Name: Author Spotlight: Insight Into Advances in Prion Diseases Research
Uploaded: 2023-08-11T13:00:00
Description: Watch this Scientific Journal Video about Adipose-Derived Mesenchymal Stromal Cells Co-Cultured with Primary Mixed Glia to Reduce Prion-Induced Inflammation at JoVE.com

The authors thank Lab Animal Resources for their animal husbandry. Our funding sources for this manuscript include the Boettcher Fund, the Murphy Turner Fund, CSU College of Veterinary Medicine, and the Biomedical Sciences College Research Council. Figure 2A, Figure 2C, and Figure 3A were created with BioRender.com.

Mice were bred and maintained at Colorado State&#39;s Lab Animal Resources, accredited by the Association for Assessment and Accreditation of Lab Animal Care International, in accordance with protocol #1138, approved by the Institutional Animal Care and Use Committee at Colorado State University.
1. Isolating and infecting primary cortical mixed glia with prions
<ol>
	<li>To isolate primary mixed glia containing both astrocytes and microglia, obtain C57Bl/6 mouse pups aged z...

Coculturing AdMSCs and Glia to Reduce Prion-Induced Inflammation

<ol>
	<li>Liddelow, S. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurotoxic+reactive+astrocytes+are+induced+by+activated+microglia.">Neurotoxic reactive astrocytes are induced by activated microglia.</a> Nature. 541 (7638), 481-487 (2017).</li><li>Smith, H. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Astrocyte+unfolded+protein+response+induces+a+specific+reactivity+state+that+causes+non-cell-autonomous+neuronal+degeneration.">Astrocyte unfolded protein response induces a specific reactivity state that causes non-cell-autonomous neuronal degeneration.</a> Neuron. 105 (5), 855-866 (2020).</li><li>Hong, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Complement+and+microglia+mediate+early+synapse+loss+in+Alzheimer+mouse+models.">Complement and microglia mediate early synapse loss in Alzheimer mouse models.</a> Science. 352 (6286), 712-716 (2016).</li><li>Collinge, J., Clarke, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+general+model+of+prion+strains+and+their+pathogenicity.">A general model of prion strains and their pathogenicity.</a> Science. 318 (5852), 930-936 (2007).</li><li>Gajdusek, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transmissible+and+non-transmissible+amyloidoses:+autocatalytic+post-translational+conversion+of+host+precursor+proteins+to+beta-pleated+sheet+configurations.">Transmissible and non-transmissible amyloidoses: autocatalytic post-translational conversion of host precursor proteins to beta-pleated sheet configurations.</a> J Neuroimmunol. 20 (2-3), 95-110 (1988).</li><li>Come, J. H., Fraser, P. E., Lansbury, P. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+kinetic+model+for+amyloid+formation+in+the+prion+diseases:+importance+of+seeding.">A kinetic model for amyloid formation in the prion diseases: importance of seeding.</a> Proceedings of the National Academy of Sciences of the United States of America. 90 (13), 5959-5963 (1993).</li><li>Hartmann, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Complement+3(+)-astrocytes+are+highly+abundant+in+prion+diseases,+but+their+abolishment+led+to+an+accelerated+disease+course+and+early+dysregulation+of+microglia.">Complement 3(+)-astrocytes are highly abundant in prion diseases, but their abolishment led to an accelerated disease course and early dysregulation of microglia.</a> Acta Neuropathologica Communications. 7 (1), 83 (2019).</li><li>Carroll, J. A., Race, B., Williams, K., Striebel, J., Chesebro, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microglia+are+critical+in+host+defense+against+prion+disease.">Microglia are critical in host defense against prion disease.</a> Journal of Virology. 92 (15), e00549 (2018).</li><li>Bradford, B. M., McGuire, L. I., Hume, D. A., Pridans, C., Mabbott, N. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microglia+deficiency+accelerates+prion+disease+but+does+not+enhance+prion+accumulation+in+the+brain.">Microglia deficiency accelerates prion disease but does not enhance prion accumulation in the brain.</a> Glia. 70 (11), 2169-2187 (2022).</li><li>Li, M., Chen, H., Zhu, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mesenchymal+stem+cells+for+regenerative+medicine+in+central+nervous+system.">Mesenchymal stem cells for regenerative medicine in central nervous system.</a> Frontiers in Neuroscience. 16, 1068114 (2022).</li><li>Sanchez-Castillo, A. I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Switching+roles:+beneficial+effects+of+adipose+tissue-derived+mesenchymal+stem+cells+on+microglia+and+their+implication+in+neurodegenerative+diseases.">Switching roles: beneficial effects of adipose tissue-derived mesenchymal stem cells on microglia and their implication in neurodegenerative diseases.</a> Biomolecules. 12 (2), 219 (2022).</li><li>Fu, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mesenchymal+stem+cell+migration+and+tissue+repair.">Mesenchymal stem cell migration and tissue repair.</a> Cells. 8 (8), 784 (2019).</li><li>Xiao, Q., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TNF-alpha+increases+bone+marrow+mesenchymal+stem+cell+migration+to+ischemic+tissues.">TNF-alpha increases bone marrow mesenchymal stem cell migration to ischemic tissues.</a> Cell Biochemistry and Biophysics. 62 (3), 409-414 (2012).</li><li>Hay, A. J. D., Murphy, T. J., Popichak, K. A., Zabel, M. D., Moreno, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adipose-derived+mesenchymal+stromal+cells+decrease+prion-induced+glial+inflammation+in+vitro.">Adipose-derived mesenchymal stromal cells decrease prion-induced glial inflammation in vitro.</a> Scientific Reports. 12 (1), 22567 (2022).</li><li>Kirkley, K. S., Popichak, K. A., Afzali, M. F., Legare, M. E., Tjalkens, R. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microglia+amplify+inflammatory+activation+of+astrocytes+in+manganese+neurotoxicity.">Microglia amplify inflammatory activation of astrocytes in manganese neurotoxicity.</a> Journal of Neuroinflammation. 14 (1), 99 (2017).</li><li>Popichak, K. A., Afzali, M. F., Kirkley, K. S., Tjalkens, R. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glial-neuronal+signaling+mechanisms+underlying+the+neuroinflammatory+effects+of+manganese.">Glial-neuronal signaling mechanisms underlying the neuroinflammatory effects of manganese.</a> Journal of Neuroinflammation. 15 (1), 324 (2018).</li><li>Livak, K. J., Schmittgen, T. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+relative+gene+expression+data+using+real-time+quantitative+PCR+and+the+2(-Delta+Delta+C(T))+Method.">Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method.</a> Methods. 25 (4), 402-408 (2001).</li><li>Hass, R., Otte, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mesenchymal+stem+cells+as+all-round+supporters+in+a+normal+and+neoplastic+microenvironment.">Mesenchymal stem cells as all-round supporters in a normal and neoplastic microenvironment.</a> Cell Communication and Signaling: CCS. 10 (1), 26 (2012).</li><li>Carroll, J. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prion+strain+differences+in+accumulation+of+PrPSc+on+neurons+and+glia+are+associated+with+similar+expression+profiles+of+neuroinflammatory+genes:+comparison+of+three+prion+strains.">Prion strain differences in accumulation of PrPSc on neurons and glia are associated with similar expression profiles of neuroinflammatory genes: comparison of three prion strains.</a> PLoS Pathogens. 12 (4), 1005551 (2016).</li><li>Carroll, J. A., Race, B., Williams, K., Chesebro, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Toll-like+receptor+2+confers+partial+neuroprotection+during+prion+disease.">Toll-like receptor 2 confers partial neuroprotection during prion disease.</a> PLoS One. 13 (12), e0208559 (2018).</li><li>Yu, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hypoxia+and+low-dose+inflammatory+stimulus+synergistically+enhance+bone+marrow+mesenchymal+stem+cell+migration.">Hypoxia and low-dose inflammatory stimulus synergistically enhance bone marrow mesenchymal stem cell migration.</a> Cell Proliferation. 50 (1), e12309 (2017).</li><li>Hay, A. J. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intranasally+delivered+mesenchymal+stromal+cells+decrease+glial+inflammation+early+in+prion+disease.">Intranasally delivered mesenchymal stromal cells decrease glial inflammation early in prion disease.</a> Frontiers in Neuroscience. 17, 1158408 (2023).</li><li>English, K., Barry, F. P., Field-Corbett, C. P., Mahon, B. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=IFN-gamma+and+TNF-alpha+differentially+regulate+immunomodulation+by+murine+mesenchymal+stem+cells.">IFN-gamma and TNF-alpha differentially regulate immunomodulation by murine mesenchymal stem cells.</a> Immunology Letters. 110 (2), 91-100 (2007).</li><li>Hemeda, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interferon-gamma+and+tumor+necrosis+factor-alpha+differentially+affect+cytokine+expression+and+migration+properties+of+mesenchymal+stem+cells.">Interferon-gamma and tumor necrosis factor-alpha differentially affect cytokine expression and migration properties of mesenchymal stem cells.</a> Stem Cells and Development. 19 (5), 693-706 (2010).</li><li>Carta, M., Aguzzi, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+foundations+of+prion+strain+diversity.">Molecular foundations of prion strain diversity.</a> Current Opinion in Neurobiology. 72, 22-31 (2022).</li><li>Yu, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phagocytic+microglia+and+macrophages+in+brain+injury+and+repair.">Phagocytic microglia and macrophages in brain injury and repair.</a> CNS Neuroscience and Therapeutics. 28 (9), 1279-1293 (2022).</li><li>Sinha, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phagocytic+activities+of+reactive+microglia+and+astrocytes+associated+with+prion+diseases+are+dysregulated+in+opposite+directions.">Phagocytic activities of reactive microglia and astrocytes associated with prion diseases are dysregulated in opposite directions.</a> Cells. 10 (7), 1728 (2021).</li><li>Stansley, B., Post, J., Hensley, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+comparative+review+of+cell+culture+systems+for+the+study+of+microglial+biology+in+Alzheimer's+disease.">A comparative review of cell culture systems for the study of microglial biology in Alzheimer's disease.</a> Journal of Neuroinflammation. 9, 115 (2012).</li><li>Shan, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Therapeutic+effect+of+autologous+compact+bone-derived+mesenchymal+stem+cell+transplantation+on+prion+disease.">Therapeutic effect of autologous compact bone-derived mesenchymal stem cell transplantation on prion disease.</a> Journal of General Virology. 98 (10), 2615-2627 (2017).</li><li>Johnson, T. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monitoring+immune+cells+trafficking+fluorescent+prion+rods+hours+after+intraperitoneal+infection.">Monitoring immune cells trafficking fluorescent prion rods hours after intraperitoneal infection.</a> Journal of Visualized Experiments. (45), e2349 (2010).</li><li>Liu, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MSC-secreted+TGF-beta+regulates+lipopolysaccharide-stimulated+macrophage+M2-like+polarization+via+the+Akt/FoxO1+pathway.">MSC-secreted TGF-beta regulates lipopolysaccharide-stimulated macrophage M2-like polarization via the Akt/FoxO1 pathway.</a> Stem Cell Research and Therapy. 10, 345 (2019).</li></ol>

Here we demonstrate a reliable and relatively inexpensive protocol for assessing the effects of adipose-derived mesenchymal stromal cells (AdMSCs) in decreasing prion-induced inflammation in a glial cell model. AdMSCs can easily be isolated and expanded in culture for use in as little as 1 week. This protocol consistently produces a heterologous population of cells that express markers consistent with those of mesenchymal stromal cells by immunofluorescence and flow cytometry, and retain immunological function when intro...

Glial inflammation plays a key role in a variety of neurodegenerative diseases, including Parkinson&#39;s, Alzheimer&#39;s, and prion disease. Although abnormal protein aggregation is attributed to much of disease pathogenesis and neurodegeneration, glial cells also play a part in exacerbating this 1,2,3. Therefore, targeting glial-induced inflammation is a promising therapeutic approach. In prion disease, the cellular prion protein (PrPC) misfolds to the disease-associated prion protein (PrPSc), which forms oligomers and aggregates and disrupts homeostasis in the brain 4,5,6.
One of the earliest signs of prion disease is an inflammatory response from astrocytes and microglia. Studies suppressing this response, either by removal of microglia or modification of astrocytes, have generally shown no improvement on, or worsened, disease pathogenesis in animal models 7,8,9. Modulating glial inflammation without eliminating it is an intriguing alternative as a therapeutic.
Mesenchymal stromal cells (MSCs) have taken the stage as a treatment for a variety of inflammatory diseases, due to their ability to modulate inflammation in a paracrine manner 10,11. They have shown the ability to migrate to sites of inflammation and respond to signaling molecules in these environments by secreting anti-inflammatory molecules, growth factors, microRNAs, and more 10,12,13. We have previously demonstrated that MSCs derived from adipose tissue (denoted AdMSCs) are able to migrate toward prion-infected brain homogenate and respond to this brain homogenate by upregulating gene expression for anti-inflammatory cytokines and growth factors.
Moreover, AdMSCs can decrease the expression of genes associated with Nuclear Factor-kappa B (NF-&#954;B), the Nod-Like Receptor family pyrin domain containing 3 (NLRP3) inflammasome signaling, and glial activation, in both BV2 microglia and primary mixed glia 14. Here, we provide protocols on how to isolate both AdMSCs and primary mixed glia from mice, stimulate AdMSCs to upregulate modulatory genes, assess AdMSC migration, and co-culture AdMSCs with prion-infected glia. We hope that these procedures can provide a foundation for further investigation of the role of MSCs in regulating glial-induced inflammation in neurodegenerative and other diseases.

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			<td>0.25% Trypsin</td>
			<td>Cytiva</td>
			<td>SH30042.01</td>
			<td></td>
		</tr>
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			<td>5 mL serological pipets</td>
			<td>Celltreat</td>
			<td>229005B</td>
			<td></td>
		</tr>
		<tr>
			<td>6-well tissue culture plates</td>
			<td>Celltreat</td>
			<td>229106</td>
			<td></td>
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			<td>10 cm cell culture dishes</td>
			<td>Peak Serum</td>
			<td>PS-4002</td>
			<td></td>
		</tr>
		<tr>
			<td>10 ml serological pipets</td>
			<td>Celltreat</td>
			<td>229210</td>
			<td></td>
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			<td>15 mL conical tubes</td>
			<td>Celltreat</td>
			<td>667015B</td>
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			<td>50 mL conical tubes</td>
			<td>Celltreat</td>
			<td>667050B</td>
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			<td>BV2 microglia cell line</td>
			<td>AcceGen Biotech</td>
			<td>ABC-TC212S</td>
			<td></td>
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			<td>Cell lifter</td>
			<td>Biologix Research Company</td>
			<td>70-2180</td>
			<td></td>
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			<td>Crystal violet</td>
			<td>Electron Microscopy Sciences</td>
			<td>&#160;12785</td>
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			<td>Dispase</td>
			<td>Thermo Scientific</td>
			<td>17105041</td>
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			<td>DMEM/F12</td>
			<td>Caisson Labs</td>
			<td>DFL14-500ML</td>
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			<td>DNase-I</td>
			<td>Sigma Aldrich</td>
			<td>11284932001</td>
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			<td>Essential amino acids</td>
			<td>Thermo Scientific</td>
			<td>11130051</td>
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			<td>Fetal bovine serum (heat inactivated)</td>
			<td>Peak Serum</td>
			<td>PS-FB4</td>
			<td>Can be purchased as heat inactivated or inactivated in the laboratory</td>
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			<td>Formaldehyde</td>
			<td>EMD Millipore</td>
			<td>1.04003.1000</td>
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			<td>Glass 10 mL serological pipet</td>
			<td>Corning</td>
			<td>&#160;7077-10N</td>
			<td></td>
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			<td>Hank&#8217;s Balances Salt Solution</td>
			<td>Sigma Aldrich</td>
			<td>H8264-500ML</td>
			<td></td>
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			<td>Hemocytometer/Neubauer Chamber</td>
			<td>Daigger</td>
			<td>HU-3100</td>
			<td></td>
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			<td>High Glucose DMEM</td>
			<td>Cytiva</td>
			<td>SH30022.01</td>
			<td></td>
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			<td>low glucose DMEM containing L-glutamine</td>
			<td>Cytiva</td>
			<td>SH30021.01</td>
			<td></td>
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			<td>MEM/EBSS</td>
			<td>Cytiva</td>
			<td>SH30024.FS</td>
			<td></td>
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			<td>non-essential amino acids</td>
			<td>Sigma-Aldrich</td>
			<td>M7145-100M</td>
			<td></td>
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			<td>Paraformaldehyde (16%)</td>
			<td>MP Biomedicals</td>
			<td>219998320</td>
			<td></td>
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			<td>Penicillin/streptomycin/neomycin</td>
			<td>Sigma-Aldrich</td>
			<td>P4083-100ML</td>
			<td></td>
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			<td>Phosphate buffered saline</td>
			<td>Cytiva</td>
			<td>&#160;SH30256.01</td>
			<td></td>
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			<td>Recombinant Mouse IFN-gamma Protein</td>
			<td>R&#38;D Systems</td>
			<td>485-MI</td>
			<td></td>
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			<td>Recombinant Mouse TNF-alpha (aa 80-235) Protein, CF</td>
			<td>R&#38;D Systems</td>
			<td>410-MT</td>
			<td></td>
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			<td>RNeasy mini kit</td>
			<td>Qiagen</td>
			<td>74104</td>
			<td></td>
		</tr>
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			<td>Sigmacote</td>
			<td>Sigma Aldrich</td>
			<td>SL2-100ML</td>
			<td>Coat inside of glass pipets by aspirating up and down twice in Sigmacote and allowing to dry thoroughly. Wrap in aluminum foil and autoclave pipets 24 h later.</td>
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		<tr>
			<td>Stemxyme</td>
			<td>Worthington Biochemical Corporation</td>
			<td>LS004106</td>
			<td>Collagenase/Dispase mixture</td>
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			<td>Sterile, individually wrapped cotton swab</td>
			<td>Puritan Medical</td>
			<td>&#160;25-8061WC</td>
			<td></td>
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			<td>Thincert Tissue Culture Inserts, 24 well, Pore Size=8 &#181;m</td>
			<td>Greiner Bio-One</td>
			<td>662638</td>
			<td></td>
		</tr>
		<tr>
			<td>Thincert Tissue Culture Inserts, 6 well, Pore Size=0.4 &#181;m</td>
			<td>Greiner Bio-One</td>
			<td>657641</td>
			<td></td>
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adipose-derived mesenchymal stromal cells co-cultured with primary mixed glia to reduce prion-induced inflammation

Mesenchymal stromal cells (MSCs) are potent regulators of inflammation through the production of anti-inflammatory cytokines, chemokines, and growth factors. These cells show an ability to regulate neuroinflammation in the context of neurodegenerative diseases such as prion disease and other protein misfolding disorders. Prion diseases can be sporadic, acquired, or genetic; they can result from the misfolding and aggregation of the prion protein in the brain. These diseases are invariably fatal, with no available treatments.
One of the earliest signs of disease is the activation of astrocytes and microglia and associated inflammation, which occurs prior to detectable prion aggregation and neuronal loss; thus, the anti-inflammatory and regulatory properties of MSCs can be harvested to treat astrogliosis in prion disease. Recently, we showed that adipose-derived MSCs (AdMSCs) co-cultured with BV2 cells or primary mixed glia reduce prion-induced inflammation through paracrine signaling. This paper describes a reliable treatment using stimulated AdMSCs to decrease prion-induced inflammation.
A heterozygous population of AdMSCs can easily be isolated from murine adipose tissue and expanded in culture. Stimulating these cells with inflammatory cytokines enhances their ability to both migrate toward prion-infected brain homogenate and produce anti-inflammatory modulators in response. Together, these techniques can be used to investigate the therapeutic potential of MSCs on prion infection and can be adapted for other protein misfolding and neuroinflammatory diseases.

Author Spotlight: Insight Into Advances in Prion Diseases Research

Adipose-Derived Mesenchymal Stromal Cells Co-Cultured with Primary Mixed Glia to Reduce Prion-Induced Inflammation

Our work focuses on prion diseases, which are fatal neurodegenerative diseases caused by the misfolding of the prion protein in the brain. Few studies have focused on the impact of glial cells on neuronal death. We want to understand if halting inflammation caused by glial cells can be a therapeutic approach.There has been a recent discovery in the mesenchymal stromal cell field, revealing that the protective properties stem from the extracellular vesicles they release. We aim to delve deeper into the contents of these vesicles and understand how they provide protection against prion-induced glial inflammation. Currently in the field of neurodegeneration, there are a few avenues that are successful at halting neuroinflammation.These include small molecules and genetic modulation using adeno or lentiviruses. However, none have been able to fully reduce inflammation, specifically in large areas of the brain. The most significant finding that we have been able to establish is that through cell therapy, we are able to slow prion-disease progression through inhibition of glial inflammation.The use of a combined with the primary glial-cell model of disease will allow us to continue to ask about cellular stress pathways important in neuroprotection, in a more efficient and direct matter than the use of solely in-vivo models.

Stimulating AdMSCs with TNF&#945; or interferon-gamma (IFN&#947;) for 24 h induces changes in the expression of anti-inflammatory molecules and growth factors. Treating AdMSCs with TNF&#945; or interferon-gamma (IFN&#947;) increases TNF-stimulated gene 6 (TSG-6) mRNA, whereas TNF&#945;, but not IFN&#947;, causes an increase in transforming growth factor beta-1 (TGF&#946;-1)&#160;mRNA. Stimulation with TNF&#945; or IFN&#947; induces an increase in vascular endothelial growth factor (VEGF)mRNA, but no cha...

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Adipose-derived mesenchymal stromal cells (AdMSCs) have potent immunomodulatory properties useful for treating diseases associated with inflammation. We demonstrate how to isolate and culture murine AdMSCs and primary mixed glia, stimulate AdMSCs to upregulate anti-inflammatory genes and growth factors, assess migration of AdMSCs, and co-culture AdMSCs with primary mixed prion-infected glia.

Mesenchymal stromal cells (MSCs) are potent regulators of inflammation through the production of anti-inflammatory cytokines, chemokines, and growth ...