Neural Cell Transplantation Research Articles

IgG Response to Intracerebral Xenotransplantation: Specificity and Role in the Rejection of Porcine Neurons

Xenogenic fetal neuroblasts are considered as a potential source of transplantable cells for the treatment of neurodegenerative diseases, but immunological barriers limit their use in the clinic. While considerable work has been performed to decipher the role of the cellular immune response in the rejection of intracerebral xenotransplants, there is much still to learn about the humoral reaction. To this end, the IgG response to the transplantation of fetal porcine neural cells (PNC) into the rat brain was analyzed. Rat sera did not contain preformed antibodies against PNC, but elicited anti-porcine IgG was clearly detected in the host blood once the graft was rejected. Only the IgG1 and IgG2a subclasses were up-regulated, suggesting a T-helper 2 immune response. The main target of these elicited IgG antibodies was porcine neurons, as determined by double labeling in vitro and in vivo. Complement and anti-porcine IgG were present in the rejecting grafts, suggesting an active role of the host humoral response in graft rejection. This hypothesis was confirmed by the prolonged survival of fetal porcine neurons in the striatum of immunoglobulin-deficient rats. These data suggest that the prolonged survival of intracerebral xenotransplants relies on the control of both cell-mediated and humoral immune responses.

Long-term fate of grafted hippocampal neural progenitor cells following ischemic injury.

The adult CNS has a very limited capacity to regenerate neurons after insult. To overcome this limitation, the transplantation of neural progenitor cells (NPCs) has developed into a key strategy for neuronal replacement. This study assesses the long-term survival, migration, differentiation, and functional outcome of NPCs transplanted into the ischemic murine brain. Hippocampal neural progenitors were isolated from FVB-Cg-Tg(GFPU)5Nagy/J transgenic mice expressing green fluorescent protein (GFP). Syngeneic GFP-positive NPCs were stereotactically transplanted into the hippocampus of FVB mice following a transient global cerebral ischemia model. Behavioral tests revealed that ischemia/reperfusion induced spatial learning disturbances in the experimental animals. The NPC transplantation promoted cognitive function recovery after ischemic injury. To study the long-term fate of grafted GFP-positive NPCs in a host brain, immunohistochemical approaches were applied. Confocal microscopy revealed that grafted cells survived in the recipient tissue for 90 days following transplantation and differentiated into mature neurons with extensive dendritic trees and apparent spines. Immunoelectron microscopy confirmed the formation of synapses between the transplanted GFP-positive cells and host neurons that may be one of the factors underlying cognitive function recovery. Repair and functional recovery following brain damage represent a major challenge for current clinical and basic research. Our results provide insight into the therapeutic potential of transplanted hippocampal progenitor cells following ischemic brain injury.

Human induced pluripotent stem cells are a novel source of neural progenitor cells (iNPCs) that migrate and integrate in the rodent spinal cord

Transplantation of human neural progenitor cells (NPCs) into the brain or spinal cord to replace lost cells, modulate the injury environment, or create a permissive milieu to protect and regenerate host neurons is a promising therapeutic strategy for neurological diseases. Deriving NPCs from human fetal tissue is feasible, although problematic issues include limited sources and ethical concerns. Here we describe a new and abundant source of NPCs derived from human induced pluripotent stem cells (iPSCs). A novel chopping technique was used to transform adherent iPSCs into free-floating spheres that were easy to maintain and were expandable (EZ spheres) (Ebert et al. [2013] Stem Cell Res 10:417-427). These EZ spheres could be differentiated towards NPC spheres with a spinal cord phenotype using a combination of all-trans retinoic acid (RA) and epidermal growth factor (EGF) and fibroblast growth factor-2 (FGF-2) mitogens. Suspension cultures of NPCs derived from human iPSCs or fetal tissue have similar characteristics, although they were not similar when grown as adherent cells. In addition, iPSC-derived NPCs (iNPCs) survived grafting into the spinal cord of athymic nude rats with no signs of overgrowth and with a very similar profile to human fetal-derived NPCs (fNPCs). These results suggest that human iNPCs behave like fNPCs and could thus be a valuable alternative for cellular regenerative therapies of neurological diseases.

Comparative Study of Efficacy of Dopaminergic Neuron Differentiation between Embryonic Stem Cell and Protein-Based Induced Pluripotent Stem Cell

In patients with Parkinson's disease (PD), stem cells can serve as therapeutic agents to restore or regenerate injured nervous system. Here, we differentiated two types of stem cells; mouse embryonic stem cells (mESCs) and protein-based iPS cells (P-iPSCs) generated by non-viral methods, into midbrain dopaminergic (mDA) neurons, and then compared the efficiency of DA neuron differentiation from these two cell types. In the undifferentiated stage, P-iPSCs expressed pluripotency markers as ES cells did, indicating that protein-based reprogramming was stable and authentic. While both stem cell types were differentiated to the terminally-matured mDA neurons, P-iPSCs showed higher DA neuron-specific markers' expression than ES cells. To investigate the mechanism of the superior induction capacity of DA neurons observed in P-iPSCs compared to ES cells, we analyzed histone modifications by genome-wide ChIP sequencing analysis and their corresponding microarray results between two cell types. We found that Wnt signaling was up-regulated, while SFRP1, a counter-acting molecule of Wnt, was more suppressed in P-iPSCs than in mESCs. In PD rat model, transplantation of neural precursor cells derived from both cell types showed improved function. The present study demonstrates that P-iPSCs could be a suitable cell source to provide patient-specific therapy for PD without ethical problems or rejection issues.

Transplanting neural progenitors into a complete transection model of spinal cord injury

Neural progenitor cell (NPC) transplantation is a promising therapeutic strategy for spinal cord injury (SCI) because of the potential for cell replacement and restoration of connectivity. Our previous studies have shown that transplants of NPC, composed of neuron- and glia-restricted progenitors derived from the embryonic spinal cord, survived well in partial lesion models and generated graft-derived neurons, which could be used to form a functional relay. We have now examined the properties of a similar NPC transplant using a complete transection model in juvenile and adult rats. We found poor survival of grafted cells despite using a variety of lesion methods, matrices, and delays of transplantation. If, instead of cultured progenitor cells, the transplants were composed of segmental or dissociated segments of fetal spinal cord (FSC) derived from similar-staged embryos, grafted cells survived and integrated well with host tissue in juvenile and adult rats. FSC transplants differentiated into neurons and glial cells, including astrocytes and oligodendrocytes. Graft-derived neurons expressed glutaminergic and GABAergic markers. Grafted cells also migrated and extended processes into host tissue. Analysis of axon growth from the host spinal cord showed serotonin-positive fibers and biotinylated dextran amine-traced propriospinal axons growing into the transplants. These results suggest that in treating severe SCI, such as complete transection, NPC grafting faces major challenges related to cell survival and formation of a functional relay. Lessons learned from the efficacy of FSC transplants could be used to develop a therapeutic strategy based on neural progenitor cells for severe SCI.

A Novel In Vivo Model of Permanent Intestinal Aganglionosis

Hirschsprung’s Disease is characterized by absent ganglia in the distal gastrointestinal tract. Surgical correction may incur significant morbidity. Alternatively, neural progenitor cell transplantation may regenerate the enteric nervous system. Existing aganglionosis model systems are limited by swift animal demise or by spontaneous regeneration of native ganglia. We propose a novel protocol to induce permanent aganglionosis in a segment of rat jejunum, which may serve as an experimental transplantation target for cellular therapy.

Exogenous BDNF and Chondroitinase ABC Consisted Biomimetic Microenvironment Regulates Survival, Migration and Differentiation of Human Neural Progenitor Cells Transplanted into a Rat Auditory Nerve

Current putative regeneration oriented studies express possible role of stem cell based implantation strategy in the restoration of fundamental perception of hearing. The present work utilizes a rat auditory nerve (AN) directed transplantation of human neural progenitor cells (HNPCs) as a cell replacement therapy for impaired auditory function. Groups of b-bungarotoxin induced auditory function compromised female rats were used to transplant HNPCs in the nerve trunk. In the treatment groups, brain derived neurotrophic factor (BDNF), peptide amphiphile nanofiber bioactive gel (Bgel) and Chondroitinase ABC (ChABC), a digestive enzyme that cleaves the core of chondroitin sulphate proteoglycans, were added along with HNPCs while the control groups were with PA inert gel (Igel) and devoid of ChABC. Six weeks post transplantation survival, migration, and differentiation of HNPCs were studied and compared. The groups treated with BDNF and Bgel showed improved survival and differentiation of transplanted HNPCs while the ChABC treated group showed significant migration of HNPCs along the AN and elongation of neuronal fibers along the nerve towards the cochlear nucleus (CN) which was characterized by immunocytochemical markers for human Nuclei (HuN), human mitochondria (HuM) and neuronal β-tubulin (Tuj1). These findings show that addition of BDNF and ChABC consisted Bgel environment facilitated HNPC survival, migration and differentiation along the transplanted rat AN towards the CN. This transplantation strategy provides unique experimental validation for futuristic role of cell based biomaterial consisted neurotrophic factor application in clinically transferable treatment of sensorineural hearing loss (SNHL) along with cochlear implants (CI).

Spinal root avulsion: an excellent model for studying motoneuron degeneration and regeneration after severe axonal injury

Spinal root avulsion: an excellent model for studying motoneuron degeneration and regeneration after severe axonal injury

Improvements in biomaterial matrices for neural precursor cell transplantation.

Progress is being made in developing neuroprotective strategies for traumatic brain injuries; however, there will never be a therapy that will fully preserve neurons that are injured from moderate to severe head injuries. Therefore, to restore neurological function, regenerative strategies will be required. Given the limited regenerative capacity of the resident neural precursors of the CNS, many investigators have evaluated the regenerative potential of transplanted precursors. Unfortunately, these precursors do not thrive when engrafted without a biomaterial scaffold. In this article we review the types of natural and synthetic materials that are being used in brain tissue engineering applications for traumatic brain injury and stroke. We also analyze modifications of the scaffolds including immobilizing drugs, growth factors and extracellular matrix molecules to improve CNS regeneration and functional recovery. We conclude with a discussion of some of the challenges that remain to be solved towards repairing and regenerating the brain.

Magnetic nanoparticles for oligodendrocyte precursor cell transplantation therapies: progress and challenges.

Oligodendrocyte precursor cells (OPCs) have shown high promise as a transplant population to promote regeneration in the central nervous system, specifically, for the production of myelin – the protective sheath around nerve fibers. While clinical trials for these cells have commenced in some areas, there are currently key barriers to the translation of neural cell therapies. These include the ability to (a) image transplant populations in vivo; (b) genetically engineer transplant cells to augment their repair potential; and (c) safely target cells to sites of pathology. Here, we review the evidence that magnetic nanoparticles (MNPs) are a ‘multifunctional nanoplatform’ that can aid in safely addressing these translational challenges in neural cell/OPC therapy: by facilitating real-time and post-mortem assessment of transplant cell biodistribution, and biomolecule delivery to transplant cells, as well as non-invasive ‘magnetic cell targeting’ to injury sites by application of high gradient fields. We identify key issues relating to the standardization and reporting of physicochemical and biological data in the field; we consider that it will be essential to systematically address these issues in order to fully evaluate the utility of the MNP platform for neural cell transplantation, and to develop efficacious neurocompatible particles for translational applications.Electronic supplementary materialThe online version of this article (doi:10.1186/2052-8426-2-23) contains supplementary material, which is available to authorized users.

Transplantation of GABAergic Cells Derived from Bioreactor-Expanded Human Neural Precursor Cells Restores Motor and Cognitive Behavioral Deficits in a Rodent Model of Huntington's Disease

Huntington's disease (HD) is a neurodegenerative disorder that is characterized by progressive dementia, choreiform involuntary movements, and emotional deterioration. Neuropathological features include the progressive degeneration of striatal γ-aminobutyric acid (GABA) neurons. New therapeutic approaches, such as the transplantation of human neural precursor cells (hNPCs) to replace damaged or degenerated cells, are currently being investigated. The aim of this study was to investigate the potential for utilizing telencephalic hNPCs expanded in suspension bioreactors for cell restorative therapy in a rodent model of HD. hNPCs were expanded in a hydrodynamically controlled and homogeneous environment under serum-free conditions. In vitro analysis revealed that the bioreactor-expanded telencephalic (BET)-hNPCs could be differentiated into a highly enriched population of GABAergic neurons. Behavioral assessments of unilateral striatal quinolinic acid-lesioned rodents revealed a significant improvement in motor and memory deficits following transplantation with GABAergic cells differentiated from BET-hNPCs. Immunohistochemical analysis revealed that transplanted BET-hNPCs retained a GABAergic neuronal phenotype without aberrant transdifferentiation or tumor formation, indicating that BET-hNPCs are a safe source of cells for transplantation. This preclinical study has important implications as the transplantation of GABAergic cells derived from predifferentiated BET-hNPCs may be a safe and feasible cell replacement strategy to promote behavioral recovery in HD.

Administration of mesenchymal stem cells and ziprasidone enhanced amelioration of ischemic brain damage in rats.

Ziprasidone is a benzisothiazolyl piperazine derivative that was developed from the chemically related antipsychotic drug tiospirone, and it improves neurological functions of the ischemic brain and is effective in treatment of schizophrenia. Mesenchymal stem cells (MSCs) are considered as a leading candidate for neurological regenerative therapy because of their neural differentiation properties in damaged brain. We investigated whether the transplantation of neural progenitor cells (NPCs) derived from adipose mesenchymal stem cells combined with ziprasidone enhances neuroprotective effects in an animal model of focal cerebral ischemia. In combination therapy groups, significant reduction of infarct volume and improvement of neurological functions were observed at 3 days after middle cerebral artery occlusion (MCAO) compared with monotherapy. Co-administration of ziprasidone and NPCs enhanced the anti-apoptotic effect and reduced the number of terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)-positive apoptotic cells compared with the NPCs alone group at 7 days after MCAO. Ziprasidone or the combination of ziprasidone and NPCs induced the expression of endogenous neurotrophic factor gene brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF), and glial cell-derived neurotrophic factor (GDNF). The immunohistochemical investigation revealed that the ziprasidone and NPCs attenuated the increased intensity of microglial marker (Iba-1) in the infarcted cortical area. Moreover, the number of transplanted NPCs on day 7 with combination therapy was significantly higher than with NPCs alone. These effects might be responsible for improved functional behavior and increased survival of NPCs. Our finding indicates that combination therapy of ziprasidone and NPCs enhances neuroprotection against ischemic brain injury.

Bridging defects in chronic spinal cord injury using peripheral nerve grafts combined with a chitosan-laminin scaffold and enhancing regeneration through them by co-transplantation with bone-marrow-derived mesenchymal stem cells: Case series of 14 patients

ObjectiveTo investigate the effect of bridging defects in chronic spinal cord injury using peripheral nerve grafts combined with a chitosan-laminin scaffold and enhancing regeneration through them by co-transplantation with bone-marrow-derived mesenchymal stem cells.MethodsIn 14 patients with chronic paraplegia caused by spinal cord injury, cord defects were grafted and stem cells injected into the whole construct and contained using a chitosan-laminin paste. Patients were evaluated using the International Standards for Classification of Spinal Cord Injuries.ResultsChitosan disintegration leading to post-operative seroma formation was a complication. Motor level improved four levels in 2 cases and two levels in 12 cases. Sensory-level improved six levels in two cases, five levels in five cases, four levels in three cases, and three levels in four cases. A four-level neurological improvement was recorded in 2 cases and a two-level neurological improvement occurred in 12 cases. The American Spinal Impairment Association (ASIA) impairment scale improved from A to C in 12 cases and from A to B in 2 cases. Although motor power improvement was recorded in the abdominal muscles (2 grades), hip flexors (3 grades), hip adductors (3 grades), knee extensors (2–3 grades), ankle dorsiflexors (1–2 grades), long toe extensors (1–2 grades), and plantar flexors (0–2 grades), this improvement was too low to enable them to stand erect and hold their knees extended while walking unaided.ConclusionMesenchymal stem cell-derived neural stem cell-like cell transplantation enhances recovery in chronic spinal cord injuries with defects bridged by sural nerve grafts combined with a chitosan-laminin scaffold.

The experimental research between magnetic resonance spectroscopy and changes of behavior for neural stem cell transplantation in APP/PS1 transgenic mice

Objective To explore the effect of neural stem cell(NSCs) transplantation on proton magnetic resonance spectroscopy (1H-MRS) and the behavior in APP/PS1 double transgenic AD mice. Methods NSCs from C57BL/6 mice were cultured and amplified. APP/PS1 double transgenic AD mice (n=30) aged 12 months were used as the study group, and mild-type mice (n=15) were used as the control group(group C ). Animals in the study group were randomly divided into two subgroups: one receiving NSCs (group A) and the other receiving PBS transplantation (group B) in bilateral hippocampal CA1 of the AD model mice. Animals in the group C were not treated. 1H-MRS and Morris water maze (MWM) were performed before transplantation and 4 weeks after transplantation, and compared with the histopathological results. Results 1H-MRS showed that there was no significant change in NAA/ Cr(1.01±0.08 and 1.03±0.05) and mI/Cr (0.69±0.05 and 0.71±0.06) ratios between group A and group B before transplantation (P> 0.05), but the changes were significant compared with the group C(NAA/ Cr: 1.21±0.05; mI/Cr: 0.58±0.06) (P<0.05). Four weeks after transplantation, NAA/ Cr ratio(1.18±0.09) was increased and mI/ Cr ratio (0.53±0.04) was decreased in group A. The difference was significant compared with the group B at the same time points (P<0.05). MWM showed the escape latency in group A was significantly shorter than that in group B after transplantation (P<0.05). In addition, group A also showed an exclusive preference for the target quadrant, and spent more time((35.21±5.44)s) in the 3rd quadrant compared with group B (P<0.05). For number of platform crossings, similar results were also shown(5.75±3.23). Nissl's staining showed that the number of neurons in the hippocampal area increased more significantly in group A than those in group B(P<0. 05). Conclusion NSCs transplantation can improve spatial learning and memory via neurons regeneration in APP/PS1 double transgenic AD mice, and 1H-MRS is able to display intracranial metabolite changes after NSCs transplantation. Key words: Neural stem cell; Alzheimer disease; Magnetic resonance spectroscopy; Transplantation

Effect of neural stem cell transplantation on cognitive functions of mice after cerebral ischemia-reperfusion

This study is aimed to determine the effect of transplantation of neural progenitor cells (NPCs) isolated from fetal hippocampus on cognitive functions of experimental animals after short-term global cerebral ischemia. NPCs were isolated from hippocampus of FVB-Cg-Tg(GFPU)5Nagy/J mice, transgenic by the GFP. Ischemic brain injury in FVB “wild” type mice was modeled by bilateral occlusion of the common carotid arteries for 20 min. GFP-positive NPCs were stereotaxically transplanted into the hippocampus of experimental animals in 24 hours after ischemia-reperfusion. Cognitive functions were evaluated using Morris water maze. Results of this study showed that global short-term cerebral ischemia resulted into cognitive impairments in mice. Stereotaxic transplantation of NPCs promoted the cognitive function recovery in experimental animals after ischemic brain injury. Thus, the data indicates that transplantation of NPCs may have a therapeutic effect in treating of ischemic stroke.

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.

Progenitor Cells in Chromospheres: In Response to Arthur S. Tischler

This is in response to the letter “What Happens in ‘Chromospheres’?” by Arthur S. Tischler, commenting on our article titled “Isolation, Characterization, and Differentiation of Progenitor Cells From Human Adult Adrenal Medulla” [1]. We would like to acknowledge the comments, the interesting discussion, and potential other point of view on our results. We agree with Dr. Tischler about the fact that human chromaffin cells have the capacity to acquire a neuron-like phenotype in monolayer cultures without a “chromosphere” stage. We also observed this phenomenon in a previous study using human adrenal chromaffin cells in monolayer cultures [2]. Since the cell culture conditions of human chromaffin cells in monolayer cultures and suspension chromosphere cultures are different, careful investigation should be made before it is assumed that the same changes are occurring. Therefore, as suggested by Dr. Tischler, it will be important to compare the two cell culture types in terms of cell proliferation capacity, expression of progenitor cell markers, duration of cell culture, regulatory peptide expression, and catecholamine content. This new set of experiments will provide information on whether the mechanisms required for chromaffin cell transdifferentiation will involve a dedifferentiation or an unnatural intermediate step [3]. Even if we do not know the mechanism, and even if we did not directly evaluate the proliferation capacity, human chromospheres, like bovine chromospheres [4], express nine markers of progenitor cells (nestin, CD133, Notch1, nerve growth factor receptor, Snai2, Sox 9, Sox 10, Phox2b, and Ascl1), and more importantly, nestin-positive cells are present in human adult adrenal medulla, suggesting the presence of progenitor cells in human adult adrenal medulla. The possibility that a “priming” process, with an upregulation of genes required for transdifferentiation, also occurs during chromosphere culture cannot be discarded. Nevertheless, we think that this could also represent an advantage of the use of chromospheres as a source of cells for clinical purposes. The survival rate of chromosphere cells after grafting is currently being investigated in animal experiments. From data available on the transplantation of neural progenitor cells, we expect that the number of spheres isolated from one adrenal gland (27,000–280,000) should give rise to enough cells for grafting. Moreover, one of the major problems of using embryonic stem cells and induced pluripotent stem cells still is their tumorigenicity. Therefore, if future data demonstrate that human chromospheres have a low cell proliferation capacity, this should emphasize the interest in their use for regenerative medicine.

Optimizing a multifunctional microsphere scaffold to improve neural precursor cell transplantation for traumatic brain injury repair.

Tissue engineering using stem cells is widely used to repair damaged tissues in diverse biological systems; however, this approach has met with less success in regenerating the central nervous system (CNS). In this study we optimized and characterized the surface chemistry of chitosan-based scaffolds for CNS repair. To maintain radial glial cell (RGC) character of primitive neural precursors, fibronectin was adsorbed to chitosan. The chitosan was further modified by covalently linking heparin using genipin, which then served as a linker to immobilize fibroblast growth factor-2 (FGF-2), creating a multifunctional film. Fetal rat neural precursors plated onto this multifunctional film proliferated and remained multipotent for at least 3 days without providing soluble FGF-2. Moreover, they remained less mature and more highly proliferative than cells maintained on fibronectin-coated substrates in culture medium supplemented with soluble FGF-2. To create a vehicle for cell transplantation, a 3% chitosan solution was electrosprayed into a coagulation bath to generate microspheres (range 30-100 µm, mean 64 µm) that were subsequently modified. Radial glial cells seeded onto these multifunctional microspheres proliferated for at least 7 days in culture and the microspheres containing cells were small enough to be injected, using 23 Gauge Hamilton syringes, into the brains of adult rats that had previously sustained cortical contusion injuries. When analysed 3 days later, the transplanted RGCs were positive for the stem cell/progenitor marker Nestin. These results demonstrate that this multifunctional scaffold can be used as a cellular and growth factor delivery vehicle for the use in developing cell transplantation therapies for traumatic brain injuries. Copyright © 2013 John Wiley & Sons, Ltd.

Transplantation of Neural Crest-Like Cells Derived from Induced Pluripotent Stem Cells Improves Diabetic Polyneuropathy in Mice

Impaired vascularity and nerve degeneration are the most important pathophysiological abnormalities of diabetic polyneuropathy (DPN). Therefore, regeneration of both the vascular and nervous systems is required for the treatment of DPN. The neural crest (NC) is a transient embryonic structure in vertebrates that differentiates into a vast range of cells, including peripheral neurons, Schwann cells, and vascular smooth muscle cells. In this study, we investigated the ability of transplantation of NC-like (NCL) cells derived from aged mouse induced pluripotent stem (iPS) cells in the treatment of DPN. iPS cells were induced to differentiate into neural cells by stromal cell-derived inducing activity (SDIA) and subsequently supplemented with bone morphogenetic protein 4 to promote differentiation of NC lineage. After the induction, p75 neurotrophin receptor-positive NCL cells were purified using magnetic-activated cell sorting. Sorted NCL cells differentiated to peripheral neurons, glial cells, and smooth muscle cells by additional SDIA. NCL cells were transplanted into hind limb skeletal muscles of 16-week streptozotocin-diabetic mice. Nerve conduction velocity, current perception threshold, intraepidermal nerve fiber density, sensitivity to thermal stimuli, sciatic nerve blood flow, plantar skin blood flow, and capillary number-to-muscle fiber ratio were evaluated. Four weeks after transplantation, the engrafted cells produced growth factors: nerve growth factor, neurotrophin 3, vascular endothelial growth factor, and basic fibroblast growth factor. It was also confirmed that some engrafted cells differentiated into vascular smooth muscle cells or Schwann cell-like cells at each intrinsic site. The transplantation improved the impaired nerve and vascular functions. These results suggest that transplantation of NCL cells derived from iPS cells could have therapeutic effects on DPN through paracrine actions of growth factors and differentiation into Schwann cell-like cells and vascular smooth muscle cells.

Improved Method of Producing Human Neural Progenitor Cells of High Purity and in Large Quantities from Pluripotent Stem Cells for Transplantation Studies

Transplantation of human neural progenitor cells (hNPCs) is a promising therapeutic approach for various diseases of the central nervous system (CNS). Reliable testing of hNPC transplantation in animal models of neurological diseases requires that these cells can be produced in sufficient amounts, show consistent homogeneity as a neural cell population, and be reliably labeled for in vivo tracking. In addition, the cells should be characterized as being at the optimal state of differentiation favoring successful engraftment. Here, we show that high numbers of purified hNPCs can be produced from human embryonic stem cells (hESCs) by manually selecting specifically sized and shaped spheres followed by fluorescence-activated cell sorting based on the relative cell size. In addition, we report that labeling of hNPCs with ultra-small superparamagnetic iron oxide (USPIO) particles does not affect the cellular morphology or growth. More importantly, we show that the transduction with lentiviral vector encoding green fluorescent protein (GFP) decreases the neurality of the cell population. We conclude that our cost-effective protocol of generating hNPCs is widely applicable for preclinical studies on CNS disorders. This improved method of producing large quantities of high-purity hNPCs maybe useful also when generating hNPCs from human induced pluripotent stem (hiPS) cell lines. However, caution should be used when lenti-GFP transduction is applied for hNPC labeling.