Human Neural Stem/progenitor Cells Research Articles

53BP1 regulates the self-renewal ability of neural stem/progenitor cells through modulating mitochondrial homeostasis

The regulation of intracellular reactive oxygen species (ROS) levels is important for maintaining the self-renewal ability of neural stem/progenitor cells (NSCs). In this study, we demonstrate that 53BP1, a DNA damage response factor known to facilitate the repair of DNA double-strand breaks, supports the maintenance of NSC stemness. ReNcell VM human NSCs with depleted 53BP1 exhibited reduced self-renewal ability compared with control NSCs, as revealed by a decrease in neurosphere size and an increase in differentiation into neural or glial cells within an NSC culture. Furthermore, 53BP1 depletion elevated cellular ROS levels, accompanied by mitochondrial abnormalities. The reduced self-renewal ability and elevated ROS levels in 53BP1-deficient NSCs were restored with the treatment of a radical scavenger, N-acetyl-l-cysteine. In addition, we investigated the functional relationship in the NSC self-renewal ability between 53BP1 and ataxia-telangiectasia mutated (ATM) or forkhead box O3a (FOXO3a), factors required for mitochondrial homeostasis, and the maintenance of NSC stemness. We found that ATM inhibition or FOXO3a deficiency, in addition to 53BP1 deficiency, did not induce further NSC stemness impairment. Collectively, our findings show that 53BP1, by cooperatively functioning with ATM and FOXO3a, supports the maintenance of NSC stemness by modulating mitochondrial homeostasis.

372 Differences in Oligodendrogenesis Between Human and Rodent Spinal Cord Stem Cells

INTRODUCTION: Research in pre-clinical rodent models suggests that targeting the endogenous neural stem/progenitor cells (NSPCs) in the spinal cord may hold the key to stimulating oligodendrocyte regeneration. However, it remains to be seen whether human spinal cord NSPCs possess the same regenerative potential as their rat counterparts. METHODS: To assess the potential for oligodendrogenesis between human and rat spinal cord NSPCs, primary tissue from adult donors and female rats was harvested and cultured using the Neurosphere assay. To induce spontaneous differentiation, primary-derived NSPCs were treated with 1% fetal bovine serum (n = 15 humans, n = 10 rats). To further promote oligodendrocyte differentiation, pdNSPCs were treated with 40 ng/µL or 100 ng/µL of platelet-derived growth factor AA (PDGF-AA) (n = 3 humans, n = 3 rats). Immunocytochemistry and RNA sequencing were then employed to compare the transcriptomes, with the differentially expressed genes and gene sets analyzed via DESeq and GSEA. RESULTS: Human NSPCs showed a reduced potential for oligodendrocyte generation compared to rat NSPCs (0.013 ± 0.01% and 0.029 ± 0.01% O4+ after one and two weeks in humans; 4.9 ± 0.4% and 6.3 ± 0.6% O4+ after one and two weeks in rats). PDGF-AA treatment at 40 ng/µL for one week was able to effectively promote oligodendrocyte differentiation in rat NSPCs, but had a minimal effect on human NSPCs (8.5 ± 1.4 fold increase in O4+). OLIG1/2, SOX10, and CNP gene transcripts were enriched in rat NSPCs. CONCLUSIONS: We compared the potential of oligodendrogenesis between primary human and rat spinal cord NSPCs and found a significantly lower capacity in the human NSPCs both functionally and transcriptionally, which could be an obstacle to the successful application of myelinating techniques.

Leukemia inhibitory factor shortens primary cilia by upregulating C-C motif chemokine 2 in human neural stem/progenitor cells.

Primary cilia on neural stem/progenitor cells (NSPCs) play an important role in determining cell fate, although the regulatory mechanisms involved in the ciliogenesis remain largely unknown. In this study, we analyzed the effect of the leukemia inhibitory factor (LIF) for the primary cilia in immortalized human NSPCs. LIF withdrawal elongated the primary cilia length, whereas the addition of LIF shortened it. Microarray gene expression analysis revealed that differentially expressed genes (DEGs) associated with LIF treatment were related with the multiple cytokine signaling pathways. Among the DEGs, C-C motif chemokine 2 (CCL2) had the highest ranking and its increase in the protein concentration in the NSPCs-conditioned medium after the LIF treatment was confirmed by ELISA. Interestingly, we found that CCL2 was a negative regulator of cilium length, and LIF-induced shortening of primary cilia was antagonized by CCL2-specific antibody, suggesting that LIF could influence cilia length via upregulating CCL2. The shortening effect of LIF and CCL2 on primary cilia was also observed in SH-SY5Y cells. The results of the study suggested that the LIF-CCL2 axis may well be a regulator of NSPCs and its primary cilia length, which could affect multiple cellular processes, including NSPC proliferation and differentiation.

Neural stem/progenitor cells from adult canine cervical spinal cord have the potential to differentiate into neural lineage cells

Background• Neural stem/progenitor cells (NSPCs) are multipotent self-renewing cells that can be isolated from the brain or spinal cord. As they need to be isolated from neural tissues, it is difficult to study human NSPCs. To facilitate NSPC research, we attempted to isolate NSPCs from dogs, as dogs share the environment and having many similar diseases with humans. We collected and established primary cultures of ependymal and subependymal cells from the central canal of the cervical spinal cord of adult dogs. To isolate pure NSPCs, we employed the monolayer culture and selective medium culture methods. We further tested the ability of the NSPCs to form neurospheres (using the suspension culture method) and evaluated their differentiation potential.Results• The cells had the ability to grow as cultures for up to 10 passages; the growth curves of the cells at the 3rd, 6th, and 9th passages showed similar patterns. The NSPCs were able to grow as neurospheres as well as monolayers, and immunostaining at the 3rd, 6th, and 9th passages showed that these cells expressed NSPC markers such as nestin and SOX2 (immunofluorescent staining). Monolayer cultures of NSPCs at the 3rd, 6th, and 9th passages were cultured for approximately 14 days using a differentiation medium and were observed to successfully differentiate into neural lineage and glial cells (astrocytes, neurons, and oligodendrocytes) at all the three passages tested.Conclusion• It is feasible to isolate and propagate (up to at least 10 passages) canine cervical spinal cord-derived NSPCs with the capacity to differentiate into neuronal and glial cells. To the best of our knowledge this is the first study to successfully isolate, propagate, and differentiate canine NSPCs derived from cervical spinal cord in the adult canine, and we believe that these cells will contribute to the field of spinal cord regeneration in veterinary and comparative medicine.

P.145 Uncovering differences in oligodendrogenesis between human and rodent spinal cord stem cells

Background: Spinal cord regeneration in pre-clinical rodent studies is feasible by promoting oligodendrocyte regrowth, which is necessary for axon myelination. It is uncertain whether human neural stem/progenitor cells (NSPCs) are capable of differentiation into oligodendrocytes, similar to rat. In this study, we compare the functional and transcriptional features of primary spinal cord NSPCs from adult humans and rats. Methods: Oligodendrogenesis between human & rat NSPCs from adult donors and rats was cultured using the Neurosphere assay. NSPCs were exposed to 1% FBS to trigger differentiation & PDGF-AA to promote oligodendrocyte formation. Immunocytochemistry & RNA sequencing compared transcriptomes and analyzing differentially expressed genes. Results: Human NSPCs showed a reduced potential for oligodendrocyte generation compared to rat NSPCs (0.013±0.01% and 0.029±0.01% O4+ after one and two weeks in humans; 4.9±0.4% and 6.3±0.6% O4+ after one and two weeks in rats). PDGF-AA treatment at 40 ng/µL for one week was able to effectively promote oligodendrocyte differentiation in rat NSPCs, but had a minimal effect on human NSPCs (8.5±1.4 fold increase in O4+). OLIG1/2, SOX10, and CNP were enriched in rat NSPCs. Conclusions: We compared oligodendrogenesis potential between human and rat NSPCs and found significantly lower capacity in human NSPCs, possibly hindering successful myelinating techniques.

Simulated Microgravity Modulates Focal Adhesion Gene Expression in Human Neural Stem Progenitor Cells.

We analyzed the morphology and the transcriptomic changes of human neural stem progenitor cells (hNSPCs) grown on laminin in adherent culture conditions and subjected to simulated microgravity for different times in a random positioning machine apparatus. Low-cell-density cultures exposed to simulated microgravity for 24 h showed cell aggregate formation and significant modulation of several genes involved in focal adhesion, cytoskeleton regulation, and cell cycle control. These effects were much more limited in hNSPCs cultured at high density in the same conditions. We also found that some of the genes modulated upon exposure to simulated microgravity showed similar changes in hNSPCs grown without laminin in non-adherent culture conditions under normal gravity. These results suggest that reduced gravity counteracts the interactions of cells with the extracellular matrix, inducing morphological and transcriptional changes that can be observed in low-density cultures.

NSCs are permissive to oncolytic Myxoma virus and provide a delivery method for targeted ovarian cancer therapy.

Despite the development of many anticancer agents over the past 20 years, ovarian cancer remains the most lethal gynecologic malignancy. Due to a lack of effective screening, the majority of patients with ovarian cancer are diagnosed at an advanced stage, and only ~20% of patients are cured. Thus, in addition to improved screening methods, there is an urgent need for novel anticancer agents that are effective against late-stage, metastatic disease. Oncolytic virotherapy is a promising approach; unfortunately, systemic delivery of viruses to tumors remains a major challenge. In this regard, neural stem/progenitor cells (NSCs) with well-established tumor-homing properties may serve as an effective delivery platform for oncolytic viruses. In this study, we tested the efficacy of myxoma virus (MYXV), a rabbit-specific poxvirus that has demonstrated efficacy against a variety of tumors, using human and mouse ovarian cancer cell lines. We showed that MYXV effectively lysed ovarian cancer cells in vitro, reducing their viability. We also demonstrated that MYXV can infect human NSCs, specifically the clonal HB1.F3.CD21 NSC line. Taken together, these results suggest that NSC-mediated delivery of MYXV may be a promising strategy for achieving more selectively targeted anti-tumor efficacy.

A Guide to Extract Spinal Cord for Translational Stem Cell Biology Research: Comparative Analysis of Adult Human, Porcine, and Rodent Spinal Cord Stem Cells.

Improving the clinical translation of animal-based neural stem/progenitor cell (NSPC) therapies to humans requires an understanding of intrinsic human and animal cell characteristics. We report a novel in vitro method to assess spinal cord NSPCs from a small (rodent) and large (porcine) animal model in comparison to human NSPCs. To extract live adult human, porcine, and rodent spinal cord tissue, we illustrate a strategy using an anterior or posterior approach that was simulated in a porcine model. The initial expansion of primary NSPCs is carried out using the neurosphere assay followed by a pharmacological treatment phase during which NSPCs derived from humans, porcines, and rodents are assessed in parallel using the same defined parameters. Using this model, NSPCs from all species demonstrated multi-lineage differentiation and self-renewal. Importantly, these methods provide conditions to enable the direct comparison of species-dependent cell behavior in response to specific exogenous signals.

Exosomes derived from neural progenitor cells preserve photoreceptors during retinal degeneration by inactivating microglia

ABSTRACT Retinal degeneration (RD) is one of the most common causes of visual impairment and blindness and is characterized by progressive degeneration of photoreceptors. Transplantation of neural stem/progenitor cells (NPCs) is a promising treatment for RD, although the mechanisms underlying the efficacy remain unclear. Accumulated evidence supports the notion that paracrine effects of transplanted stem cells is likely the major approach to rescuing early degeneration, rather than cell replacement. NPC-derived exosomes (NPC-exos), a type of extracellular vesicles (EVs) released from NPCs, are thought to carry functional molecules to recipient cells and play therapeutic roles. In present study, we found that grafted human NPCs (hNPCs) secreted EVs and exosomes in the subretinal space (SRS) of RCS rats, an RD model. And direct administration of mouse neural progenitor cell-derived exosomes (mNPC-exos) delayed photoreceptor degeneration, preserved visual function, prevented thinning of the outer nuclear layer (ONL), and decreased apoptosis of photoreceptors in RCS rats. Mechanistically, mNPC-exos were specifically internalized by retinal microglia and suppressed their activation in vitro and in vivo. RNA sequencing and miRNA profiling revealed a set of 17 miRNAs contained in mNPC-exos that markedly inhibited inflammatory signal pathways by targeting TNF-α, IL-1β, and COX-2 in activated microglia. The exosomes derived from hNPC (hNPC-exos) contained similar miRNAs to mNPC-exos that inhibited microglial activation. We demonstrated that NPC-exos markedly suppressed microglial activation to protect photoreceptors from apoptosis, suggesting that NPC-exos and their contents may be the mechanism of stem cell therapy for treating RD.

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 line-derived 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 GDNF-hNSPCs 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.

GENE-10. A SIMPLE AND EFFICIENT METHOD FOR GENERATING MULTI-ALLELIC INSERTION-DELETION MUTATIONS AND PRECISE DELETIONS IN PRIMARY HUMAN CELL CULTURES

Abstract CRISPR-Cas9-based technologies have revolutionized experimental manipulation of the human genome by enabling generation of site-specific genetic alterations, such as insertion-deletion (indel) mutations. None-the-less, limitations of the delivery and efficacy of these technologies restrict their application in primary human cells. Here, we present a simple and effective method for fast and penetrant induction of multi-allelic indels and near-precise deletions (ranging from ~50bp to >50kbp) in primary human neural stem/progenitor cell (NPC) and brain tumor-derived stem-like cell cultures using the CRISPR-Cas9 system. We report that mono-, bi-, or multi-allelic (depending on ploidy) indel efficiencies of >90% can be routinely achieved within 3 days, without the need for pre-engineering cells to express Cas9 or isolating clones. As a result, this method enables the quick generation of a series of knockouts at different genomic loci, which we illustrate by successively targeting TP53, CDKN2A, PTEN, and NF1 in human NPC populations, where ³95% loss of protein expression is observed. RNA-seq analysis of these NPC knockout pools confirmed gene expression changes including p53 transcriptional and Rb-axis targets, as well as NF1-dependent repression of major histocompatibility complex class II gene expression. Given its simplicity, this method is readily adaptable to other primary mammalian cell types.

368 - Direct comparison of adult human and rat spinal cord stem cell behavior

Background: Spinal cord (SC) injury is a debilitating disease during which endogenous neural stem/progenitor cells (NSPCs) are activated but fail to restore functionality. These NSPCs have been targeted to promote neurological recovery in animal models. However, it remains unclear if human SC NSPCs can acquire similarly instructed fates during injury and regeneration.

Attenuation of reactive gliosis in stroke-injured mouse brain does not affect neurogenesis from grafted human iPSC-derived neural progenitors.

Induced pluripotent stem cells (iPSCs) or their progeny, derived from human somatic cells, can give rise to functional improvements after intracerebral transplantation in animal models of stroke. Previous studies have indicated that reactive gliosis, which is associated with stroke, inhibits neurogenesis from both endogenous and grafted neural stem/progenitor cells (NSPCs) of rodent origin. Here we have assessed whether reactive astrocytes affect the fate of human iPSC-derived NSPCs transplanted into stroke-injured brain. Mice with genetically attenuated reactive gliosis (deficient for GFAP and vimentin) were subjected to cortical stroke and cells were implanted adjacent to the ischemic lesion one week later. At 8 weeks after transplantation, immunohistochemical analysis showed that attenuated reactive gliosis did not affect neurogenesis or commitment towards glial lineage of the grafted NSPCs. Our findings, obtained in a human-to-mouse xenograft experiment, provide evidence that the reactive gliosis in stroke-injured brain does not affect the formation of new neurons from intracortically grafted human iPSC-derived NSPCs. However, for a potential clinical translation of these cells in stroke, it will be important to clarify whether the lack of effect of reactive gliosis on neurogenesis is observed also in a human-to-human experimental setting.

Extracellular vesicles are independent metabolic units with asparaginase activity.

Extracellular vesicles (EVs) are membrane particles involved in the exchange of a broad range of bioactive molecules between cells and the microenvironment. While it has been shown that cells can traffic metabolic enzymes via EVs much remains to be elucidated with regard to their intrinsic metabolic activity. Accordingly, herein we assessed the ability of neural stem/progenitor cell (NSC)-derived EVs to consume and produce metabolites. Both our metabolomics and functional analyses revealed that EVs harbour L-asparaginase activity catalysed by the enzyme Asparaginase-like protein 1 (Asrgl1). Critically, we show that Asrgl1 activity is selective for asparagine and is devoid of glutaminase activity. We found that mouse and human NSC-derived EVs traffic ASRGL1. Our results demonstrate for the first time that NSC EVs function as independent, extracellular metabolic units able to modify the concentrations of critical nutrients, with the potential to affect the physiology of their microenvironment.

Enhancing oligodendrocyte differentiation by transient transcription activation via DNA nanoparticle-mediated transfection.

Current protocols to derive oligodendrocytes from human pluripotent stem cells (hPSCs) require lengthy culture time with low generation efficiencies of mature oligodendrocytes. We described a new approach to enhance oligodendrocyte differentiation through nanoparticle-mediated transcription modulation. We tested an effective transfection method using cell-compatible poly (β-amino ester) (PBAE)/DNA nanoparticles as gene carrier to deliver transcription factor Olig1 and Olig2 into human fetal tissue-derived neural stem/progenitor cells, and showed efficient oligodendrocyte differentiation following transgene expression of Olig1 and Olig2. We believe that this translatable approach can be applied to many other cell-based regenerative therapies as well.

Cross-species analyses unravel the complexity of H3K27me3 and H4K20me3 in the context of neural stem progenitor cells

Neural stem progenitor cells (NSPCs) in the human subventricular zone (SVZ) potentially contribute to lifelong neurogenesis, yet subtypes of glioblastoma multiforme (GBM) contain NSPC signatures that highlight the importance of cell fate regulation. Among numerous regulatory mechanisms, the posttranslational methylations onto histone tails are crucial regulator of cell fate. The work presented here focuses on the role of 2 repressive chromatin marks trimethylations on histone H3 lysine 27 (H3K27me3) and histone H4 lysine 20 (H4K20me3) in the adult NSPC within the SVZ. To best model healthy human NSPCs as they exist in vivo for epigenetic profiling of H3K27me3 and H4K20me3, we used NSPCs isolated from the adult SVZ of baboon brain (Papio anubis) with brain structure and genomic level similar to human. The putative role of H3K27me3 in normal NSPCs predominantly falls into the regulation of gene expression, cell cycle, and differentiation, whereas H4K20me3 is involved in DNA replication/repair, metabolism, and cell cycle. Using conditional knockout mouse models to diminish Ezh2 and Suv4-20h responsible for H3K27me3 and H4K20me3, respectively, we found that both repressive marks have irrefutable function for cell cycle regulation in the NSPC population. Although both EZH2/H3K27me3 and Suv4-20h/H4K20me3 have implication in cancers, our comparative genomics approach between healthy NSPCs and human GBM specimens revealed that substantial sets of genes enriched with H3K27me3 and H4K20me3 in the NSPCs are altered in the human GBM. In sum, our integrated analyses across species highlight important roles of H3K27me3 and H4K20me3 in normal and disease conditions in the context of NSPC.

Inhibition of Neurosphere Formation in Neural Stem/Progenitor Cells by Acrylamide

Previous studies showed that transplantation of cultured neural stem/progenitor cells (NSPCs) could improve functional recovery for various neurological diseases. This study aims to develop a stem cell-based model for predictive toxicology of development in the neurological system after acrylamide exposure. Treatment of mouse (KT98/F1B-GFP) and human (U-1240 MG/F1B-GFP) NSPCs with 0.5 mM acrylamide resulted in the inhibition of neurosphere formation (definition of self-renewal ability in NSPCs), but not inhibition of cell proliferation. Apoptosis and differentiation of KT98 (a precursor of KT98/F1B-GFP) and KT98/F1B-GFP are not observed in acrylamide-treated neurospheres. Analysis of secondary neurosphere formation and differentiation of neurons and glia illustrated that acrylamide-treated KT98 and KT98/F1B-GFP neurospheres retain the NSPC properties, such as self-renewal and differentiation capacity. Correlation of acrylamide-inhibited neurosphere formation with cell-cell adhesion was observed in mouse NSPCs by live cell image analysis and the presence of acrylamide. Protein expression levels of cell adhesion molecules [neural cell adhesion molecule (NCAM) and N-cadherin] and extracellular signal-regulated kinases (ERK) in acrylamide-treated KT98/F1B-GFP and U-1240 MG/F1B-GFP neurospheres demonstrated that NCAM decreased and phospho-ERK (pERK) increased, whereas expression of N-cadherin remained unchanged. Analysis of AKT (protein kinase B, PKB)/β-catenin pathway showed decrease in phospho-AKT (p-AKT) and cyclin D1 expression in acrylamide-treated neurospheres of KT98/F1B-GFP. Furthermore, PD98059, an ERK phosphorylation inhibitor, attenuated acrylamide-induced ERK phosphorylation, indicating that pERK contributed to the cell proliferation, but not in neurosphere formation in mouse NSPCs. Coimmunoprecipitation results of KT98/F1B-GFP cell lysates showed that the complex of NCAM and fibroblast growth factor receptor 1 (FGFR1) is present in the neurosphere, and the amount of this complex decreases after acrylamide treatment. Our results reveal that acrylamide inhibits neurosphere formation through the disruption of the neurosphere architecture in NSPCs. The downregulation of cell-cell adhesion resulted from decreasing the levels of NCAM as well as the formation of NCAM/FGFR complex.

Interplay between human microglia and neural stem/progenitor cells in an allogeneic co‐culture model

Experimental neural cell therapies, including donor neural stem/progenitor cells (NPCs) have been reported to offer beneficial effects on the recovery after an injury and to counteract inflammatory and degenerative processes in the central nervous system (CNS). The interplay between donor neural cells and the host CNS still to a large degree remains unclear, in particular in human allogeneic conditions. Here, we focused our studies on the interaction of human NPCs and microglia utilizing a co-culture model. In co-cultures, both NPCs and microglia showed increased survival and proliferation compared with mono-cultures. In the presence of microglia, a larger subpopulation of NPCs expressed the progenitor cell marker nestin, whereas a smaller group of NPCs expressed the neural markers polysialylated neural cell adhesion molecule, A2B5 and glial fibrillary acidic protein compared with NPC mono-cultures. Microglia thus hindered differentiation of NPCs. The presence of human NPCs increased microglial phagocytosis of latex beads. Furthermore, we observed that the expression of CD200 molecules on NPCs and the CD200 receptor protein on microglia was enhanced in co-cultures, whereas the release of transforming growth factor-β was increased suggesting anti-inflammatory features of the co-cultures. To conclude, the interplay between human allogeneic NPCs and microglia, significantly affected their respective proliferation and phenotype. Neural cell therapy including human donor NPCs may in addition to offering cell replacement, modulate host microglial phenotypes and functions to benefit neuroprotection and repair.

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

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

Intranasal Delivery of Neural Stem/Progenitor Cells: A Noninvasive Passage to Target Intracerebral Glioma

Stem cell-based therapies for neurological disorders, including brain tumors, advance continuously toward clinical trials. Optimized cell delivery to the central nervous system remains a challenge since direct intracerebral injection is an invasive method with low transplantation efficiency. We investigated the feasibility of intranasal administration of neural stem/progenitor cells (NSPCs) as an alternative, noninvasive, and direct passage for the delivery of stem cells to target malignant gliomas. Tumor-targeting and migratory pathways of murine and human NSPCs were investigated by intravital magnetic resonance imaging and in histological time course analyses in the intracerebral U87, NCE-G55T2, and syngenic Gl261 glioblastoma models. Intranasally administered NSPCs displayed a rapid, targeted tumor tropism with significant numbers of NSPCs accumulating specifically at the intracerebral glioma site within 6 hours after intranasal delivery. Histological time series analysis revealed that NSPCs migrated within the first 24 hours mainly via olfactory pathways but also by systemic distribution via the microvasculature of the nasal mucosa. Intranasal application of NSPCs leads to a rapid, targeted migration of cells toward intracerebral gliomas. The directional distribution of cells accumulating intra- and peritumorally makes the intranasal delivery of NSPCs a promising noninvasive and convenient alternative delivery method for the treatment of malignant gliomas with the possibility of multiple dosing regimens.