Cell-derived Neural Progenitor Cells Research Articles

A switch from canonical to noncanonical Wnt signaling mediates early differentiation of human neural stem cells.

Wnt/β-catenin signaling is essential for neurogenesis but less is known about β-catenin-independent Wnt signals. We show here that Wnt/activator protein-1 (AP-1) signaling drives differentiation of human embryonic stem cell and induced pluripotent stem cell-derived neural progenitor cells. Neuronal differentiation was accompanied by a reduction in β-catenin/Tcf-dependent transcription and target gene expression, increased levels and/or phosphorylation of activating transcription factor 2 (ATF2), cyclic AMP response element-binding protein, and c-Jun, and increased AP-1-dependent transcription. Inhibition of Wnt secretion using the porcupine inhibitors IWP-2 and Wnt-C59 blocked neuronal differentiation, while activation or inhibition of Wnt/β-catenin signaling had no effect. Neuronal differentiation increased expression of several Wnt genes, including WNT3A, silencing of which reduced differentiation. Addition of recombinant Wnt-3a to cells treated with IWP-2 or Wnt-C59 increased AP-1 levels and restored neuronal differentiation. The effects of Wnt-3a could not be blocked by addition of Dkk-1 or IWR-1, suggesting the involvement of noncanonical signaling. Consistent with this, restoration of neuronal differentiation by Wnt-3a was reduced by inhibition of Jun N-terminal kinase (JNK) and by gene silencing of ATF2. Together, these observations suggest that β-catenin-independent Wnt signals promote neural stem/progenitor cell differentiation in a signaling pathway involving Wnt-3a, JNK, and ATF2.

Neuronal NF1/RAS regulation of cyclic AMP requires atypical PKC activation.

Neurofibromatosis type 1 (NF1) is a common neurodevelopmental disorder in which affected individuals are prone to learning, attention and behavioral problems. Previous studies in mice and flies have yielded conflicting results regarding the specific effector pathways responsible for NF1 protein (neurofibromin) regulation of neuronal function, with both cyclic AMP (cAMP)- and RAS-dependent mechanisms described. Herein, we leverage a combination of induced pluripotent stem cell-derived NF1 patient neural progenitor cells and Nf1 genetically engineered mice to establish, for the first time, that neurofibromin regulation of cAMP requires RAS activation in human and mouse neurons. However, instead of involving RAS-mediated MEK/AKT signaling, RAS regulation of cAMP homeostasis operates through the activation of atypical protein kinase C zeta, leading to GRK2-driven Gαs inactivation. These findings reveal a novel mechanism by which RAS can regulate cAMP levels in the mammalian brain.

Differentiation, polarization, and migration of human induced pluripotent stem cell-derived neural progenitor cells co-cultured with a human glial cell line with radial glial-like characteristics

Here we established a unique human glial cell line, GDC90, derived from a human glioma and demonstrated its utility as a glial scaffold for the polarization and differentiation of human induced pluripotent stem cell-derived neural progenitor cells (iPSC-NPCs). When co-cultured with GDC90 cells, iPSC-NPCs underwent rapid polarization and neurite extension along the radially spreading processes of the GDC90 cells, and showed migratory behavior. This method is potentially useful for detailed examination of neurites or for controlling neurites behavior for regenerative medicine.

Human Cytomegalovirus Infection of Human Embryonic Stem Cell-Derived Primitive Neural Stem Cells Is Restricted at Several Steps but Leads to the Persistence of Viral DNA

Congenital human cytomegalovirus (HCMV) infection is a major cause of central nervous system structural anomalies and sensory impairments. It is likely that the stage of fetal development, as well as the state of differentiation of susceptible cells at the time of infection, affects the severity of the disease. We used human embryonic stem (ES) cell-derived primitive prerosette neural stem cells (pNSCs) and neural progenitor cells (NPCs) maintained in chemically defined conditions to study HCMV replication in cells at the early stages of neural development. In contrast to what was observed previously using fetus-derived NPCs, infection of ES cell-derived pNSCs with HCMV was nonprogressive. At a low multiplicity of infection, we observed only a small percentage of cells expressing immediate-early genes (IE) and early genes. IE expression was found to be restricted to cells negative for the anterior marker FORSE-1, and treatment of pNSCs with retinoic acid restored IE expression. Differentiation of pNSCs into NPCs restored IE expression but not the transactivation of early genes. Virions produced in NPCs and pNSCs were exclusively cell associated and were mostly non-neural tropic. Finally, we found that viral genomes could persist in pNSC cultures for up to a month after infection despite the absence of detectable IE expression by immunofluorescence, and infectious virus could be produced upon differentiation of pNSCs to neurons. In conclusion, our results highlight the complex array of hurdles that HCMV must overcome in order to infect primitive neural stem cells and suggest that these cells might act as a reservoir for the virus. Human cytomegalovirus (HCMV) is a betaherpesvirus that is highly prevalent in the population. HCMV infection is usually asymptomatic but can lead to severe consequences in immunosuppressed individuals. HCMV is also the most important infectious cause of congenital developmental birth defects. Manifestations of fetal HCMV disease range from deafness and learning disabilities to more severe symptoms such as microcephaly. In this study, we have used embryonic stem cells to generate primitive neural stem cells and have used these to model HCMV infection of the fetal central nervous system (CNS) in vitro. Our results reveal that these cells, which are similar to those present in the developing neural tube, do not support viral replication but instead likely constitute a viral reservoir. Future work will define the effect of viral persistence on cellular functions as well as the exogenous signals leading to the reactivation of viral replication in the CNS.

The ROCK Inhibitor Y-26732 Enhances the Survival and Proliferation of Human Embryonic Stem Cell-Derived Neural Progenitor Cells upon Dissociation

Human neural progenitor cells (hNPCs) are the starting material required for neuronal subtype differentiation. Proliferation of hNPCs allows researchers to study the mechanistic complexities and microenvironments present during neural differentiation and to explore potential applications for hNPCs in cell therapies. The use of enzymatic dissociation during hNPC proliferation causes dissociation-induced apoptosis; therefore, in the present study, we examined the effect of the p-160-Rho-associated coiled-coil kinase (ROCK) inhibitor Y-26732 on dissociation-induced apoptosis of hNPCs. We generated hNPCs via embryoid body formation using serum-free culture medium supplemented with noggin. The established hNPCs were characterized and the effect of the ROCK inhibitor on hNPC dissociation was studied. We demonstrated that supplementation of the culture media with 10 μM Y-26732 efficiently reduced apoptosis of dissociated hNPCs; this supplementation was effective when the inhibitor was applied either at (i) 24 h before dissociation of the cells and at 24 h after plating the cells or (ii) at 24 h after plating of the cells only. In addition to reducing apoptosis, both supplementation conditions with Y-26732 enhanced the proliferation of dissociated hNPCs. Our findings provide the optimal time window for ROCK treatment of hNPC dissociation in respect to apoptosis and cell proliferation.

Transplantation of Bone Marrow Stromal Cell-Derived Neural Precursor Cells Ameliorates Deficits in a Rat Model of Complete Spinal Cord Transection

After severe spinal cord injury, spontaneous functional recovery is limited. Numerous studies have demonstrated cell transplantation as a reliable therapeutic approach. However, it remains unknown whether grafted neuronal cells could replace lost neurons and reconstruct neuronal networks in the injured spinal cord. To address this issue, we transplanted bone marrow stromal cell-derived neural progenitor cells (BM-NPCs) in a rat model of complete spinal cord transection 9 days after the injury. BM-NPCs were induced from bone marrow stromal cells (BMSCs) by gene transfer of the Notch-1 intracellular domain followed by culturing in the neurosphere method. As reported previously, BM-NPCs differentiated into neuronal cells in a highly selective manner in vitro. We assessed hind limb movements of the animals weekly for 7 weeks to monitor functional recovery after local injection of BM-NPCs to the transected site. To test the sensory recovery, we performed functional magnetic resonance imaging (fMRI) using electrical stimulation of the hind limbs. In the injured spinal cord, transplanted BM-NPCs were confirmed to express neuronal markers 7 weeks following the transplantation. Grafted cells successfully extended neurites beyond the transected portion of the spinal cord. Adjacent localization of synaptophysin and PSD-95 in the transplanted cells suggested synaptic formations. These results indicated survival and successful differentiation of BM-NPCs in the severely injured spinal cord. Importantly, rats that received BM-NPCs demonstrated significant motor recovery when compared to the vehicle injection group. Volumes of the fMRI signals in somatosensory cortex were larger in the BM-NPC-grafted animals. However, neuronal activity was diverse and not confined to the original hind limb territory in the somatosensory cortex. Therefore, reconstruction of neuronal networks was not clearly confirmed. Our results indicated BM-NPCs as an effective method to deliver neuronal lineage cells in a severely injured spinal cord. However, reestablishment of neuronal networks in completed transected spinal cord was still a challenging task.

MicroRNA-195 targets ADP-ribosylation factor-like protein 2 to induce apoptosis in human embryonic stem cell-derived neural progenitor cells

Neural progenitor cells (NPCs) derived from human embryonic stem cells (hESCs) have great potential in cell therapy, drug screening and toxicity testing of neural degenerative diseases. However, the molecular regulation of their proliferation and apoptosis, which needs to be revealed before clinical application, is largely unknown. MicroRNA miR-195 is known to be expressed in the brain and is involved in a variety of proapoptosis or antiapoptosis processes in cancer cells. Here, we defined the proapoptotic role of miR-195 in NPCs derived from two independent hESC lines (human embryonic stem cell-derived neural progenitor cells, hESC-NPCs). Overexpression of miR-195 in hESC-NPCs induced extensive apoptotic cell death. Consistently, global transcriptional microarray analyses indicated that miR-195 primarily regulated genes associated with apoptosis in hESC-NPCs. Mechanistically, a small GTP-binding protein ADP-ribosylation factor-like protein 2 (ARL2) was identified as a direct target of miR-195. Silencing ARL2 in hESC-NPCs provoked an apoptotic phenotype resembling that of miR-195 overexpression, revealing for the first time an essential role of ARL2 for the survival of human NPCs. Moreover, forced expression of ALR2 could abolish the cell number reduction caused by miR-195 overexpression. Interestingly, we found that paraquat, a neurotoxin, not only induced apoptosis but also increased miR-195 and reduced ARL2 expression in hESC-NPCs, indicating the possible involvement of miR-195 and ARL2 in neurotoxin-induced NPC apoptosis. Notably, inhibition of miR-195 family members could block neurotoxin-induced NPC apoptosis. Collectively, miR-195 regulates cell apoptosis in a context-dependent manner through directly targeting ARL2. The finding of the critical role of ARL2 for the survival of human NPCs and association of miR-195 and ARL2 with neurotoxin-induced apoptosis have important implications for understanding molecular mechanisms that control NPC survival and would facilitate our manipulation of the neurological pathogenesis.

Vector-Free and Transgene-Free Human iPS Cells Differentiate into Functional Neurons and Enhance Functional Recovery after Ischemic Stroke in Mice

Stroke is a leading cause of human death and disability in the adult population in the United States and around the world. While stroke treatment is limited, stem cell transplantation has emerged as a promising regenerative therapy to replace or repair damaged tissues and enhance functional recovery after stroke. Recently, the creation of induced pluripotent stem (iPS) cells through reprogramming of somatic cells has revolutionized cell therapy by providing an unlimited source of autologous cells for transplantation. In addition, the creation of vector-free and transgene-free human iPS (hiPS) cells provides a new generation of stem cells with a reduced risk of tumor formation that was associated with the random integration of viral vectors seen with previous techniques. However, the potential use of these cells in the treatment of ischemic stroke has not been explored. In the present investigation, we examined the neuronal differentiation of vector-free and transgene-free hiPS cells and the transplantation of hiPS cell-derived neural progenitor cells (hiPS-NPCs) in an ischemic stroke model in mice. Vector-free hiPS cells were maintained in feeder-free and serum-free conditions and differentiated into functional neurons in vitro using a newly developed differentiation protocol. Twenty eight days after transplantation in stroke mice, hiPS-NPCs showed mature neuronal markers in vivo. No tumor formation was seen up to 12 months after transplantation. Transplantation of hiPS-NPCs restored neurovascular coupling, increased trophic support and promoted behavioral recovery after stroke. These data suggest that using vector-free and transgene-free hiPS cells in stem cell therapy are safe and efficacious in enhancing recovery after focal ischemic stroke in mice.

Phenotype and Stability of Neural Differentiation of Androgenetic Murine ES Cell-Derived Neural Progenitor Cells.

Uniparental zygotes with two paternal (androgenetic, AG) or two maternal genomes (gynogenetic, GG) cannot develop into viable offsprings but form blastocysts from which pluripotent embryonic stem (ES) cells can be derived. For most organs, it is unclear whether uniparental ES cells can give rise to stably expandable somatic stem cells that can repair injured tissues. Even if previous reports indicated that the capacity of AG ES cells to differentiate in vitro into pan-neural progenitor cells (pNPCs) and into cells expressing neural markers is similar to biparental [normal fertilized (N)] ES cells, their potential for functional neurogenesis is not known. Here we show that murine AG pNPCs give rise to neuron-like cells, which then generate sodium-driven action potentials while maintaining fidelity of imprinted gene expression. Neural engraftment after intracerebral transplantation was achieved only by late (22 days) AG and N pNPCs with in vitro low colony-forming cell (CFC) capacity. However, persisting CFC formation seen, in particular, in early (13 or 16 days) differentiation cultures of N and AG pNPCs correlated with a high incidence of trigerm layer teratomas. As AG ES cells display functional neurogenesis and in vivo stability similar to N ES cells, they represent a unique model system to study the roles of paternal and maternal genomes on neural development and on the development of imprinting-associated brain diseases.

Coordinated Development of Voltage-Gated Na+and K+Currents Regulates Functional Maturation of Forebrain Neurons Derived from Human Induced Pluripotent Stem Cells

Like embryonic stem (ES) cells, human induced pluripotent stem (hiPS) cells can differentiate into neuronal cells. However, it is unclear how their exquisite neuronal function is electrophysiologically coordinated during differentiation and whether they are functionally identical to human ES cell-derived neurons. In this study, we differentiated hiPS and ES cells into pyramidal-like neurons and conducted electrophysiological characterization over the 4-week terminal differentiation period. The human neuron-like cells express forebrain pyramidal cell markers NeuN, neurofilament, the microtubule-associated protein 2 (MAP2), the paired box protein Pax-6 (PAX6), Tuj1, and the forkhead box protein G1 (FoxG1). The size of developing neurons increased continuously during the 4-week culture, and cell-resting membrane potentials (RMPs) underwent a negative shift from -40 to -70 mV. Expression of the muscarinic receptor-modulated K(+) currents (IM) participated in the development of cell RMPs and controlled excitability. Immature neurons at week 1 could only fire abortive action potentials (APs) and the frequency of AP firing progressively increased with neuronal maturation. Interestingly, the developmental change of voltage-gated Na(+) current (INa) did not correlate with the change in the AP firing frequency. On the other hand, the transient outward K(+) current (IA), but not the delayed rectifier current (IK) contributed to the high frequency firing of APs. Synaptic activities were observed throughout the 4-week development. These morphological and electrophysiological features were almost identical between iPS and ES cell-derived neurons. This is the first systematic investigation showing functional evidence that hiPS cell-derived neurons possess similar neuronal activities as ES cell-derived neurons. These data support that iPS cell-derived neural progenitor cells have the potential for replacing lost neurons in cell-based therapy.

Cortical region-specific engraftment of embryonic stem cell-derived neural progenitor cells restores axonal sprouting to a subcortical target and achieves motor functional recovery in a mouse model of neonatal hypoxic-ischemic brain injury

Hypoxic-ischemic encephalopathy (HIE) at birth could cause cerebral palsy (CP), mental retardation, and epilepsy, which last throughout the individual's lifetime. However, few restorative treatments for ischemic tissue are currently available. Cell replacement therapy offers the potential to rescue brain damage caused by HI and to restore motor function. In the present study, we evaluated the ability of embryonic stem cell-derived neural progenitor cells (ES-NPCs) to become cortical deep layer neurons, to restore the neural network, and to repair brain damage in an HIE mouse model. ES cells stably expressing the reporter gene GFP are induced to a neural precursor state by stromal cell co-culture. Forty-hours after the induction of HIE, animals were grafted with ES-NPCs targeting the deep layer of the motor cortex in the ischemic brain. Motor function was evaluated 3 weeks after transplantation. Immunohistochemistry and neuroanatomical tracing with GFP were used to analyze neuronal differentiation and axonal sprouting. ES-NPCs could differentiate to cortical neurons with pyramidal morphology and expressed the deep layer-specific marker, Ctip2. The graft showed good survival and an appropriate innervation pattern via axonal sprouting from engrafted cells in the ischemic brain. The motor functions of the transplanted HIE mice also improved significantly compared to the sham-transplanted group. These findings suggest that cortical region specific engraftment of preconditioned cortical precursor cells could support motor functional recovery in the HIE model. It is not clear whether this is a direct effect of the engrafted cells or due to neurotrophic factors produced by these cells. These results suggest that cortical region-specific NPC engraftment is a promising therapeutic approach for brain repair.

Generation of Tripotent Neural Progenitor Cells from Rat Embryonic Stem Cells

Rat is a valuable model for pharmacological and physiological studies. Germline-competent rat embryonic stem (rES) cell lines have been successfully established and the molecular networks maintaining the self-renewing, undifferentiated state of rES cells have also been well uncovered. However, little is known about the differentiation strategies and the underlying mechanisms of how these authentic rat pluripotent stem cells give rise to specific cell types. The aim of this study is to investigate the neural differentiation capacity of rES cells. By means of a modified procedure based on previous publications - combination of mitogen-activated protein kinase (MAPK) and glycogen synthase kinase 3 (GSK3) inhibitors (two inhibitors, "2i") with feeder-conditioned medium, we successfully obtained high-quality rat embryoid bodies (rEBs) from rES cells and then differentiated them to tripotent neural progenitors. These rES cell-derived neural progenitor cells (rNPCs) were capable of self-renewing and giving rise to all three neural lineages, including astrocytes, oligodendrocytes, and neurons. Besides, these rES cell-derived neurons stained positive for γ-aminobutyric acid (GABA) and tyrosine hydroxylase (TH). In summary, we develop an experimental system for differentiating rES cells to tripotent neural progenitors, which may provide a powerful tool for pharmacological test and a valuable platform for studying the pathogenesis of many neurodegenerative disorders such as Parkinson's disease and the development of rat nervous system.

Genetic risk for Parkinson's disease correlates with alterations in neuronal manganese sensitivity between two human subjects

Manganese (Mn) is an environmental risk factor for Parkinson's disease (PD). Recessive inheritance of PARK2 mutations is strongly associated with early onset PD (EOPD). It is widely assumed that the influence of PD environmental risk factors may be enhanced by the presence of PD genetic risk factors in the genetic background of individuals. However, such interactions may be difficult to predict owing to the complexities of genetic and environmental interactions. Here we examine the potential of human induced pluripotent stem (iPS) cell-derived early neural progenitor cells (NPCs) to model differences in Mn neurotoxicity between a control subject (CA) with no known PD genetic risk factors and a subject (SM) with biallelic loss-of-function mutations in PARK2 and family history of PD but no evidence of PD by neurological exam. Human iPS cells were generated from primary dermal fibroblasts of both subjects. We assessed several outcome measures associated with Mn toxicity and PD. No difference in sensitivity to Mn cytotoxicity or mitochondrial fragmentation was observed between SM and CA NPCs. However, we found that Mn exposure was associated with significantly higher reactive oxygen species (ROS) generation in SM compared to CA NPCs despite significantly less intracellular Mn accumulation. Thus, this report offers the first example of human subject-specific differences in PD-relevant environmental health related phenotypes that are consistent with pathogenic interactions between known genetic and environmental risk factors for PD.

In Vitro Maturation of Dopaminergic Neurons Derived from Mouse Embryonic Stem Cells: Implications for Transplantation

The obvious motor symptoms of Parkinson's disease result from a loss of dopaminergic neurons from the substantia nigra. Embryonic stem cell-derived neural progenitor or precursor cells, adult neurons and fetal midbrain tissue have all been used to replace dying dopaminergic neurons. Transplanted cell survival is compromised by factors relating to the new environment, for example; hypoxia, mechanical trauma and excitatory amino acid toxicity. In this study we investigate, using live-cell fluorescence Ca2+ and Cl− imaging, the functional properties of catecholaminergic neurons as they mature. We also investigate whether GABA has the capacity to act as a neurotoxin early in the development of these neurons. From day 13 to day 21 of differentiation [Cl−]i progressively dropped in tyrosine hydroxylase positive (TH+) neurons from 56.0 (95% confidence interval, 55.1, 56.9) mM to 6.9 (6.8, 7.1) mM. At days 13 and 15 TH+ neurons responded to GABA (30 µM) with reductions in intracellular Cl− ([Cl−]i); from day 21 the majority of neurons responded to GABA (30 µM) with elevations of [Cl−]i. As [Cl−]i reduced, the ability of GABA (30 µM) to elevate intracellular Ca2+ ([Ca2+]i) did also. At day 13 of differentiation a three hour exposure to GABA (30 µM) or L-glutamate (30 µM) increased the number of midbrain dopaminergic (TH+ and Pitx3+) neurons labeled with the membrane-impermeable nuclear dye TOPRO-3. By day 23 cultures were resistant to the effects of both GABA and L-glutamate. We believe that neuronal susceptibility to amino acid excitotoxicity is dependent upon neuronal maturity, and this should be considered when isolating cells for transplantation studies.

Distinct generation, pharmacology, and distribution of sphingosine 1-phosphate and dihydrosphingosine 1-phosphate in human neural progenitor cells

In vivo and in vitro studies suggest a crucial role for Sphingosine 1-phosphate (S1P) and its receptors in the development of the nervous system. Dihydrosphingosine 1-phosphate (dhS1P), a reduced form of S1P, is an agonist at S1P receptors, but the pharmacology and physiology of dhS1P has not been widely studied. The mycotoxin fumonisin B1 (FB 1) is a potent inhibitor of ceramide synthases and causes selective accumulation of dihydrosphingosine and dhS1P. Recent studies suggest that maternal exposure to FB 1 correlates with the development of neural tube defects (NTDs) in which the neural epithelial progenitor cell layers of the developing brain fail to fuse. We hypothesize that the altered balance of S1P and dhS1P in neural epithelial cells contributes to the developmental effects of FB 1. The goal of this work was first to define the effect of FB 1 exposure on levels of sphingosine and dh-sphingosine and their receptor-active 1-phosphate metabolites in human embryonic stem cell-derived neural epithelial progenitor (hES-NEP) cells; and second, to define the relative activity of dhS1P and S1P in hES-NEP cells. We found that dhS1P is a more potent stimulator of inhibition of cAMP and Smad phosphorylation than is S1P in neural progenitors, and this difference in apparent potency may be due, in part, to more persistent presence of extracellular dhS1P applied to human neural progenitors rather than a higher activity at S1P receptors. This study establishes hES-NEP cells as a useful human in vitro model system to study the mechanism of FB 1 toxicity and the molecular pharmacology of sphingolipid signaling. This article is part of a Special Issue entitled ‘Post-Traumatic Stress Disorder’.

Mitochondrial gene replacement in human pluripotent stem cell-derived neural progenitors.

Human pluripotent stem cell-derived neural progenitor (hNP) cells are an excellent resource for understanding early neural development and neurodegenerative disorders. Given that many neurodegenerative disorders can be correlated with defects in the mitochondrial genome, optimal utilization of hNP cells requires an ability to manipulate and monitor changes in the mitochondria. Here, we describe a novel approach that uses recombinant human mitochondrial transcription factor A (rhTFAM) protein to transfect and express a pathogenic mitochondrial genome (mtDNA) carrying the G11778A mutation associated with Leber's hereditary optic neuropathy (LHON) disease, into dideoxycytidine (ddC)-treated hNPs. Treatment with ddC reduced endogenous mtDNA and gene expression, without loss of hNP phenotypic markers. Entry of G11778A mtDNA complexed with the rhTFAM was observed in mitochondria of ddC-hNPs. Expression of the pathogenic RNA was confirmed by restriction enzyme analysis of the SfaN1-digested cDNA. On the basis of the expression of neuron-specific class III beta-tubulin, neuronal differentiation occurred. Our results show for the first time that pathogenic mtDNA can be introduced and expressed into hNPs without loss of phenotype or neuronal differentiation potential. This mitochondrial gene replacement technology allows for creation of in vitro stem cell-based models useful for understanding neuronal development and treatment of neurodegenerative disorders.

Combined Transplantation of Bone Marrow Stromal Cell-Derived Neural Progenitor Cells with a Collagen Sponge and Basic Fibroblast Growth Factor Releasing Microspheres Enhances Recovery After Cerebral Ischemia in Rats

Bone marrow stromal cells (MSCs) are a useful source of cells because of their abundant supply and few associated ethical problems. We have previously reported that neural progenitor cells (NS-MSCs) can be effectively induced from MSCs and differentiate into neurons to contribute to functional recovery when transplanted into the rat stroke model. In this study, we attempted to enhance the therapeutic effects of NS-MSCs with a collagen sponge and basic fibroblast growth factor (bFGF) releasing microspheres. NS-MSCs were generated from MSCs by transfection of Notch-1 intracellular domain followed by culturing the cells in a free-floating culture system. The resulting NS-MSCs were transplanted into the rats with induced brain ischemia by using collagen sponges as scaffolds for transplanted cells, and with bFGF incorporated into gelatin microspheres to aid neovascularization around the transplanted region and proliferation of neural stem cells/neural progenitor cells. In culture, NS-MSCs successfully formed spheres containing cells highly expressing neural progenitor markers. Cell survival, neovascularization, and proliferation of host neural stem cells/neural progenitor cells were improved in animals that received NS-MSCs together with these biomaterials. Behavioral analysis also revealed significant functional recovery. These observations demonstrate that transplantation of NS-MSCs in combination with a collagen sponge and bFGF releasing microspheres significantly improves histological and functional recovery in the rat stroke model. When used with these biomaterials, NS-MSCs would be a promising cell source for treating stroke and neurodegenerative diseases.

296 PRODUCTION OF HEMIZYGOUS AND HOMOZYGOUS EMBRYONIC STEM CELL-DERIVED NEURAL PROGENITOR CELLS FROM THE TRANSGENIC ALZHEIMER GÖTTINGEN MINIPIG

Alzheimer’s disease is the most prevalent cause of dementia and afflicts ∼26 million people worldwide. There are currently no cures for this disease. Production of in vitro models of the disease would be extremely useful for studying disease mechanisms and for potential screening of novel drugs. In this study we produced 2 hemizygote and 2 homozygote embryonic stem cell-derived neural progenitor cell lines from Day 8 transgenic blastocysts carrying a human gene linked to early-onset Alzheimer’s disease [Swedish mutation of the amyloid precursor protein (hAPPsw)]. Following onset of spontaneous oestrus, a mating of hAPPsw± × hAPPsw± Göttingen transgenic progeny was performed. Eight days after the first of 2 matings, embryos were flushed from the tip of both cornuas of the gilt under surgical anaesthesia. A total of 6 blastocysts were obtained and 7 corpora lutei recorded. Blastocysts were transported for 4 h in porcine zygote medium 3 (PZM-3) in hypoxic, humidified conditions at 39°C to the cell laboratory. Compact epiblasts were mechanically isolated from the embryo using insulin needles and cultured on inactivated mouse embryonic fibroblasts in embryonic stem cell medium, supplemented with 20 ng mL–1 human recombinant basic fibroblast growth factor (Prospec) and 20 ng mL–1 human recombinant Activin A (Prospec), for a period of 5 days in hypoxic conditions at 39°C. Five of the 6 epiblasts expanded to form embryonic stem-cell-like outgrowth colonies. These were cut into small colonies and plated on MS5 murine stromal cells to induce spontaneous neural differentiation in DMEM medium containing 15% knockout serum replacement. Neuronal rosette-like structures were identified from Day 10 of differentiation onward. Six rosette structures were mechanically isolated from 4 outgrowths and plated in serum-free conditions on Matrigel-coated dishes. Two of the 6 lines failed to proliferate beyond passage 2. The 4 remaining cell lines have currently been cultured to passage 7. These lines were analysed at passage 5 by comparative real-time PCR and found to be positive for the neural progenitor markers VIMENTIN, SOX2, NESTIN PAX6, MUSASHI; other neural markers BETAIIITUBULIN and NCAM; and the astrocyte marker, GFAP. These lines were also subjected to analysis by immunocytochemistry and found to express SOX2, VIMENTIN, and NESTIN. Further genotyping by comparative real-time PCR using primers designed to target the hAPPsw gene revealed that 2 lines carried a single copy of hAPPsw and 2 lines carried 2 copies of hAPPsw. The expression levels of the hAPPsw transgene in these cell lines were determined using quantitative PCR. These cell lines are currently being investigated for their ability to differentiate into cholinergic neurons and for their expression of hyperphosphorylated TAU and β-Amyloid secretion. These cell lines will be potentially relevant for the in vitro study of amyloid precursor protein accumulation in neural cells and its role in cell death, as well as for potential screening of novel drugs for Alzheimer’s disease.

Controlled Release of Neurotrophin-3 and Platelet-Derived Growth Factor from Fibrin Scaffolds Containing Neural Progenitor Cells Enhances Survival and Differentiation into Neurons in a Subacute Model of SCI

A consistent problem with stem/neural progenitor cell transplantation following spinal cord injury (SCI) is poor cell survival and uncontrolled differentiation following transplantation. The current study evaluated the feasibility of enhancing embryonic stem cell-derived neural progenitor cell (ESNPC) viability and directing their differentiation into neurons and oligodendrocytes by embedding the ESNPCs in fibrin scaffolds containing growth factors (GF) and a heparin-binding delivery system (HBDS) in a subacute rat model of SCI. Mouse ESNPCs were generated from mouse embryonic stem cells (ESCs) using a 4-/4+ retinoic acid (RA) induction protocol. The ESNPCs were then transplanted as embryoid bodies (EBs, 70% neural progenitor cells) into the subacute model of SCI. ESNPCs (10 EBs per animal) were implanted directly into the SCI lesion, encapsulated in fibrin scaffolds, encapsulated in fibrin scaffolds containing the HBDS, neurotrophin-3 (NT-3), and platelet-derived growth factor (PDGF), or encapsulated in fibrin scaffolds with NT-3 and PDGF with no HBDS. We report here that the combination of the NT-3, PDGF, and fibrin scaffold (with or without HBDS) enhanced the total number of ESNPCs present in the spinal cord lesion 2 weeks after injury. In addition, the inclusion of the HBDS with growth factor resulted in an increase in the number of ESNPC-derived NeuN-positive neurons. These results demonstrate the ability of fibrin scaffolds and the controlled release of growth factors to enhance the survival and differentiation of neural progenitor cells following transplantation into a SCI model.

Tissue-engineered fibrin scaffolds containing neural progenitors enhance functional recovery in a subacute model of SCI.

Two recurring problems with stem/neural progenitor cell (NPC) transplantation therapies for spinal cord injury (SCI) are poor cell survival and uncontrolled cell differentiation. The current study evaluated the viability and differentiation of embryonic stem cell-derived neural progenitor cells (ESNPCs) transplanted within fibrin scaffolds containing growth factors (GFs) and a heparin-binding delivery system (HBDS) to enhance cell survival and direct differentiation into neurons. Mouse ESNPCs were generated from mouse embryonic stem cells (ESCs) using a 4-/4+ retinoic acid (RA) induction protocol that resulted in a population of cells that was 70% nestin positive NPCs. The ESNPCs were transplanted directly into a rat subacute dorsal hemisection lesion SCI model. ESNPCs were either encapsulated in a fibrin scaffold; encapsulated in fibrin containing the HBDS, neurotrophin-3 (NT-3) and platelet derived growth factor (PDGF-AA); or encapsulated in fibrin scaffolds with NT-3 and PDGF-AA without the HBDS. We report that the combination of GFs and fibrin scaffold (without HBDS) enhanced the total number of ESNPCs present in the treated spinal cords and increased the number of ESNPC-derived NeuN positive neurons 8 weeks after transplantation. All experimental groups treated with ESNPCs exhibited an increase in behavioral function 4 weeks after transplantation. In a subset of animals, the ESNPCs over-proliferated as evidenced by SSEA-1 positive/Ki67 positive ESCs found at 4 and 8 weeks. These results demonstrate the potential of tissue-engineered fibrin scaffolds to enhance the survival of NPCs and highlight the need to purify cell populations used in therapies for SCI.