Neuronal Cell Lineages Research Articles

Trimethylation and Acetylation of β-Catenin at Lysine 49 Represent Key Elements in ESC Pluripotency

Wnt/β-catenin signaling is required for embryonic stem cell (ESC) pluripotency by inducing mesodermal differentiation and inhibiting neuronal differentiation; however, how β-catenin counter-regulates these differentiation pathways is unknown. Here, we show that lysine 49 (K49) of β-catenin is trimethylated (β-catMe3) by Ezh2 or acetylated (β-catAc) by Cbp. Significantly, β-catMe3 acts as a transcriptional co-repressor of the neuronal differentiation genes sox1 and sox3, whereas β-catAc acts as a transcriptional co-activator of the key mesodermal differentiation gene t-brachyury (t-bra). Furthermore, β-catMe3 and β-catAc are alternatively enriched on repressed or activated genes, respectively, during ESC and adult stem cell differentiation into neuronal or mesodermal progenitor cell lineages. Importantly, expression of a β-catenin K49A mutant results in major defects in ESC differentiation. We conclude that β-catenin K49 trimethylation and acetylation are key elements in regulating ESC pluripotency and differentiation potential.

Bovine herpesviruses induce different cell death forms in neuronal and glial-derived tumor cell cultures.

Oncolytic viruses have the ability to infect tumor cells and leave healthy cells intact. In this study, bovine herpesvirus 1 (BHV1; Los Angeles, Cooper, and SV56/90 strains) and bovine herpesvirus 5 (BHV5; SV507/99 and GU9457818 strains) were used to infect two neuronal tumor cell lineages: neuro2a (mouse neuroblastoma cells) and C6 (rat glial cells). BHV1 and BHV5 strains infected both cell lines and positively correlated with viral antigen detection (p < 0.005). When neuro2a cells were infected by Los Angeles, SV507/99, and GU9457818 strains, 40% of infected cells were under early apoptosis and necroptosis pathways. Infected C6 cells were >40% in necroptosis phase when infected by BHV5 (GU9457818 strain). Blocking caspase activation did not interfere with cell death. However, when necroptosis was blocked, 60-80% of both infected cells with either virus switched to early apoptosis pathway with no interference with virus replication. Moreover, reactive oxygen species production and mitochondrial membrane dysfunction were detected at high levels in both infected cell lines. In spite of apoptosis and necroptosis blockage, tumor necrosis factor alpha (TNFA) and virus transcription were positively correlated for all viral strains studied. Thus, these results contribute to the characterization of BHV1 and BHV5 as potential oncolytic viruses for non-human cells. Nonetheless, the mechanisms underlying their oncolytic activity in human cells are still to be determined.

Oral mucosal progenitor cell clones resist in vitro myogenic differentiation

Progenitor cells derived from the oral mucosa lamina propria (OMLP-PCs) demonstrate an ability to differentiate into tissue lineages removed from their anatomical origin. This clonally derived population of neural-crest cells have demonstrated potential to differentiate along mesenchymal and neuronal cell lineages. ObjectiveSignificant efforts are being made to generate functioning muscle constructs for use in research and clinical tissue engineering. In this study we aimed to determine the myogenic properties of clonal populations of expanded OMLP-PCs. DesignPCs were subject to several in vitro culture conditions in an attempt to drive myogenic conversion. Methodologies include use of demethylation gene-modifying reagents, mechanical conditioning of tissue culture substrates, tuneable polyacrylamide gels and a 3-dimensional construct as well as published myogenic media compositions. PCR and immunostaining for the muscle cell markers Desmin and MyoD1 were used to assess muscle differentiation. ResultsThe clones tested did not intrinsically express myogenic lineage markers. Despite use of two and 3-dimensional pre-published in vitro culture protocols OMLP clones could not be differentiated down a myogenic lineage. ConclusionsWithin the confines of these experimental parameters it was not possible to generate identifiable muscle using the clonal populations. When reviewing the previously successful reports of myogenic conversion, cells utilised have either been derived from tissues that are already ‘primed’ with the requisite myogenic genetic potential or have undergone specific genetic reprogramming to enhance the myogenic conversion rate. This, along with as yet unidentified stromal interplay, may therefore be required for positive myogenic differentiation to be realised.

Prostaglandin E2 promotes neural proliferation and differentiation and regulates Wnt target gene expression.

Prostaglandin E2 (PGE2 ) is an endogenous lipid molecule that regulates important physiological functions, including calcium signaling, neuronal plasticity, and immune responses. Exogenous factors such as diet, exposure to immunological agents, toxic chemicals, and drugs can influence PGE2 levels in the developing brain and have been associated with autism disorders. This study seeks to determine whether changes in PGE2 level can alter the behavior of undifferentiated and differentiating neuroectodermal (NE-4C) stem cells and whether PGE2 signaling impinges on the Wnt/β-catenin pathways. We show that PGE2 increases proliferation of undifferentiated NE-4C stem cells. PGE2 also promotes the progression of NE-4C stem cell differentiation into neuronal-lineage cells, which is apparent by accelerated appearance of neuronal clusters (neurospheres) and earlier expression of the neuronal marker microtubule-associated protein tau. Furthermore, PGE2 alters the expression of downstream Wnt-regulated genes previously associated with neurodevelopmental disorders. In undifferentiated stem cells, PGE2 downregulates Ptgs2 expression and upregulates Mmp9 and Ccnd1 expression. In differentiating neuronal cells, PGE2 causes upregulation of Wnt3, Tcf4, and Ccnd1. The convergence of the PGE2 and the Wnt pathways is also apparent through increased expression of active β-catenin, a key signaling component of the Wnt/β-catenin pathways. This study provides novel evidence that PGE2 influences progression of neuronal development and influences Wnt target gene expression. We discuss how these findings could have potential implications for neurodevelopmental disorders such as autism. © 2016 Wiley Periodicals, Inc.

Ex vivo differentiation of human bone marrow-derived stem cells into neuronal cell-like lineages

Ex vivo differentiation of human bone marrow-derived stem cells into neuronal cell-like lineages Adeeb Al-Zoubi,1,2 Feras Altwal,3 Farah Khalifeh,2 Jamil Hermas,4 Ziad Al-Zoubi,5 Emad Jafar,5 Mohammed El-Khateeb,6,7 1Department of Surgery, University of Illinois College of Medicine at Peoria, Peoria, IL, USA; 2Stem Cells of Arabia, Amman, Jordan; 3Department of Neuroscience, School of Graduate and Postdoctoral Studies, Rosalind Franklin University of Medicine and Science, North Chicago, IL, USA; 4Stem Cell Division, Al-Yamama Company, 5Jordan Orthopedic and Spinal Center, 6National Center for Diabetes, Endocrinology and Genetics, 7Department of Pathology, Faculty of Medicine, University of Jordan, Amman, Jordan Background: Methods to obtain safe and practical populations of stem cells (SCs) at a clinical grade that are able to differentiate into neuronal cell lineages are yet to be developed. In a previous study, we showed that mouse bone marrow-derived SCs (BM-SCs) differentiated into neuronal cell-like lineages when put in a neuronal-like environment, which is a special media supplemented with the necessary growth factors needed for the differentiation of SCs into neuronal cell-like lineages. Aim: In this study, we aim to assess the potentials of adult human CD34+ and CD133+ SCs to differentiate into neuronal cell-like lineages ex vivo when placed in a neuronal-like microenvironment. Methods: The neuronal-like microenvironment was created by culturing cells in nonhematopoietic expansion media (NHEM) supplemented with growth factors that favor differentiation into neuronal cell lineages (low-affinity nerve growth factor [LNGF], mouse spinal cord extract [mSpE], or both). Cultured cells were assessed for neuronal differentiation by cell morphologies and by expression of GFAP. Results: Our results show that culturing unpurified human BM-derived mononuclear cells (hBM-MNCs) in NHEM+LNGF+mSpE did not lead to neuronal differentiation. In contrast, culturing of purified CD34+ hBM-SCs in NHEM+LNGF+mSpE favored their differentiation into astrocyte-like cells, whereas culturing of purified CD133+ hBM-SCs in the same media favored their differentiation into neuronal-like cells. Interestingly, coculturing of CD34+ and CD133+ hBM-SCs in the same media enhanced the differentiation into astrocyte-like cells and neuronal-like cells, in addition to oligodendrocyte-like cells. Conclusion: These results suggest that a mixture of purified CD34+ and CD133+ cells may enhance the differentiation into neuronal cell-like lineages and give broader neuronal cell lineages than when each of these cell types is cultured alone. This method opens the window for the utilization of specific populations of hBM-SCs to be delivered in a purified form for the potential treatment of neurodegenerative diseases in the future. Keywords: cell therapy, neurodegenerative disease, neurons, astrocyes, CD34+, CD133+, MACS

Gelatin Directly Enhances Neurogenic Differentiation Potential in Bone Marrow-Derived Mesenchymal Stem Cells Without Stimulation of Neural Progenitor Cell Proliferation

Gelatin has been reported to induce generation of mesenchymal stem cells (MSCs) with enhanced potential of differentiation into neuronal lineage cells. However, the presence of various cell types besides MSCs in bone marrow has raised doubts about the effects of gelatin. In the following report, we determined whether gelatin can directly enhance neurogenic differentiation potential in MSCs without proliferation of neural progenitor cells (NPCs). MSCs comprised a high proportion of bone marrow-derived primary cells (BMPCs) and gelatin induced significant increases in MSC proliferation during primary culture, and the proportion of MSCs was maintained at more than 99% throughout the subculture. However, NPCs comprised a low percentage of BMPCs and a decrease in proliferation was detected despite gelatin treatment during the primary culture, and the proportion of subcultured NPCs gradually decreased. In a similar manner, MSCs exposed to gelatin during primary culture showed more enhanced neurogenic differentiation ability than those not exposed to gelatin. Together, these results demonstrate that gelatin directly enhances neurogenic differentiation in bone marrow-derived MSCs without stimulating NPC proliferation.

Induction of the type I interferon response in neurological forms of Gaucher disease

BackgroundNeuroinflammation is a key phenomenon in the pathogenesis of many neurodegenerative diseases. Understanding the mechanisms by which brain inflammation is engaged and delineating the key players in the immune response and their contribution to brain pathology is of great importance for the identification of novel therapeutic targets for these devastating diseases. Gaucher disease, the most common lysosomal storage disease, is caused by mutations in the GBA1 gene and is a significant risk factor for Parkinson’s disease; in some forms of Gaucher disease, neuroinflammation is observed.MethodsAn unbiased gene profile analysis was performed on a severely affected brain area of a neurological form of a Gaucher disease mouse at a pre-symptomatic stage; the mouse used for this study, the Gbaflox/flox; nestin-Cre mouse, was engineered such that GBA1 deficiency is restricted to cells of neuronal lineage, i.e., neurons and macroglia.ResultsThe 10 most up-regulated genes in the ventral posteromedial/posterolateral region of the thalamus were inflammatory genes, with the gene expression signature significantly enriched in interferon signaling genes. Interferon β levels were elevated in neurons, and interferon-stimulated genes were elevated mainly in microglia. Interferon signaling pathways were elevated to a small extent in the brain of another lysosomal storage disease mouse model, Krabbe disease, but not in Niemann-Pick C or Sandhoff mouse brain. Ablation of the type I interferon receptor attenuated neuroinflammation but had no effect on GD mouse viability.ConclusionsOur results imply that the type I interferon response is involved in the development of nGD pathology, and possibly in other lysosomal storage diseases in which simple glycosphingolipids accumulate, and support the notion that interferon signaling pathways play a vital role in the sterile inflammation that often occurs during chronic neurodegenerative diseases in which neuroinflammation is present.Electronic supplementary materialThe online version of this article (doi:10.1186/s12974-016-0570-2) contains supplementary material, which is available to authorized users.

293 cells express both epithelial as well as mesenchymal cell adhesion molecules.

The 293 cell line, used extensively in various types of studies due to the ease with which these cells can be transfected, was thought to be derived by the transformation of primary cultures of human embryonic kidney cells with sheared adenovirus type 5 DNA. Although the 293 cells were assumed to originate from epithelial cells, the exact origin of these cells remains unknown. Previous attempts to characterize these cells combined immunostaining, immunoblot analysis and microarray analysis to demonstrate that 293 cells express neurofilament subunits, α-internexin, and several other proteins typically found in neurons. These findings raised the possibility that the 293 cell line may have originated from human neuronal lineage cells. Contrary to this suggestion, in this study, we found that the 293 cells expressed N-cadherin and vimentin, which are marker proteins expressed in mesenchymal cells. Furthermore, the 293 cells also expressed E-cadherin, cytokeratins 5/8 and desmoglein 2, which are epithelial cell markers. When the cells, primarily cultured from the kidneys of Clawn miniature swine and passaged 10–15 generations [termed porcine kidney epithelial (PKE) cells] were examined, they were found to be positive for the expression of both mesenchymal and epithelial markers. Thus, transformation by adenovirus was not necessary for the cells to express N-cadherin. Occludin and zonula occludens (ZO)-1, two components of tight junctions in epithelial and endothelial cells, were detected in the 293 and the PKE cells. Thus, the findings of the present study demonstrate that 293 cells retain several characteristics of epithelial cells.

Lysine-specific demethylase-1 (LSD1) is compartmentalized at nuclear chromocenters in early post-mitotic cells of the olfactory sensory neuronal lineage.

Mammalian olfaction depends on the development of specialized olfactory sensory neurons (OSNs) that each express one odorant receptor (OR) protein from a large family of OR genes encoded in the genome. The lysine-specific demethylase-1 (LSD1) protein removes activating H3K4 or silencing H3K9 methylation marks at gene promoters and is required for proper OR regulation. We show that LSD1 protein exhibits variable organization within nuclei of developing OSNs, and tends to consolidate into a single dominant compartment at the edges of chromocenters within nuclei of early post-mitotic cells of the mouse olfactory epithelium (MOE). Using an immortalized cell line derived from developing olfactory placode, we show that consolidation of LSD1 appears to be cell-cycle regulated, with a peak occurrence in early G1. LSD1 co-compartmentalizes with CoREST, a protein known to collaborate with LSD1 to carry out a variety of chromatin-modifying functions. We show that LSD1 compartments co-localize with 1-3 OR loci at the exclusion of most OR genes, and commonly associate with Lhx2, a transcription factor involved in OR regulation. Together, our data suggests that LSD1 is sequestered into a distinct nuclear space that might restrict a histone-modifying function to a narrow developmental time window and/or range of OR gene targets.

A mouse embryonic stem cell culture system with stable and regulatable expression miR-3099: an in vitro approach towards functional genomics study

Event Abstract Back to Event A mouse embryonic stem cell culture system with stable and regulatable expression miR-3099: an in vitro approach towards functional genomics study Shahidee Zainal Abidin1, 2, 3*, Jia-Wen Leong1, 2, 4, Norshariza Nordin2, 3, Syahril Abdullah2, 3, Pike-See Cheah1, 2, 4 and King-Hwa Ling1, 2, 3* 1 Universiti Putra Malaysia, NeuroBiology and Genetics Group, Faculty of Medicine and Health Sciences, Malaysia 2 Universiti Putra Malaysia, Genetics and Regenerative Medicine Research Center, Faculty of Medicine and Health Sciences, Malaysia 3 Universiti Putra Malaysia, Medical Genetics Unit, Department of Biomedical Sciences, Faculty of Medicine and Health Sciences, Malaysia 4 Universiti Putra Malaysia, Department of Human Anatomy, Faculty of Medicine and Health Sciences, Malaysia miR-3099 is a small non-coding RNA that is highly expressed throughout embryogenesis and is localised in cortical plate between E13.5 and E17.5 in developing mouse brain. Majority of the cells in the brain at these time-points are committed to neuronal cell lineage. To understand more on the role of miR-3099 during neurogenesis, this study aims to characterise the role of miR-3099 via in vitro approach with respect to functional genomic study. The bioinformatics study was used to predict the downstream-targeted genes by using four independent prediction software (miRDB, miRanda, TargetScan and DIANA micro-T CDS). The target genes that were predicted by at least three different databases were subjected to DAVID bioinformatics analysis to understand their function in biological processes followed by Allen Brain Atlas expression profile analysis. Based on the analysis, it was suggested that miR-3099 was potentially involved in neural stem cell (NSC) fate determination pathway. This warrants further characterisation by generating mouse embryonic stem (mES) cell with regulatable expression miR-3099 to perform function study at molecular level. Interestingly, miR-3099 seed region was found to be 100% identical with chr21:40558602-40558624 in human genome. This indicates that, by extrapolating the finding from animal model will contribute to a more detailed knowledge of the molecular mechanisms of NSC fate determination toward specific lineages. Acknowledgements The study was supported by Malaysian Ministry of Higher Education's Fundamental Research Grant Scheme awarded to P.-S. Cheah (FRGS/1/2015/SKK08/UPM/02/1) and Malaysian Ministry of Science, Technology and Innovation's Sciencefund grant awarded to K.-H. Ling (02-01-04-SF2336). Keywords: Cerebral Cortex, Genomics, miRNA, in vitro, embryonic stem cell Conference: 14th Meeting of the Asian-Pacific Society for Neurochemistry, Kuala Lumpur, Malaysia, 27 Aug - 30 Aug, 2016. Presentation Type: O03: Postgraduate Travel Awardees Oral Session 3 Topic: 14th Meeting of the Asian-Pacific Society for Neurochemistry Citation: Zainal Abidin S, Leong J, Nordin N, Abdullah S, Cheah P and Ling K (2016). A mouse embryonic stem cell culture system with stable and regulatable expression miR-3099: an in vitro approach towards functional genomics study. Conference Abstract: 14th Meeting of the Asian-Pacific Society for Neurochemistry. doi: 10.3389/conf.fncel.2016.36.00091 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 04 Aug 2016; Published Online: 11 Aug 2016. * Correspondence: Mr. Shahidee Zainal Abidin, Universiti Putra Malaysia, NeuroBiology and Genetics Group, Faculty of Medicine and Health Sciences, Serdang, Selangor, Malaysia, shah.68000@gmail.com Dr. King-Hwa Ling, Universiti Putra Malaysia, NeuroBiology and Genetics Group, Faculty of Medicine and Health Sciences, Serdang, Selangor, Malaysia, lkh@upm.edu.my Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Shahidee Zainal Abidin Jia-Wen Leong Norshariza Nordin Syahril Abdullah Pike-See Cheah King-Hwa Ling Google Shahidee Zainal Abidin Jia-Wen Leong Norshariza Nordin Syahril Abdullah Pike-See Cheah King-Hwa Ling Google Scholar Shahidee Zainal Abidin Jia-Wen Leong Norshariza Nordin Syahril Abdullah Pike-See Cheah King-Hwa Ling PubMed Shahidee Zainal Abidin Jia-Wen Leong Norshariza Nordin Syahril Abdullah Pike-See Cheah King-Hwa Ling Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

SC-02 * MODELING PEDIATRIC GBM USING NEURAL STEM CELL-DERIVED TUMORS WITH DIVERGENT BEHAVIOR

Pediatric GBM can exhibit a diversity of cell behaviors and responses to therapy despite similar oncogenic drivers. The reasons for this are often unclear. Using a murine model for GBM based on the oncogenic transformation of neural progenitor cells we sought to generate tumors with diverse cell behaviors despite being driven by a common oncogene. Using in vivo tumor phenotype as evidence of divergent tumor behavior, we isolated two tumor lines with different phenotypes. Tumor progenitor cell (TPC) line-A was highly proliferative and resulted in rapid lethality (median overall survival of 22 days) vs. TPC-B that was less proliferative and exhibited a more prolonged survival (median overall survival of 33 days). Interestingly, TPC-A invaded in perivascular fashion in vivo. In contrast, TPC-B tumors invaded diffusely, as single cells. Transcriptional profiling revealed an enrichment of mesenchymal GBM subtype genes in TPC-A (normalized enrichment score of 1.5) and proneural GBM subtype genes in TPC-A (normalized enrichment score of 2.6). Consistent with the gene expression data, TPC-A exhibited increased activation of Stat3 (pY705) relative to TPC-B. Furthermore, while both clones expressed hEGFRvIII, TPC-A cells showed higher levels of EGFR activation (pY1173 and pY1068), than TPC-B cells. In vitro, TPC-A expanded at a higher rate than TPC-B and expressed Prominin-1 (CD133). TPC-B cells were Prominin-1 negative, but more prone to adhere to ECM components than TPC-A, and invaded more extensively in vitro. Moreover, under differentiation conditions, TPC-A failed to differentiate and continuously proliferated. TPC-B differentiated to cells of neuronal, astrocytic, and oligodendrocytic lineages. Interestingly, these lines also exhibited differential response to therapy. In the current project we demonstrate how transduction of neurospheres can be used to generate tumor lines with vastly different cell behaviors, reflective of different pediatric GBM, and that these lines are useful for both mechanistic and preclinical studies.

Selection for tameness, a key behavioral trait of domestication, increases adult hippocampal neurogenesis in foxes.

Work on laboratory and wild rodents suggests that domestication may impact on the extent of adult hippocampal neurogenesis and its responsiveness to regulatory factors. There is, however, no model of laboratory rodents and their nondomesticated conspecifics that would allow a controlled comparison of the effect of domestication. Here, we present a controlled within-species comparison of adult hippocampal neurogenesis in farm-bred foxes (Vulpes vulpes) that differ in their genetically determined degree of tameness. Quantitative comparisons of cell proliferation (Ki67) and differentiating cells of neuronal lineage (doublecortin, DCX) in the hippocampus of foxes were performed as a proxy for neurogenesis. Higher neurogenesis was observed in tameness-selected foxes, notably in an extended subgranular zone of the middle and temporal compartments of the hippocampus. Increased neurogenesis is negatively associated with aggressive behavior. Across all animals, strong septotemporal gradients were found, with higher numbers of proliferating cells and young neurons relative to resident granule cells in the temporal than in the septal hippocampus. The opposite gradient was found for the ratio of DCX/Ki67- positive cells. When tameness-selected and unselected foxes are compared with rodents and primates, proliferation is similar, while the number of young neurons is higher. The difference may be mediated by an extended period of differentiation or higher rate of survival. On the background of this species-specific neurogenic pattern, selection of foxes for a single behavioral trait key to domestication, i.e., genetic tameness, is accompanied by global and region-specific increases in neurogenesis.

Differentiation of Wharton's jelly mesenchymal stem cells into neurons in alginate scaffold.

Alginate scaffold has been considered as an appropriate biomaterial for promoting the differentiation of embryonic stem cells toward neuronal cell lineage. We hypothesized that alginate scaffold is suitable for culturing Wharton's jelly mesenchymal stem cells (WJMSCs) and can promote the differentiation of WJMSCs into neuron-like cells. In this study, we cultured WJMSCs in a three-dimensional scaffold fabricated by 0.25% alginate and 50 mM CaCl2 in the presence of neurogenic medium containing 10 μM retinoic acid and 20 ng/mL basic fibroblast growth factor. These cells were also cultured in conventional two-dimensional culture condition in the presence of neurogenic medium as controls. After 10 days, immunofluorescence staining was performed for detecting β-tubulin (marker for WJMSCs-differentiated neuron) and CD271 (motor neuron marker). β-Tubulin and CD271 expression levels were significantly greater in the WJMSCs cultured in the three-dimensional alginate scaffold than in the conventional two-dimensional culture condition. These findings suggest that three-dimensional alginate scaffold cell culture system can induce neuronal differentiation of WJMSCs effectively.

An agarose gel-based neurosphere culture system leads to enrichment of neuronal lineage cells in vitro.

Stem cell-based therapy holds great potential especially for neurological disorders. However, clinical applications await further understanding of many aspects of stem cell differentiation and development of technology enabling manipulation of stem cells into desired cell types in the central nervous system. Here, we developed a new method that leads to enrichment of neuronal lineage cells in neural stem cell cultures. The protocol involves cultivation of primary cells derived from the forebrains of rat E18 embryos above a layer of nonadhesive hard agarose gel in the form of neurospheres. In contrast to the neurospheres that were cultured above an anti-adhesive hydrogel layer, the primary cells that were cultured above a layer of agarose gel preferentially differentiated into β-III tubulin-positive neurons when allowed to undergo differentiation in vitro.In an effort to investigate the mechanism behind this observation, we found that the gene expression of a vertebrate neuronal determination gene (neurogenin1) was enhanced in the neurospheres that proliferated above a layer of agarose gel as compared with the control, and the gene expression level of neurogenin1 was quite well correlated with the rigidity of agarose gel. These results indicate that agarose gel can contribute, at least in part, to enrich neuronal progenitors and immature postmitotic neurons during neurosphere formation and may provide additional information to establish efficient protocols for the neural stem cell-based study.

Chemically defined serum-free and xeno-free media for multiple cell lineages.

Cell culture is one of the most common methods used to recapitulate a human disease environment in a laboratory setting. Cell culture techniques are used to grow and maintain cells of various types including those derived from primary tissues, such as stem cells and cancer tumors. However, a major confounding factor with cell culture is the use of serum and animal (xeno) products in the media. The addition of animal products introduces batch and lot variations that lead to experimental variability, confounds studies with therapeutic outcomes for cultured cells, and represents a major cost associated with cell culture. Here we report a commercially available serum-free, albumin-free, and xeno free (XF) media (Neuro-Pure(TM)) that is more cost-effective than other commercial medias. Neuro-Pure was used to maintain and differentiate various cells of neuronal lineages, fibroblasts, as well as specific cancer cell lines; without the use of contaminants such serum, albumin, and animal products. Neuro-Pure allows for a controlled and reproducible cell culture environment that is applicable to translational medicine and general tissue culture.

GENERATION AND UTILIZATION OF HUMAN UMBILICAL VEIN ENDOTHELIAL CELLS-DERIVED-IPS FOR STUDYING ENDOTHELIAL GENE REGULATION

Damage to vascular endothelial cells (EC) is the core of various vascular diseases such as stroke and cardiovascular complications. Understanding molecular mechanisms that govern endothelial gene expression and function is crucial for developing novel therapeutic approaches for vascular endothelial dysfunction-related complications. Induced pluripotent stem cells (iPS) provide a valuable in vitro system to study the molecular processes, including EC-specific gene regulation, that contribute to establishment of EC phenotype. We aimed to study endothelial gene regulation by generating iPS from human umbilical vein EC (HUVECs) and differentiating the resulting iPS back into EC. This model provides a system with a homogenous genetic Background to explore how EC phenotype is revoked (HUVEC to iPS), and reestablished (iPS to EC). Colonies (iPS) were generated using originally reported transcription factors. Quantitative RT-PCR and immunofluorescence analysis were used to characterize iPS and cell lineages derived from iPS. Pluripotency of the iPS were demonstrated by generation of embryoid bodies (EB) and detection of the three germ layer markers. Additionally a novel method was used to directly differentiate iPS into neuronal cell lineages with high efficiency without the use of EB. Next EB generated from iPS were induced to differentiate into EC, thus establishing the EC to iPS back to EC system for analyses. In this system we explored the pattern of activation and repression of a highly EC-specific gene, von Willebrand factor (VWF), as well as transacting factors and cofactors that regulate the VWF promoter activity. Our analyses demonstrated that expression of transacting factors that function as activators of the VWF promoter mirrored that of VWF gene, while those functioning as repressors where either reversed or not significantly altered. Temporal expression of activators were observed during various stages of differentiation. Specifically during the progression from EB to EC we observed upregulation of GATA6 followed by Ets transactivators. Furthermore, the ability of iPS to incorporate into microvasculature was demonstrated in a mouse model of kidney vascular injury. Generation of iPS from EC and differentiation of iPS back to EC provides a unique opportunity to explore the molecular mechanisms that are involved in establishment of EC specific gene regulation and consequently the establishment of EC phenotype. This system will also provide an opportunity for exploring the potential of these EC derived iPS and their differentiated EC towards developing cell- therapy approaches for vascular diseases.

A novel in vivo model of permanent intestinal aganglionosis

BackgroundEnteric neuromuscular disease is a characteristic of several disease states, including Hirschsprung disease, esophageal achalasia, Chagas disease, and gastroparesis. Medical therapy for these conditions is limited, and surgical intervention may incur significant morbidity. Alternatively, transplantation of neural progenitor cells may regenerate enteric ganglia. Existing aganglionosis model systems are limited by swift animal demise or by spontaneous regeneration of native ganglia. We propose a novel protocol to induce permanent aganglionosis in a segment of rat jejunum, which may serve as an experimental transplantation target for cellular therapy. Materials and methodsThis protocol was performed in 17 adult female Sprague–Dawley rats. A laparotomy was performed and a 1-cm segment of jejunum was isolated from continuity. Among 14 rats, the isolated segments were treated with benzalkonium chloride (BAC) for 20 min to induce aganglionosis. Jejunal segment isolation was performed without BAC treatment in three rats. The animals were euthanized at posttreatment days 21–166. Muscle layer diameter was compared among normal, isolated, and BAC-treated isolated jejunal segments. The presence of jejunal ganglia was documented by immunohistochemical staining (IHC) for beta-III tubulin (TUJ1) and S100, markers of neuronal and glial cell lineages, respectively. ResultsGanglia were identified by IHC in normal and isolated jejunal segments. Isolated segments had significantly hypertrophied smooth muscle layers compared with normal jejunum (diameter 343 ± 53 μm versus 211 ± 37 μm, P < 0.0001). BAC-treated jejunal segments had no IHC evidence of ganglionic structures. Aganglionosis was persistent in all specimens up to 166 days after treatment. ConclusionsThe exclusion of a jejunal segment from continuity and concurrent treatment with BAC results in an effective, reproducible, and permanent model of aganglionosis. Muscular hypertrophy and aganglionosis in the isolated jejunal segment make it an ideal recipient site for transplantation of neuroglial precursor cells.

Establishment of endogenous human tympanic membrane-derived somatic stem cells for stem cell therapy.

We examined whether somatic stem cells (SSCs) exist in human tympanis membrane (hTM) and whether they could be differentiated into neural lineage cells. The hTM-SSCs could generate neurospheres, which could differentiate into specific neural linage cells under specific differentiation conditions. Also, we conducted another experiment that led to differentiation into neurospheres and neuronal lineage cells, which occurred independent of each other. Independent of each other condition, hTM-SSCs could differentiate into neurospheres, and subsequently, into neuronal lineage cells. However, NS-NR neural differentiation rates are higher than independent of each other culture system.

The Lgr5 transgene is expressed specifically in glycinergic amacrine cells in the mouse retina

Retinal amacrine cells are a diverse set of interneurons within the inner nuclear layer. The canonical Wnt pathway is highly active within mature amacrine cells, but its role remains unclear. Leucine-rich repeat containing G-protein receptor 5 (Lgr5) is a newly identified component of the Wnt receptor complex that potentiates beta-catenin signaling. In multiple epithelial organs Lgr5 marks adult tissue stem cells. We investigated the expression of this gene using Lgr5-eGFP-IRES-CreER transgenic reporter mice. In the eye, Lgr5 was exclusively expressed in glycinergic amacrine cells in adult mice. Amacrine cells are post-mitotic and represent the first neuronal and non-stem cell lineage to express Lgr5. We further interrogated the spatiotemporal labeling of individual amacrine cells with controlled fluorophore expression. This “fluorofilling” technique provides a tool to study amacrine morphology and dissect neural networks.

Light‐controlled astrocytes promote human mesenchymal stem cells toward neuronal differentiation and improve the neurological deficit in stroke rats

Astrocytes are key components of the central nervous system (CNS) and release factors to support neural stem cell proliferation, differentiation, and migration. Adenosine 5'-triphosphate (ATP) is one of the key factors released upon activation of astrocytes that regulates the neural stem cell's function. However, it is not clear whether ATP derived from the depolarized astrocytes plays a vital role in promoting the neuronal differentiation of mesenchymal stem cells (MSCs) in vitro and in vivo. Herein, for the first time, we co-cultured MSCs with light-stimulated-channelrhodopsin-2 (ChR2)-astrocytes, and observed that the neuronal differentiation of MSCs was enhanced by expressing more neuronal markers, Tuj1 and NeuN. The ChR2-astrocyte-conditioned medium also stimulated MSCs differentiating into neuronal lineage cells by expressing more Tuj1 and Pax6, which was blocked by the P2X receptor antagonist, TNP-ATP. Then we found that light-depolarization of astrocytes significantly increased ATP accumulation in their bathing medium without impairing the cell membrane. We further found that ATP up-regulated the Tuj1, Pax6, FZD8 and β-catenin mRNA levels of MSCs, which could be reversed by application of TNP-ATP. Together these in vitro data provided convergent evidence that ATP from light-depolarized-astrocytes activated the wnt/β-catenin signaling of MSCs through binding to the P2X receptors, and promoted the neuronal differentiation of MSCs. Finally but importantly, our study also demonstrated in stroke rats that light-controlled astrocytes stimulated endogenous ATP release into the ischemic area to influence the transplanted MSCs, resulting in promoting the MSCs towards neuronal differentiation and improvements of neurological deficit.