Neural Progenitor Cell Markers Research Articles

Particle radiation-induced dysregulation of protein homeostasis in primary human and mouse neuronal cells

Space particle radiations may cause significant damage to proteins and oxidative stress in the cells within the central nervous system and pose a potential health hazard to humans in long-term manned space explorations. Dysregulation of the ubiquitin-proteasome system as evidenced by abnormal accumulation of polyubiquitin (pUb) chain linkages has been implicated in several age-related neurodegenerative disorders by mechanisms that may involve the inter-neuronal spread of toxic misfolded proteins, the induction of chronic neuroinflammation, or the inappropriate inhibition or activation of key enzymes, which could lead to dysfunction in, for example, proteolysis, or the accumulation of post-translationally-modified substrates.In this study, we employed a quantitative proteomics method to evaluate the impact of particle-radiation induced alterations in three major pUb-linked chains at lysine residues Lys-48 (K-48), Lys-63 (K-63), and Lys-11 (K-11), and probed for global proteomic changes in mouse and human neural cells that were irradiated with low doses of 250 MeV proton, 260 MeV/u silicon or 1 GeV/u iron ions. We found significant accumulation in K-48 linkage after 1 Gy protons and K-63 linkage after 0.5 Gy iron ions in human neural cells. Cells derived from different regions of the mouse brain (cortex, striatum and mesencephalon) showed differential sensitivity to particle radiation exposure. Although none of the linkages were altered after proton exposure, both K-48 and K-63 linkages in mouse striatal neuronal cells were elevated after 0.5 Gy of silicon or iron ions. Changes were also seen in proteins commonly used as markers of neural progenitor and stem cells, in DNA binding/damage repair and cellular redox pathways. In contrast, no significant changes were observed at the same time point after proton irradiation. These results suggest that the quality of the particle radiation plays a key role in the level, linkage and cell type specificity of protein homeostasis in key populations of neuronal cells.

Corticotropin-releasing factor protects against ammonia neurotoxicity in isolated larval zebrafish brains.

The physiological roles of corticotropin-releasing factor (CRF) have recently been extended to cytoprotection. Here, to determine whether CRF is neuroprotective in fish, the effects of CRF against high environmental ammonia (HEA)-mediated neurogenic impairment and cell death were investigated in zebrafish. In vivo, exposure of 1 day post-fertilization (dpf) embryos to HEA only reduced the expression of the determined neuron marker neurod1 In contrast, in 5 dpf larvae, HEA increased the expression of nes and sox2, neural progenitor cell markers, and reduced the expression of neurog1, gfap and mbpa, proneuronal cell, radial glia and oligodendrocyte markers, respectively, and neurod1 The N-methyl-d-aspartate (NMDA) receptor inhibitor MK801 rescued the HEA-induced reduction in neurod1 in 5 dpf larvae but did not affect the HEA-induced transcriptional changes in other neural cell types, suggesting that hyperactivation of NMDA receptors specifically contributes to the deleterious effects of HEA in determined neurons. As observed in vivo, HEA exposure elicited marked changes in the expression of cell type-specific markers in isolated 5 dpf larval brains. The addition of CRF reversed the in vitro effects of HEA on neurod1 expression and prevented an HEA-induced increase in cell death. Finally, the protective effects of CRF against HEA-mediated neurogenic impairment and cell death were prevented by the CRF type 1 receptor selective antagonist antalarmin. Together, these results provide novel evidence that HEA has developmental time- and cell type-specific neurotoxic effects, that NMDA receptor hyperactivation contributes to HEA-mediated impairment of determined neurons, and that CRF has neuroprotective properties in the larval zebrafish brain.

Efficient Neural Differentiation using Single-Cell Culture of Human Embryonic Stem Cells.

In vitro differentiation of human embryonic stem cells (hESCs) has transformed the ability to study human development on both biological and molecular levels and provided cells for use in regenerative applications. Standard approaches for hESC culture using colony type culture to maintain undifferentiated hESCs and embryoid body (EB) and rosette formation for differentiation into different germ layers are inefficient and time-consuming. Presented here is a single-cell culture method using hESCs instead of a colony-type culture. This method allows maintenance of the characteristic features of undifferentiated hESCs, including expression of hESC markers at levels comparable to colony type hESCs. In addition, the protocol presents an efficient method for neural progenitor cell (NPC) generation from single-cell type hESCs that produces NPCs within 1 week. These cells highly express several NPC marker genes and can differentiate into various neural cell types, including dopaminergic neurons and astrocytes. This single-cell culture system for hESCs will be useful in investigating the molecular mechanisms of these processes, studies of certain diseases, and drug discovery screens.

Lovastatin alters neurotrophin expression in rat hippocampus-derived neural stem cells in vitro

Neural stem/progenitor cells hold valuable potential for the treatment of neurodegenerative disorders. The modulation of intrinsic growth factor expression, such as neurotrophins and their receptors, is a necessary step in achieving neural stem cells (NSCs) therapy. The statins have recently been reported to provide both anti‑inflammatory and neuroprotective effects. In the developing and mature nervous systems, neurotrophic factors are known to impact neuronal growth and survival. In this study, we investigated for a positive effect of lovastatin on the expression of neurotrophins in the neonatal rat hippocampus‑derived NSCs. NSCs were isolated and cultured up to passage three. To confirm cellular identity, immunocytochemical evaluation and flow cytometry analysis were performed using specific antibodies. To determine the optimum concentration of lovastatin, the MTT assay was used. Neurotrophin expression was evaluated using quantitative real‑time reverse transcription‑polymerase chain reaction (RT‑qPCR). Flow cytometry results demonstrated that NSCs were positive for nestin, a marker for neural progenitor cells. An increase in cellular viability was observed with a 24 h exposure of lovastatin. Moreover, results showed an increase in mRNA expression for all neurotrophins compared to the control group. Taken together, the results of this study add to the growing body of literature on the neuroprotective effects of statins in neurological disorders. Lovastatin is a promising therapeutic agent for the treatment of neurodegenerative disorders.

Expression of markers of neural stem and progenitor cells in glioblastoma multiforme in relation to tumor recurrence and overall survival.

Expression of markers of neural stem and progenitor cells in glioblastoma multiforme in relation to tumor recurrence and overall survival.

Rapid and Efficient Conversion of Human Fibroblasts into Functional Neurons by Small Molecules.

SummaryRecent studies have demonstrated that human astrocytes and fibroblasts can be directly converted into functional neurons by small molecules. However, fibroblasts, as a potentially better cell resource for transplantation, are not as easy to reprogram as astrocytes regarding their fate to neurons, and chemically induced neurons (iNs) with low efficiency from fibroblasts resulted in limited application for the treatment of neurological disorders, including depression. Here, we report that human fibroblasts can be efficiently and directly reprogrammed into glutamatergic neuron-like cells by serially exposing cells to a combination of small molecules. These iNs displayed neuronal transcriptional networks, and also exhibited mature firing patterns and formed functional synapses. Importantly, iNs could integrate into local circuits after transplantation into postnatal mouse brain. Our study provides a rapid and efficient transgene-free approach for chemically generating neuron-like cells from human fibroblasts. Furthermore, our approach offers strategies for disease modeling and drug discovery in central nervous system disorders.

Identifying Cell Surface Markers of Primary Neural Stem and Progenitor Cells by Metabolic Labeling of Sialoglycan.

Neural stem and progenitor cells (NSPCs) are the cellular basis for the complex structures and functions of the brain. They are located in specialized niches in vivo and can be isolated and expanded in vitro, serving as an important resource for cell transplantation to repair brain damage. However, NSPCs are heterogeneous and not clearly defined at the molecular level or purified due to a lack of specific cell surface markers. The protocol presented, which has been previously reported, combines a neural-endothelial co-culture system with a metabolic glycan labeling method to identify the surface sialoglycoproteome of primary NSPCs. The NSPC-endothelial co-culture system allows self-renewal and expansion of primary NSPCs in vitro, generating a sufficient number of NSPCs. Sialoglycans in cultured NSPCs are labeled using an unnatural sialic acid metabolic reporter with bioorthogonal functional groups. By comparing the sialoglycoproteome from self-renewing NSPCs expanded in an endothelial co-culture with differentiating neural culture, we identify a list of membrane proteins that are enriched in NSPCs. In detail, the protocol involves: 1) set-up of an NSPC-endothelial co-culture and NSPC differentiating culture; 2) labeling with azidosugar per-O-acetylated N-azidoacetylmannosamine (Ac4ManNAz); and 3) biotin conjugation to modified sialoglycan for imaging after fixation of neural culture or protein extraction from neural culture for mass spectrometry analysis. Then, the NSPC-enriched surface marker candidates are selected by comparative analysis of mass spectrometry data from both the expanded NSPC and differentiated neural cultures. This protocol is highly sensitive for identifying membrane proteins of low abundance in the starting materials, and it can be applied to marker discovery in other systems with appropriate modifications.

Identifying Cell Surface Markers of Primary Neural Stem and Progenitor Cells by Metabolic Labeling of Sialoglycan

Identifying Cell Surface Markers of Primary Neural Stem and Progenitor Cells by Metabolic Labeling of Sialoglycan

Aluminum affects neural phenotype determination of embryonic neural progenitor cells.

Aluminum (Al) is a neurotoxin and is associated with the etiology of neurodegenerative diseases, such as Alzheimer's disease (AD). The Al-free ion (Al3+) is the biologically reactive and toxic form. However, the underlying mechanisms of Al toxicity in the brain remain unclear. Here, we evaluated the effects of Al3+ (in the chloride form-AlCl3) at different concentrations (0.1-100µM) on the morphology, proliferation, apoptosis, migration and differentiation of neural progenitor cells (NPCs) isolated from embryonic telencephalons, cultured as neurospheres. Our results reveal that Al3+ at 100µM reduced the number and diameter of neurospheres. Cell cycle analysis showed that Al3+ had a decisive function in proliferation inhibition of NPCs during neural differentiation and induced apoptosis on neurospheres. In addition, 1µM Al3+ resulted in deleterious effects on neural phenotype determination. Flow cytometry and immunocytochemistry analysis showed that Al3+ promoted a decrease in immature neuronal marker β3-tubulin expression and an increase in co-expression of the NPC marker nestin and glial fibrillary acidic protein. Thus, our findings indicate that Al3+ caused cellular damage and reduced proliferation and migration, resulting in global inhibition of NPC differentiation and neurogenesis.

Abstract 2869: Zika virus is a potent oncolytic agent against aggressive human ependymoma

Abstract Ependymoma is a metastatic CNS tumor responsible for 10% of all pediatric brain tumors. Displaying a poor survival rate of only 32.5%, the conventional ependymoma treatment is surgery and radiation therapy associated to significant morbidity. Previous studies found significantly increased expression of NESTIN, a neural progenitor cell (NPC) marker, in aggressive ependymoma. Zika virus (ZIKV) has recently emerged as an oncolytic therapy against adult and pediatric CNS tumors due to its ability to infect NPCs and neural stem-like tumor cells. We showed for the first time that Brazilian Zika virus strain efficiently destroyed pediatric CNS tumor cells in vitro and in vivo, resulting in significantly mice longer survival and fewer metastasis. Once tumor cells overexpressing NPCs markers are more susceptible to ZIKV infection, here we evaluated the oncolytic properties of Brazilian ZIKV against human ependymoma. A patient-derived ependymoma cell line (USP21-EPE) has been established and found to abnormally express the pluripotency markers CD133, SOX2 and NESTIN. USP21-EPE monolayer culture showed massive cell death three days after ZIKV infection in four tested MOIs: 0.01, 0.1, 1 and 2. At MOI 0,1 50% of cell population decreased viability while at higher MOIs was observed 100% of cell death 72 hours post ZIKV infection. Moreover, ZIKV significantly disrupted CNS ependymoma tumorspheres reinforcing its oncolytic effect against the ependymoma tumor cells. Considering the poor effectiveness and severe side effects of available treatments for ependymoma and that most ZIKV infections are associated to very mild or no symptoms, our findings open new avenues for novel therapies against this aggressive pediatric tumor. Citation Format: Carolini Kaid, Patrícia Semedo-Kuriki, Ernesto Goulart, Luiz Caires-Júnior, Renato Astray, Oswaldo Keith Okamoto, Mayana Zatz. Zika virus is a potent oncolytic agent against aggressive human ependymoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2869.

LncRNA Rik-203 contributes to anesthesia neurotoxicity via microRNA-101a-3p and GSK-3\u03b2-mediated neural differentiation

The mechanism of anesthesia neurotoxicity remains largely to be determined. The effects of long noncoding RNAs (LncRNAs) on neural differentiation and the underlying mechanisms are unknown. We thus identified LncRNA Rik-203 (C130071C03Rik) and studied its role on neural differentiation and its interactions with anesthetic sevoflurane, miRNA and GSK-3β. We found that levels of Rik-203 were higher in hippocampus than other tissues and increased during neural differentiation. Sevoflurane decreased the levels of Rik-203. Rik-203 knockdown reduced mRNA levels of Sox1 and Nestin, the markers of neural progenitor cells, and decreased the count of Sox1 positive cells. RNA-RNA pull-down showed that miR-101a-3p was highly bound to Rik-203. Finally, sevoflurane, knockdown of Rik-203, and miR-101a-3p overexpression all decreased GSK-3β levels. These data suggest that Rik-203 facilitates neural differentiation by inhibiting miR-101a-3p’s ability to reduce GSK-3β levels and that LncRNAs would serve as the mechanism of the anesthesia neurotoxicity.

Identification of CD24 as a marker of Patched1 deleted medulloblastoma-initiating neural progenitor cells.

High morbidity and mortality are common traits of malignant tumours and identification of the cells responsible is a focus of on-going research. Many studies are now reporting the use of antibodies specific to Clusters of Differentiation (CD) cell surface antigens to identify tumour-initiating cell (TIC) populations in neural tumours. Medulloblastoma is one of the most common malignant brain tumours in children and despite a considerable amount of research investigating this tumour, the identity of the TICs, and the means by which such cells can be targeted remain largely unknown. Current prognostication and stratification of medulloblastoma using clinical factors, histology and genetic profiling have classified this tumour into four main subgroups: WNT, Sonic hedgehog (SHH), Group 3 and Group 4. Of these subgroups, SHH remains one of the most studied tumour groups due to the ability to model medulloblastoma formation through targeted deletion of the Shh pathway inhibitor Patched1 (Ptch1). Here we sought to utilise CD antibody expression to identify and isolate TIC populations in Ptch1 deleted medulloblastoma, and determine if these antibodies can help classify the identity of human medulloblastoma subgroups. Using a fluorescence-activated cell sorted (FACS) CD antibody panel, we identified CD24 as a marker of TICs in Ptch1 deleted medulloblastoma. CD24 expression was not correlated with markers of astrocytes or oligodendrocytes, but co-labelled with markers of neural progenitor cells. In conjunction with CD15, proliferating CD24+/CD15+ granule cell precursors (GCPs) were identified as a TIC population in Ptch1 deleted medulloblastoma. On human medulloblastoma, CD24 was found to be highly expressed on Group 3, Group 4 and SHH subgroups compared with the WNT subgroup, which was predominantly positive for CD15, suggesting CD24 is an important marker of non-WNT medulloblastoma initiating cells and a potential therapeutic target in human medulloblastoma. This study reports the use of CD24 and CD15 to isolate a GCP-like TIC population in Ptch1 deleted medulloblastoma, and suggests CD24 expression as a marker to help stratify human WNT tumours from other medulloblastoma subgroups.

Neural induction of porcine-induced pluripotent stem cells and further differentiation using glioblastoma-cultured medium.

Prior to transplantation, preclinical study of safety and efficacy of neural progenitor cells (NPCs) is needed. Therefore, it is important to generate an efficient in vitro platform for neural cell differentiation in large animal models such as pigs. In this study, porcine‐induced pluripotent stem cells (iPSCs) were seeded at high cell density to a neural induction medium containing the dual Sma‐ and Mad‐related protein (SMAD) inhibitors, a TGF‐β inhibitor and BMP4 inhibitor. The dSMADi‐derived NPCs showed NPC markers such as PLAG1, NESTIN and VIMENTIN and higher mRNA expression of Sox1 compared to the control. The mRNA expression of HOXB4 was found to significantly increase in the retinoic acid‐treated group. NPCs propagated in vitro and generated neurospheres that are capable of further differentiation in neurons and glial cells. Gliobalstoma‐cultured medium including injury‐related cytokines treated porcine iPSC‐NPCs survive well in vitro and showed more neuronal marker expression compared to standard control medium. Collectively, the present study developed an efficient method for production of neural commitment of porcine iPSCs into NPCs.

Spheroids Spontaneously Generated In Vitro from Sheep Ovarian Cortical Cells Contain Integrating Cells That Exhibit Hallmarks of Neural Stem/Progenitor Cells.

Cell spheroids are inducible or spontaneously generated cell aggregates produced in vitro that can provide a valuable model for developmental biology, stem cell biology, and cancer therapy research. This investigation aimed to define the cellular identity of spheroids spontaneously generated in vitro from sheep ovarian cortical cells cultured under specific serum-free conditions. Spheroids were characterized during 21 days of culture by morphometric evaluation, detection of alkaline phosphatase (AP) activity, gene expression analyses of stemness transcription factors and several lineage markers, immunolocalization analyses, as well as assessment of self-renewal and differentiation potential. Cell aggregation, evidenced from day 3 of culture onward, resulted in efficient generation of 65-75 spheroids for every 500,000 cells seeded. The spheroids reached maximum diameter (187 ± 15.9 μm) during the second week of culture and exhibited AP activity. Sox2, Oct4, and Nanog were expressed throughout the culture period, with upregulation of Sox2. Neural lineage specification genes (eg, nestin, vimentin, Pax6, and p75NTR) were expressed from day 10 onward at levels above that of Oct4, Nanog and those for endoderm [alpha-fetoprotein (AFP)], and mesoderm (brachyury) specification. Neural stem cell (NSC)/neural progenitor cell (NPC) markers, nestin, Pax6, p75NTR, and vimentin, were extensively localized in cells on day 10, 15 (44.75% ± 5.84%; 93.54% ± 1.35%; 78.90% ± 4.80%; 73.82% ± 3.40%, respectively), and 21 (49.98% ± 5.30%; 91.84% ± 1.9%; 76.74% ± 11.0%; 95.80% ± 3.60%, respectively). Spheroid cell self-renewal was evidenced by cell proliferation and the generation of new spheroids during two consecutive expansion periods. Culture of spheroid cells under differentiation conditions gave rise to cells showing immunolocalization of the neuron-specific antigen NeuN and the astroglial antigen GFAP (glial fibrillary acidic protein). Our results indicate that spheroids spontaneously generated in this culture system were comprised of cells with molecular characteristics of NSC/NPC that can self-renew and differentiate into neurons and glia, supporting the identity of spheroids as neurospheres.

Effects of muscarinic acetylcholine receptor stimulation on the differentiation of mouse induced pluripotent stem cells into neural progenitor cells.

Muscarinic acetylcholine receptors (mAchRs), which are expressed in various embryonic cells, may regulate neuronal differentiation. In the present study, we examined the effects of mAchR stimulation on the differentiation of mouse induced pluripotent stem (iPS) cells into neural progenitor cells (NPCs). Mouse iPS cells were cultured on ultra-low attachment dishes to induce embryoid body (EB) formation. All-trans retinoic acid (ATRA, 3μmol/L) and/or pilocarpine (10 or 100μmol/L), a mAchR agonist, were added to EB cultures for 4days, following which the EBs were cultured on gelatin-coated plates for 7days. Subtype-specific antibody staining revealed that mouse iPS cells predominantly express m2 - and m4 -AchR. Treatment with pilocarpine alone did not affect the expression of Nestin (a specific marker for neural progenitor cells). However, additional treatment with pilocarpine significantly suppressed ATRA-induced Nestin expression. Pretreating EBs with either AF-DX116 (an antagonist of both m2 - and m4 -AchR) or forskolin (an activator of adenylate cyclase) significantly reversed the pilocarpine-induced suppression of Nestin expression. In addition, treatment with pilocarpine significantly suppressed ATRA-induced phosphorylation of cyclic adenosine monophosphate (cAMP) response element-binding protein (CREB). These findings suggest that the stimulation of m2 - or m4 -AchR suppresses ATRA-induced differentiation of mouse iPS cells into NPCs by inhibiting the cAMP/protein kinase A pathway and CREB activation.

Accelerated neural differentiation of mouse embryonic stem cells on aligned GYIGSR-functionalized nanofibers

Substrates for embryonic stem cell culture are typified by poorly defined xenogenic, whole proteins or cellular components that are difficult and expensive to generate, characterize, and recapitulate. Herein, the generation of well-defined scaffolds of Gly-Tyr-Ile-Gly-Ser-Arg (GYIGSR) peptide-functionalized poly(ε-caprolactone) (PCL) aligned nanofibers are used to accelerate the neural lineage commitment and differentiation of D3 mouse embryonic stem cells (mESCs). Gene expression trends and immunocytochemistry analysis were similar to laminin-coated glass, and indicated an earlier differentiation progression than D3 mESCs on laminin. Further, GYIGSR-functionalized nanofiber substrates yielded an increased gene expression of Sox1, a neural progenitor cell marker, and Tubb3, Cdh2, Syp, neuronal cell markers, at early time points. In addition, guidance of neurites was found to parallel the fiber direction. We demonstrate the fabrication of a well-defined, xeno-free functional nanofiber scaffold and demonstrates its use as a surrogate for xenogenic and complex matrixes currently used for the neural differentiation of stem cells ex vivo. Statement of SignificanceIn this paper, we report the use of GYIGSR-functionalized poly(ε-caprolactone) aligned nanofibers as a tool to accelerate the neural lineage commitment and differentiation of D3 mouse embryonic stem cells. The results indicate that functional nanofiber substrates promote faster differentiation than laminin coated substrates. The data suggest that aligned nanofibers and post-electrospinning surface modification with bioactive species can be combined to produce translationally relevant xeno-free substrates for stem cell therapy. Future development efforts are focused on additional bioactive species that are able to function as surrogates for other xenogenic factors found in differentiation media.

Highly efficient methods to obtain homogeneous dorsal neural progenitor cells from human and mouse embryonic stem cells and induced pluripotent stem cells

BackgroundEmbryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) have been widely used to generate cellular models harboring specific disease-related genotypes. Of particular importance are ESC and iPSC applications capable of producing dorsal telencephalic neural progenitor cells (NPCs) that are representative of the cerebral cortex and overcome the challenges of maintaining a homogeneous population of cortical progenitors over several passages in vitro. While previous studies were able to derive NPCs from pluripotent cell types, the fraction of dorsal NPCs in this population is small and decreases over several passages. Here, we present three protocols that are highly efficient in differentiating mouse and human ESCs, as well as human iPSCs, into a homogeneous and stable population of dorsal NPCs. These protocols will be useful for modeling cerebral cortical neurological and neurodegenerative disorders in both mouse and human as well as for high-throughput drug screening for therapeutic development.MethodsWe optimized three different strategies for generating dorsal telencephalic NPCs from mouse and human pluripotent cell types through single or double inhibition of bone morphogenetic protein (BMP) and/or SMAD pathways. Mouse and human pluripotent cells were aggregated to form embryoid bodies in suspension and were treated with dorsomorphin alone (BMP inhibition) or combined with SB431542 (double BMP/SMAD inhibition) during neural induction. Neural rosettes were then selected from plated embryoid bodies to purify the population of dorsal NPCs. We tested the expression of key dorsal NPC markers as well as nonectodermal markers to confirm the efficiency of our three methods in comparison to published and commercial protocols.ResultsSingle and double inhibition of BMP and/or SMAD during neural induction led to the efficient differentiation of dorsal NPCs, based on the high percentage of PAX6-positive cells and the NPC gene expression profile. There were no statistically significant differences in the variation of PAX6 and SOX1-positive NPCs between the two human pluripotent cell-derived methods; therefore, both methods are suitable for producing stable dorsal NPCs. When further differentiated into mature neurons, NPCs gave rise to a population of almost exclusively forebrain cortical neurons, confirming the dorsal fate commitment of the progenitors.ConclusionsThe methods described in this study show improvements over previously published studies and are highly efficient at differentiating human and mouse pluripotent cell types into dorsal PAX6-positive NPCs and eventually into forebrain cortical neurons.

Canine dorsal root ganglia satellite glial cells represent an exceptional cell population with astrocytic and oligodendrocytic properties

Dogs can be used as a translational animal model to close the gap between basic discoveries in rodents and clinical trials in humans. The present study compared the species-specific properties of satellite glial cells (SGCs) of canine and murine dorsal root ganglia (DRG) in situ and in vitro using light microscopy, electron microscopy, and immunostainings. The in situ expression of CNPase, GFAP, and glutamine synthetase (GS) has also been investigated in simian SGCs. In situ, most canine SGCs (>80%) expressed the neural progenitor cell markers nestin and Sox2. CNPase and GFAP were found in most canine and simian but not murine SGCs. GS was detected in 94% of simian and 71% of murine SGCs, whereas only 44% of canine SGCs expressed GS. In vitro, most canine (>84%) and murine (>96%) SGCs expressed CNPase, whereas GFAP expression was differentially affected by culture conditions and varied between 10% and 40%. However, GFAP expression was induced by bone morphogenetic protein 4 in SGCs of both species. Interestingly, canine SGCs also stimulated neurite formation of DRG neurons. These findings indicate that SGCs represent an exceptional, intermediate glial cell population with phenotypical characteristics of oligodendrocytes and astrocytes and might possess intrinsic regenerative capabilities in vivo.

Comparison of 2D and 3D neural induction methods for the generation of neural progenitor cells from human induced pluripotent stem cells

Neural progenitor cells (NPCs) from human induced pluripotent stem cells (hiPSCs) are frequently induced using 3D culture methodologies however, it is unknown whether spheroid-based (3D) neural induction is actually superior to monolayer (2D) neural induction. Our aim was to compare the efficiency of 2D induction with 3D induction method in their ability to generate NPCs, and subsequently neurons and astrocytes. Neural differentiation was analysed at the protein level qualitatively by immunocytochemistry and quantitatively by flow cytometry for NPC (SOX1, PAX6, NESTIN), neuronal (MAP2, TUBB3), cortical layer (TBR1, CUX1) and glial markers (SOX9, GFAP, AQP4). Electron microscopy demonstrated that both methods resulted in morphologically similar neural rosettes. However, quantification of NPCs derived from 3D neural induction exhibited an increase in the number of PAX6/NESTIN double positive cells and the derived neurons exhibited longer neurites. In contrast, 2D neural induction resulted in more SOX1 positive cells. While 2D monolayer induction resulted in slightly less mature neurons, at an early stage of differentiation, the patch clamp analysis failed to reveal any significant differences between the electrophysiological properties between the two induction methods. In conclusion, 3D neural induction increases the yield of PAX6+/NESTIN+ cells and gives rise to neurons with longer neurites, which might be an advantage for the production of forebrain cortical neurons, highlighting the potential of 3D neural induction, independent of iPSCs' genetic background.

Isolation, characterization, and differentiation of multipotent neural progenitor cells from human cerebrospinal fluid in fetal cystic myelomeningocele

Despite benefits of prenatal in utero repair of myelomeningocele, a severe type of spina bifida aperta, many of these patients will still suffer mild to severe impairment. One potential source of stem cells for new regenerative medicine-based therapeutic approaches for spinal cord injury repair is neural progenitor cells (NPCs) in cerebrospinal fluid (CSF). To this aim, we extracted CSF from the cyst surrounding the exposed neural placode during the surgical repair of myelomeningocele in 6 fetuses (20 to 26weeks of gestation). In primary cultured CSF-derived cells, neurogenic properties were confirmed by in vitro differentiation into various neural lineage cell types, and NPC markers expression (TBR2, CD15, SOX2) were detected by immunofluorescence and RT-PCR analysis. Differentiation into three neural lineages was corroborated by arbitrary differentiation (depletion of growths factors) or explicit differentiation as neuronal, astrocyte, or oligodendrocyte cell types using specific induction mediums. Differentiated cells showed the specific expression of neural differentiation markers (βIII-tubulin, GFAP, CNPase, oligo-O1). In myelomeningocele patients, CSF-derived cells could become a potential source of NPCs with neurogenic capacity. Our findings support the development of innovative stem-cell-based therapeutics by autologous transplantation of CSF-derived NPCs in damaged spinal cords, such as myelomeningocele, thus promoting neural tissue regeneration in fetuses.