postulate that NEUROG2‐expressing human NPCs facilitate functional recovery after neonatal HI brain injury via their ability to secrete multiple factors that enhance neuronal survival and neuroplasticity.

TNF-α induces human neural progenitor cell survival after oxygen-glucose deprivation by activating the NF-κB pathway

Neural progenitor cell (NPC) transplantation has been shown to be beneficial in the ischemic brain. However, the low survival rate of transplanted NPCs in an ischemic microenvironment limits their therapeutic effects. Tumor necrosis factor-alpha (TNF-α) is one of the proinflammatory cytokines involved in the pathogenesis of various injuries. On the other hand, several studies have shown that TNF-α influences the proliferation, survival, and differentiation of NPCs. Our study investigated the effect of TNF-α pretreatment on human NPCs (hNPCs) under ischemia-related conditions in vitro. hNPCs harvested from fetal brain tissue were pretreated with TNF-α before being subjected to oxygen–glucose deprivation (OGD) to mimic ischemia in vitro. TNF-α pretreatment improved the viability and reduced the apoptosis of hNPCs after OGD. At the molecular level, TNF-α markedly increased the level of NF-κB signaling in hNPCs, and an NF-κB pathway inhibitor, BAY11-7082, completely reversed the protective effects of TNF-α on hNPCs. These results suggest that TNF-α improves hNPC survival by activating the NF-κB pathway. In addition, TNF-α significantly enhanced the expression of cellular inhibitor of apoptosis 2 (cIAP2). Use of a lentivirus-mediated short hairpin RNA targeting cIAP2 mRNA demonstrated that cIAP2 protected against OGD-induced cytotoxicity in hNPCs. Our study of intracellular NF-κB signaling revealed that inhibition of NF-κB activity abolished the TNF-α-mediated upregulation of cIAP2 in hNPCs and blocked TNF-α-induced cytoprotection against OGD. Therefore, this study suggests that TNF-α pretreatment, which protects hNPCs from OGD-induced apoptosis by activating the NF-κB pathway, provides a safe and simple approach to improve the viability of transplanted hNPCs in cerebral ischemia.Stroke: creating a safe haven for neurological repairA potent “survival signal” for brain stem cells could enable effective regenerative therapies for stroke patients. Neural progenitor cells (NPCs) can develop into functional neurons and supportive glial cells, and researchers are tantalized by the prospect of using NPCs to repair damaged brain tissue. NPCs generally fail to flourish after transplantation, but a team led by Kook In Park at Yonsei University College of Medicine, South Korea, have found a signaling factor that helps these cells to survive and divide. Tumor necrosis factor-α (TNF-α) is associated with inflammation, but also protects neurons after a stroke. The researchers showed that pretreatment with TNF-α preserved NPCs exposed to starvation and oxygen-deprivation conditions in cell culture by activating critical cell survival pathways. These findings suggest that TNF-α may enable NPCs to survive long enough to repair post-stroke neurological damage.

Inverted Quasi‐Spherical Droplets on Polydopamine–TiO2 Substrates for Enhancing Gene Delivery

Devising efficient gene delivery systems is crucial to enhancing the therapeutic efficacy of gene-cell therapy approaches. Herein, inverted quasi-spherical (iQS) droplet systems, which enhance gene delivery efficiencies by reducing the path lengths of gene vectors, mediating motions of vectors at early stages, and raising the contact frequencies of vectors with cells, are developed by adopting the principle of 3D hanging-drop cell culture. Micrometer-sized polydopamine (pDA) holes are created on superhydrophobic titanium isopropoxide (TiO2 )-coated substrates by physical scraping; droplets are loaded on the pDA holes, and inversion of the substrate generates iQS droplets with large contact angles. Both human neural stem cells (hNSCs) and adeno-associated viral vectors are simultaneously incorporated into the iQS droplets to assess gene delivery efficiencies. The steep angles of iQS droplets and enhanced cell/vector contact frequencies facilitate the viral association with hNSCs and enhancing cell-cell interactions, thereby significantly promoting gene delivery efficiencies. Even with reduced viral quantities/exposure times and cell numbers, the iQS droplet systems elicit sufficient gene expression (i.e., interleukin-10). The ability of the iQS droplet systems to maximize beneficial gene delivery effects with minimal materials (e.g., medium, cells, and vectors) should enable their extensive use as a platform for preparing genetically stimulated cellular therapeutics.