Name: an enzyme- and serum-free neural stem cell culture model for emt investigation suited for drug discovery
Uploaded: 2016-08-23T13:00:00.000+00:00
Duration: 7 min 43 s
Description: Watch this Scientific Journal Video about An Enzyme- and Serum-free Neural Stem Cell Culture Model for EMT Investigation Suited for Drug Discovery at JoVE.com

The study was supported by the University of Basel Science Foundation and the Swiss National Science Foundation by a grant to MHS and AG (SNF IZLIZ3_157230). We thank: Dr. Tania Rinaldi Burkat for generously providing infrastructure; all members of the Bettler group for discussions and comments. We thank Gerhard Dorne (Leica Microsystems, Switzerland) for professional and competent installation of the Full HD MC170 video camera (Leica Microsystems, Switzerland).

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The authors declare that they have no competing financial interests.

In this study a standardized system for EMT analysis utilizing NSCs is described (summarized in Supplementary Figure 3). The standardization ensures reproducibility (Table 1 and 2). The NSCs are derived from the developing cortex, a tissue that normally does not undergo EMT. This is of advantage for the analysis of early steps in EMT. Initial steps in EMT cannot be adequately studied in tumor cells that have accumulated genetic changes and may have already adopted EMT features. Moreover, primary tumor samples are not ideal to understand EMT since most malignant tumors are heterogeneous containing both invasive and non-invasive cells. The presented protocol provides EMT researchers with a novel model to study early steps of EMT induction. Here we show that the key EMT inducers of the Zeb and Twist family are sequentially activated: during the first phase Zeb1 and Zeb2 are upregulated, during the second phase Twist2 (Figure 6). Previously, we had shown that Snail1 is already upregulated several hours after BMP4-exposure8. We had also shown that upon BMP4-exposure the neuroepithelial stem cells from the central cortex differentiate into mesenchymal cells, positive for smooth muscle actin (SMA) and calponin9. The results above complete the previous study in demonstrating that all of the three known key EMT regulators are involved in the system. In addition, we observed that TGF&#946;1 can significantly enhance the migratory effect of FGF2/BMP4 alone. These results point to a network between FGF-, BMP- and TGF&#946;-signaling during EMT induction.
The most critical steps for successful isolation of NSCs for EMT and migration analysis are: correctly identifying the embryonic age, identifying anatomical landmarks of the developing embryo, preparation of fresh culture media and (at least overnight) coated plates, use of fine-tipped instruments and proper dissection of the central portion of the developing cortex to establish cortical explants.
The presented techniques may be enhanced by some modifications. As suggested above, multiple explants are plated on a single dish with a grid. For screening of multiple compounds, however, it may be more convenient to place single explants in every well of a 96-well plate. Further, the model system can be refined for large scale drug discovery. As described above, PDPN is a valuable marker for newly acquired migratory features since PDPN is found in invasive NSCs8. A PDPN reporter can be transfected in normal NSCs before EMT induction. As a consequence PDPN expressing cells can serve as an internal control for cell transformation since PDPN is not expressed in normal NSCs (Figure 4 and Table 1). Several additional NSC markers are available, such as Nestin, that are lost after EMT induction8. As a consequence, both the initial state, &#34;non-migratory/non-invasive/Nestin+&#34;, and the final state, &#34;migratory/invasive/PDPN+&#34;, can be monitored, which enables automated drug discovery. In large cell culture plates with multiple wells (e.g., 96-well plates and larger) initial cells may be seeded and treated with established drugs or compounds without known function. Thus, multiwell plates can be automatically scanned for expression of PDPN or other migration/invasion markers. If an inhibitor is the main target of the screen, disappearance of PDPN expression can help to identify putatively interesting novel inhibitory compounds.
During testing of a novel substance the explants may not show migration and/or the explants may round up and detach from the dish. In this situation, it is best to change only half of the media every day (instead of full media change every other day). This reduces stress by surface tension when replacing with fresh media. The explants may not attach after the initial plating. In this situation, fibronectin has to be in contact with the dish surface for at least 36 h. Fibronectin used for coating should be freshly prepared without exposure to a plastic tube for more than 10 min since fibronectin may stick to the plastic. Further, the explants may settle outside of the grid. In this situation, it is advisable to swirl the dish in a slow circular motion. This allows the explants to reposition to the center by centripetal force (cf. Figure 3).
There is strong evidence that gliomas are derived from NSCs and/or OPCs derived from NSCs23,24. As a consequence other groups have established glioma growth models on the basis of NSCs: Sampetrean et al. isolated NSCs from Ink4/ARF-deficient mice and forced overexpression of H-Ras25. High-grade malignant tumors resulted that showed proliferation and invasion after transplantation25. McNeill et al. also isolated NSCs from genetically modified mice (floxed Rb1, Nf1, Kras, Pten alone and in combinations)26. The genes were inactivated by Cre-virus infection and the NSCs were transplanted26. Both these model systems have the advantage that invasion can be monitored in vivo and potential new anti-invasion drugs can be tested. Their main disadvantage is that the onset of invasion is not well defined and it is unclear whether the cells show glioma-typical EMT invasion (EMT genes) or only local migration. Further only one drug can be tested per animal and the analysis requires several weeks. To identify a novel anti-invasion drug, pharmaceutical companies need (a) to screen for several thousand compounds or more at once, (b) to replicate the tests and (c) to try different drug concentrations. To prepare several thousand transplanted animals, treat them with different drugs and finally test for effects by histochemical or bioluminescence analysis is very resource-consuming. Here a novel NSC-based model system for EMT-related invasion is described that allows the cost-efficient screening of thousands of compounds.
Although this model enables high throughput screening of compounds, it is a primary screening platform only. Once compounds of interest are identified in this model, they will require additional validation. In vivo testing is necessary for safety and efficacy parameters for any compound identified and transplantation models as described above become important25,26. The model is also limited by the fact that it is based on rodent cells. Although the model can be used to investigate rat or mouse EMT, it is unclear if the same mechanisms are in place in human cells or in the human patient. Further experiments are needed to investigate if NSCs are not only invasive in vitro, but also after transplantation. Several lines of evidence support the role of NSCs in glioma formation. Glioma progression has been linked to the following genes: Tenascin C, Hey1, SPARC, Snail1 and Snail2, FGFR+, BMPR1a, EGFR, PDGFR&#946;, Sox2, Podoplanin, Gli3 and p75NGFR8. All of these genes are also expressed during the transformation of normal non-invasive NSCs to invasive mesenchymal cells8. At the time being, it is unclear whether NSCs can be transformed into tumors by external growth factors only.
Tumor-initiating stem cells (TICs) from different tumors have been isolated and are used as models to understand tumor progression27. TICs are, however, not well suited for EMT analysis, since EMT did already occur in TICs27. To understand early and also late steps of EMT induction, a primary non-neoplastic cell population is needed. Ideally this population was not meant to undergo EMT in the first place. It had been previously shown that embryonic NSCs were suitable candidates for this purpose9,28. Namely, migration and invasion could be induced in NSCs by FGF2 and BMP49,28. FGF2/BMP4-induced migration was related to single EMT-related genes, however, complete evidence was lacking since the key EMT families Zeb and Twist had not been investigated8. The results above show that a specific combination of four factors, FGF2, BMP4, TGF&#946;1 and insulin cause a very strong and complete EMT induction, not observed before. The present study demonstrates that the key EMT genes of the Zeb- and Twist-families are also upregulated. This study therefore provides the first evidence that there is not selective upregulation of single genes, but the results show that all key EMT families are active in the proposed cell culture system.
EMT has also been observed in epithelial cancers outside of the brain, such as lung, breast, colon and gastric cancer29. Several models to study EMT are in use for these tumors30-32, which come, however, with significant limitations: (a) usage of transformed tumor cell lines carrying multiple genetic and epigenetic changes33; to identify inhibitors of EMT for early cancer stages, late-stage cancer cells are inadequate; (b) serum-dependent cultures which contain various undefined growth factors; this renders identification of critical factors very difficult. Further, experiment reproducibility is impaired since serum quality varies from batch to batch; (c) serum contains inhibitors and enzymatic activities possibly inactivating potentially useful exogenous compounds; (d) need of enzymes for cell passaging that degrade cell surface molecules; (e) to identify EMT agonists to promote physiological EMT, normal cells are needed to test induction. Tumor cell models are inadequate to find substances that promote regeneration in normal stem cells.
Migration is also required for normal processes, such as wound healing and regeneration of the injured brain18. After traumatic brain injury and stroke, NSCs are necessary to migrate to the lesion site to participate in the regeneration4,34. The current system may also be used to identify substances that promote migration and invasion. As shown above, migration is induced upon start with BMP4-treatment. If the cells are not exposed to BMPs but instead to novel compounds, these may substitute for the BMP action which will help to identify novel BMP agonists. With a reporter system that indicates PDPN expression, large scale testing for novel substances becomes feasible; if a novel compound can substitute for BMPs, PDPN expressing cells can be automatically identified.
The proposed system is also useful to investigate cell-signaling interactions. Our results show that the BMP effect on migration could be enhanced by TGF&#946;1 activation. The results uncover an additive interaction between BMP- and TGF&#946;-signaling. However, additional signaling pathways, such as Notch-, Wnt- or EGFR-/MAPK- and other signaling cascades may be involved. Therefore, further investigations on BMP/TGF&#946;-interactions and cross-talk to other pathways are needed. In addition, the responsive central cortex can be isolated from genetically modified mice, which can help to elucidate underlying mechanisms driving EMT. In summary, the EMT model system described here may be helpful in the fields of stem cell biology and regeneration, as well as in cancer research. It may be used for screening of drug libraries for substances inhibiting or enhancing migration and invasion.

During several stages of embryonic development, epithelial cells lose their strong adherence to each other (e.g., tight junctions) and acquire a migratory phenotype in a process called epithelial to mesenchymal transition (EMT)1. EMT is required for the formation of additional cell types, such as the mesenchymal neural crest cells, a population that segregates from the neuroepithelium2. EMT is not only essential during embryonic stages but also required at later stages of adult life to maintain physiological processes in the adult organism, such as wound healing3and central nervous system (CNS) regeneration in demyelinating lesions4.
Epithelial tumors are known to reactivate EMT as an initiation step for migration, invasion and metastasis, ultimately leading to cancer progression1,3. EMT is indeed centrally linked to strong migration1,3. The cellular steps of conditioning, initiating, undergoing and maintaining EMT are not fully understood and need further investigation.
Here, a standardized in vitro EMT model system based on NSCs, with defined growth factors and media (no serum and no enzyme usage) is presented. This model system is of relevance for scientists working on EMT. The Snail, Zeb and Twist protein families have been shown to be critical for EMT both in development and disease1. The Snail, Zeb and Twist families are also involved in the presented system. The system is based on a specific region of the forebrain that normally does not undergo EMT providing a particular advantage for the study of initial events during EMT induction.
The model system could potentially be applied to study EMT in epithelia outside the CNS, since key EMT inducers, such as the Snail, Zeb and Twist proteins, are also found during EMT in tissue systems outside the CNS. This model system allows the standardized isolation of NSCs from the developing cortex to study stem cell features in general and EMT in particular. Using this system, we isolated NSCs, induced EMT and studied the subsequent migration under the effect of FGF2 and BMP4. We observed that FGF- and BMP-signaling interacts with TGF&#946;-signaling to promote cell migration, thus validating the model system.

<table><tbody><tr><td>BMP4, rhBMP4</td><td>RnD Systems&amp;nbsp;</td><td>314-BP-01M</td><td></td></tr><tr><td>Bovine pancreas insulin</td><td>Sigma&amp;nbsp;</td><td>I1882</td><td></td></tr><tr><td>Boyden chamber, CytoSelect cell invasion assay</td><td>Cell Biolabs</td><td>CBA-110</td><td>24 well plate system</td></tr><tr><td>Cell culture dish with grid</td><td>Ibidi 500 mm dish, 35 mm</td><td>80156</td><td></td></tr><tr><td>CellMask Orange</td><td>Life Technologies</td><td>C10045</td><td>Plasma membrane dye, use at 1:1,000.</td></tr><tr><td>DAPI</td><td>LifeTechnologies</td><td>D1306</td><td>Stock at 5 mg/ml. Use at 1:10,000. Cancerogenic. Appropriate protection (gloves, coat, goggles) required.</td></tr><tr><td>DMEM/F12 1:1 medium bottle</td><td>Gibco Invitrogen</td><td>21331-020</td><td></td></tr><tr><td>FGF2, rhFGF2</td><td>RnD Systems</td><td>233-FB-01M</td><td></td></tr><tr><td>Fibronectine, bovine</td><td>Sigma&amp;nbsp;</td><td>F4759</td><td></td></tr><tr><td>Glutamax supplement&amp;nbsp;</td><td>Gibco Invitrogen&amp;nbsp;</td><td>35050-061</td><td></td></tr><tr><td>Graphics software with pixel measurement feature</td><td>Fiji</td><td>fiji.sc/Fiji</td><td>version 2.0.0-rc-30/1.49s</td></tr><tr><td>HBSS media</td><td>Sigma&amp;nbsp;</td><td>H9394</td><td></td></tr><tr><td>Human apo-Transferrin</td><td>Sigma</td><td>T1147</td><td>Possible lung irritant. Avoid inhalation. Use appropriate protection.</td></tr><tr><td>L-glutamine</td><td>Gibco Invitrogen&amp;nbsp;</td><td>25030-024</td><td></td></tr><tr><td>Nestin, Mouse anti Nestin antibody</td><td>Genetex</td><td>GTX26142</td><td>Use at 1:100, 4% PFA fixation, Triton X100 at 0.1%</td></tr><tr><td>Olig2, Rabbit anti Olig2 antibody</td><td>Provided by Hirohide Takebayash</td><td>Personal stock</td><td>Use at 1:2,000, 4% PFA fixation, Triton X100 at 0.1%</td></tr><tr><td>Penicillin/Streptomycin/Fungizone</td><td>Gibco Invitrogen&amp;nbsp;</td><td>15240-062</td><td></td></tr><tr><td>Podoplanin, Mouse anti Podoplanin antibody</td><td>Acris</td><td>DM3614P</td><td>Use at 1:250, 4% PFA fixation, avoid Triton X100</td></tr><tr><td>Poly-L-ornithine</td><td>Sigma&amp;nbsp;</td><td>P3655</td><td></td></tr><tr><td>Putrescine</td><td>Sigma&amp;nbsp;</td><td>P5780</td><td>Skin and eye irritant. Appropriate protection required.</td></tr><tr><td>Sodium selenite</td><td>Sigma&amp;nbsp;</td><td>S5261</td><td></td></tr><tr><td>Sox10, Rabbit anti Sox10 antibody</td><td>Millipore Chemicon</td><td>AB5774</td><td>Use at 1:200, 4% PFA fixation, Triton X100 at 0.1%</td></tr><tr><td>TGFb1, rhTGFb1</td><td>RnD Systems</td><td>240-B-010</td><td></td></tr><tr><td>Uncoated Petri dishes</td><td>Falcon Corning</td><td>351029</td><td></td></tr></tbody></table>

an enzyme- and serum-free neural stem cell culture model for emt investigation suited for drug discovery

Epithelial to mesenchymal transition (EMT) describes the process of epithelium transdifferentiating into mesenchyme. EMT is a fundamental process during embryonic development that also commonly occurs in glioblastoma, the most frequent malignant brain tumor. EMT has also been observed in multiple carcinomas outside the brain including breast cancer, lung cancer, colon cancer, gastric cancer. EMT is centrally linked to malignancy by promoting migration, invasion and metastasis formation. The mechanisms of EMT induction are not fully understood. Here we describe an in vitro system for standardized isolation of cortical neural stem cells (NSCs) and subsequent EMT-induction. This system provides the flexibility to use either single cells or explant culture. In this system, rat or mouse embryonic forebrain NSCs are cultured in a defined medium, devoid of serum and enzymes. The NSCs expressed Olig2 and Sox10, two transcription factors observed in oligodendrocyte precursor cells (OPCs). Using this system, interactions between FGF-, BMP- and TGF&#946;-signaling involving Zeb1, Zeb2, and Twist2 were observed where TGF&#946;-activation significantly enhanced cell migration, suggesting a synergistic BMP-/TGF&#946;-interaction. The results point to a network of FGF-, BMP- and TGF&#946;-signaling to be involved in EMT induction and maintenance. This model system is relevant to investigate EMT in vitro. It is cost-efficient and shows high reproducibility. It also allows for the comparison of different compounds with respect to their migration responses (quantitative distance measurement), and high-throughput screening of compounds to inhibit or enhance EMT (qualitative measurement). The model is therefore well suited to test drug libraries for substances affecting EMT.

Watch this Scientific Journal Video about An Enzyme- and Serum-free Neural Stem Cell Culture Model for EMT Investigation Suited for Drug Discovery at JoVE.com

An Enzyme- and Serum-free Neural Stem Cell Culture Model for EMT Investigation Suited for Drug Discovery

Epithelial to mesenchymal transition (EMT) allows cancers to become invasive. To investigate EMT, a neural stem cell (NSC)-based in vitro model devoid of serum and enzymes is described. This standardized system allows quantitative and qualitative assessment of cell migration, gene and protein expression. The model is suited for drug discovery.

Epithelial to mesenchymal transition (EMT) describes the process of epithelium transdifferentiating into mesenchyme. EMT is a fundamental process during ...

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Nanyang Technological University

Research

JoVE Journal

Developmental Biology

9.9K Views.  University of Basel. Epithelial to mesenchymal transition (EMT) allows cancers to become invasive. To investigate EMT, a neural stem cell (NSC)-based in vitro model devoid of serum and enzymes is described. This standardized system allows quantitative and qualitative assessment of cell migration, gene and protein expression. The model is suited for drug discovery.

Video: An Enzyme- and Serum-free Neural Stem Cell Culture Model for EMT Investigation Suited for Drug Discovery

ES Cell-derived Neuroepithelial Cell Cultures

ES cells have the potential to differentiate into cells from all germ layers, which makes them an attractive tool for the development of new therapies. In general, the differentiation of ES cells follows the concept to first generate immature progenitor cells, which then can be propagated and differentiated into mature cellular phenotypes. This also applies for ES cell-derived neurogenesis, in which the development of neural cells follows two major steps: First, the derivation and expansion of immature neuroepithelial precursors and second, their differentiation into mature neural cells. A common method to produce neural progenitors from ES cells is based on embryoid body (EB) formation, which reveals the differentiation of cells from all germ layers including neuroectoderm. An alternative and more efficient method to induce neuroepithelial cell development uses stromal cell-derived inducing activity (SDIA), which can be achieved by co-culturing ES cells with skull bone marrow-derived stromal cells (1). Both, EB formation and SDIA, reveal the development of rosette-like structures, which are thought to resemble neural tube- and/or neural crest-like progenitors. The neural precursors can be isolated, expanded and further differentiated into specific neurons and glia cells using defined culture conditions. Here, we describe the generation and isolation of such rosettes in co-culture experiments with the stromal cell line MS5 (2-5).

Derivation of neuroepithelial precursors from embryonic stem (ES) cells using stromal cell-derived inducing activity (SDIA).

ES cells have the potential to differentiate into cells from all germ layers, which makes them an attractive tool for the development of new therapies. In ...

Biology

Human Pluripotent Stem Cell Based Developmental Toxicity Assays for Chemical Safety Screening and Systems Biology Data Generation

Efficient protocols to differentiate human pluripotent stem cells to various tissues in combination with -omics technologies opened up new horizons for in vitro toxicity testing of potential drugs. To provide a solid scientific basis for such assays, it will be important to gain quantitative information on the time course of development and on the underlying regulatory mechanisms by systems biology approaches. Two assays have therefore been tuned here for these requirements. In the UKK test system, human embryonic stem cells (hESC) (or other pluripotent cells) are left to spontaneously differentiate for 14 days in embryoid bodies, to allow generation of cells of all three germ layers. This system recapitulates key steps of early human embryonic development, and it can predict human-specific early embryonic toxicity/teratogenicity, if cells are exposed to chemicals during differentiation. The UKN1 test system is based on hESC differentiating to a population of neuroectodermal progenitor (NEP) cells for 6 days. This system recapitulates early neural development and predicts early developmental neurotoxicity and epigenetic changes triggered by chemicals. Both systems, in combination with transcriptome microarray studies, are suitable for identifying toxicity biomarkers. Moreover, they may be used in combination to generate input data for systems biology analysis. These test systems have advantages over the traditional toxicological studies requiring large amounts of animals. The test systems may contribute to a reduction of the costs for drug development and chemical safety evaluation. Their combination sheds light especially on compounds that may influence neurodevelopment specifically.

The protocols describe two in vitro developmental toxicity test systems (UKK and UKN1) based on human embryonic stem cells and transcriptome studies. The test systems predict human developmental toxicity hazard, and may contribute to reduce animal studies, costs and the time required for chemical safety testing.

Efficient protocols to differentiate human pluripotent stem cells to various tissues in combination with -omics technologies opened up new horizons for in ...

Culture of Mouse Neural Stem Cell Precursors

Primary neural stem cell cultures are useful for studying the mechanisms underlying central nervous system development. Stem cell research will increase our understanding of the nervous system and may allow us to develop treatments for currently incurable brain diseases and injuries. In addition, stem cells should be used for stem cell research aimed at the detailed study of mechanisms of neural differentiation and transdifferentiation and the genetic and environmental signals that direct the specialization of the cells into particular cell types.  This video demonstrates a technique used to disaggregate cells from the embryonic day 12.5 mouse dorsal forebrain. The dissection procedure includes harvesting E12.5 mouse embryos from the uterus, removing the "skin" with fine dissecting forceps and finally isolating pieces of cerebral cortex. Following the dissection, the tissue is digested and mechanically dissociated. The resuspended dissociated cells are then cultured in "stem cell" media that favors growth of neural stem cells.

This video describes the method used for isolation of neuroprecursors from the developing cortex of embryonic mice. The procedure for removing embryos from the uterus, dissecting the cortical tissue, and digesting the isolated cerebral cortex is shown.

Primary neural stem cell cultures are useful for studying the mechanisms underlying central nervous system development. Stem cell research will increase ...

Differentiation of a Human Neural Stem Cell Line on Three Dimensional Cultures, Analysis of MicroRNA and Putative Target Genes

Neural stem cells (NSCs) are capable of self-renewal and differentiation into neurons, astrocytes and oligodendrocytes under specific local microenvironments. In here, we present a set of methods used for three dimensional (3D) differentiation and miRNA analysis of a clonal human neural stem cell (hNSC) line, currently in clinical trials for stroke disability (NCT01151124 and NCT02117635, Clinicaltrials.gov). HNSCs were derived from an ethical approved first trimester human fetal cortex and conditionally immortalized using retroviral integration of a single copy of the c-mycERTAMconstruct. We describe how to measure axon process outgrowth of hNSCs differentiated on 3D scaffolds and how to quantify associated changes in miRNA expression using PCR array. Furthermore we exemplify computational analysis with the aim of selecting miRNA putative targets. SOX5 and NR4A3 were identified as suitable miRNA putative target of selected significantly down-regulated miRNAs in differentiated hNSC. MiRNA target validation was performed on SOX5 and NR4A3 3&#8217;UTRs by dual reporter plasmid transfection and dual luciferase assay.

With the intent of characterizing changes in miRNAs on differentiated human neural stem cells (hNSCs) we describe hNSC differentiation on a three dimensional system,the evaluation of changes in microRNA expression by miRNA PCR array, and computational analysis for miRNA target prediction and its validation by dual luciferase assay.

Neural stem cells (NSCs) are capable of self-renewal and differentiation into neurons, astrocytes and oligodendrocytes under specific local ...

Neural Differentiation of Mouse Embryonic Stem Cells in Serum-free Monolayer Culture

The ability to differentiate mouse embryonic stem cells (ESC) to neural progenitors allows the study of the mechanisms controlling neural specification as well as the generation of mature neural cell types for further study. In this protocol we describe a method for the differentiation of ESC to neural progenitors using serum-free, monolayer culture. The method is scalable, efficient and results in production of ~70% neural progenitor cells within 4 - 6 days. It can be applied to ESC from various strains grown under a variety of conditions. Neural progenitors can be allowed to differentiate further into functional neurons and glia or analyzed by microscopy, flow cytometry or molecular techniques. The differentiation process is amenable to time-lapse microscopy and can be combined with the use of reporter lines to monitor the neural specification process. We provide detailed instructions on media preparation and cell density optimization to allow the process to be applied to most ESC lines and a variety of cell culture vessels.

This protocol describes in detail the method for generating neural progenitors from embryonic stem cells using a serum-free monolayer method. These progenitors can be used to derive mature neural cell types or to study the process of neural specification and is amenable to multiwell format scaling for compound screening.

The ability to differentiate mouse embryonic stem cells (ESC) to neural progenitors allows the study of the mechanisms controlling neural specification as ...

Growing Neural Stem Cells from Conventional and Nonconventional Regions of the Adult Rodent Brain

Recent work demonstrates that central nervous system (CNS) regeneration and tumorigenesis involves populations of stem cells (SCs) resident within the adult brain. However, the mechanisms these normally quiescent cells employ to ensure proper functioning of neural networks, as well as their role in recovery from injury and mitigation of neurodegenerative processes are little understood. These cells reside in regions referred to as &#34;niches&#34; that provide a sustaining environment involving modulatory signals from both the vascular and immune systems. The isolation, maintenance, and differentiation of CNS SCs under defined culture conditions which exclude unknown factors, makes them accessible to treatment by pharmacological or genetic means, thus providing insight into their in vivo behavior. Here we offer detailed information on the methods for generating cultures of CNS SCs from distinct regions of the adult brain and approaches to assess their differentiation potential into neurons, astrocytes, and oligodendrocytes in vitro. This technique yields a homogeneous cell population as a monolayer culture that can be visualized to study individual SCs and their progeny. Furthermore, it can be applied across different animal model systems and clinical samples, being used previously to predict regenerative responses in the damaged adult nervous system.

Neural stem cells harvested from the adult brain are increasing utilized in applications ranging from the basic research of nervous system development to exploring potential clinical applications in regenerative medicine. This makes rigorous control in the isolation and culturing conditions used to grow these cells critical to sound experimental outcomes.

Recent work demonstrates that central nervous system (CNS) regeneration and tumorigenesis involves populations of stem cells (SCs) resident within the ...

Neuroscience

One Mouse, Two Cultures: Isolation and Culture of Adult Neural Stem Cells from the Two Neurogenic Zones of Individual Mice

The neurosphere assay and the adherent monolayer culture system are valuable tools to determine the potential (proliferation or differentiation) of adult neural stem cells in vitro. These assays can be used to compare the precursor potential of cells isolated from genetically different or differentially treated animals&#160;to determine the effects of exogenous factors on neural precursor cell proliferation and differentiation and to generate neural precursor cell lines that can be assayed over continuous passages. The neurosphere assay is traditionally used for the post-hoc identification of stem cells, primarily due to the lack of definitive markers with which they can be isolated from primary tissue&#160;and has the major advantage of giving a quick estimate of precursor cell numbers in brain tissue derived from individual animals. Adherent monolayer cultures, in contrast, are not traditionally used to compare proliferation between individual animals, as each culture is generally initiated from the combined tissue of between 5-8 animals. However, they have the major advantage that, unlike neurospheres, they consist of a mostly homogeneous population of precursor cells and are useful for following the differentiation process in single cells. Here, we describe, in detail, the generation of neurosphere cultures and, for the first time, adherent cultures from individual animals. This has many important implications including paired analysis of proliferation and/or differentiation potential in both the subventricular zone (SVZ) and dentate gyrus (DG) of treated or genetically different mouse lines, as well as a significant reduction in animal usage.

Here we describe a detailed protocol for the simultaneous generation of neural precursor cell cultures, as either adherent monolayers or neurospheres, from the subventricular zone and dentate gyrus of individual adult mice.

The neurosphere assay and the adherent monolayer culture system are valuable tools to determine the potential (proliferation or differentiation) of adult ...

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.

Presented here is a protocol for the generation of a single-cell culture of human embryonic stem cells and their subsequent differentiation into neural progenitor cells. The protocol is simple, robust, scalable, and suitable for drug screening and regenerative medicine applications.

In vitro differentiation of human embryonic stem cells (hESCs) has transformed the ability to study human development on both biological and molecular ...

Functional Evaluation of Biological Neurotoxins in Networked Cultures of Stem Cell-derived Central Nervous System Neurons

Therapeutic and mechanistic studies of the presynaptically targeted clostridial neurotoxins (CNTs) have been limited by the need for a scalable, cell-based model that produces functioning synapses and undergoes physiological responses to intoxication. Here we describe a simple and robust method to efficiently differentiate murine embryonic stem cells (ESCs) into defined lineages of synaptically active, networked neurons. Following an 8 day differentiation protocol, mouse embryonic stem cell-derived neurons (ESNs) rapidly express and compartmentalize neurotypic proteins, form neuronal morphologies and develop intrinsic electrical responses. By 18 days after differentiation (DIV 18), ESNs exhibit active glutamatergic and &#947;-aminobutyric acid (GABA)ergic synapses and emergent network behaviors characterized by an excitatory:inhibitory balance. To determine whether intoxication with CNTs functionally antagonizes synaptic neurotransmission, thereby replicating the in vivo pathophysiology that is responsible for clinical manifestations of botulism or tetanus, whole-cell patch clamp electrophysiology was used to quantify spontaneous miniature excitatory post-synaptic currents (mEPSCs) in ESNs exposed to tetanus neurotoxin (TeNT) or botulinum neurotoxin (BoNT) serotypes /A-/G. In all cases, ESNs exhibited near-complete loss of synaptic activity within 20 hr. Intoxicated neurons remained viable, as demonstrated by unchanged resting membrane potentials and intrinsic electrical responses. To further characterize the sensitivity of this approach, dose-dependent effects of intoxication on synaptic activity were measured 20 hr after addition of BoNT/A. Intoxication with 0.005 pM BoNT/A resulted in a significant decrement in mEPSCs, with a median inhibitory concentration (IC50) of 0.013 pM. Comparisons of median doses indicate that functional measurements of synaptic inhibition are faster, more specific and more sensitive than SNARE cleavage assays or the mouse lethality assay. These data validate the use of synaptically coupled, stem cell-derived neurons for the highly specific and sensitive detection of CNTs.

A custom protocol is described to differentiate mouse ES cells into defined populations of highly pure neurons exhibiting functioning synapses and emergent network behavior. Electrophysiological analysis demonstrates the loss of synaptic transmission following exposure to botulinum neurotoxin serotypes /A-/G and tetanus neurotoxin.

Therapeutic and mechanistic studies of the presynaptically targeted clostridial neurotoxins (CNTs) have been limited by the need for a scalable, ...

Human Neural Organoids for Studying Brain Cancer and Neurodegenerative Diseases

The lack of relevant in vitro neural models is an important obstacle on medical progress for neuropathologies. Establishment of relevant cellular models is crucial both to better understand the pathological mechanisms of these diseases and identify new therapeutic targets and strategies. To be pertinent, an in vitro model must reproduce the pathological features of a human disease. However, in the context of neurodegenerative disease, a relevant in vitro model should provide neural cell replacement as a valuable therapeutic opportunity. 
Such a model would not only allow screening of therapeutic molecules but also can be used to optimize neural protocol differentiation [for example, in the context of transplantation in Parkinson's disease (PD)]. This study describes two in vitro protocols of 1) human glioblastoma development within a human neural organoids (NO) and 2) neuron dopaminergic (DA) differentiation generating a three-dimensional (3D) organoid. For this purpose, a well-standardized protocol was established that allows the production of size-calibrated neurospheres derived from human embryonic stem cell (hESC) differentiation. The first model can be used to reveal molecular and cellular events occurring during in glioblastoma development within the neural organoid, while the DA organoid not only represents a suitable source of DA neurons for cell therapy in Parkinson's disease but also can be used for drug testing.

This study introduces and describes protocols to derive two specific human neural organoids&#160;as a relevant and accurate model for studying 1) human glioblastoma development within human neural organoids exclusively in humans and 2) neuron dopaminergic differentiation generating a three-dimensional organoid.

The lack of relevant in vitro neural models is an important obstacle on medical progress for neuropathologies. Establishment of relevant cellular models ...

An Enzyme- and Serum-free Neural Stem Cell Culture Model for EMT Investigation Suited for Drug Discovery

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