Adipose-Derived Mesenchymal Stromal Cells Co-Cultured with Primary Mixed Glia to Reduce Prion-Induced Inflammation
Summary
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.
Abstract
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.
Introduction
Glial inflammation plays a key role in a variety of neurodegenerative diseases, including Parkinson's, Alzheimer'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-κB), 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.
Protocol
Representative Results
Discussion
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…
Disclosures
The authors have nothing to disclose.
Acknowledgements
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.
Materials
| 0.25% Trypsin | Cytiva | SH30042.01 | |
| 5 mL serological pipets | Celltreat | 229005B | |
| 6-well tissue culture plates | Celltreat | 229106 | |
| 10 cm cell culture dishes | Peak Serum | PS-4002 | |
| 10 ml serological pipets | Celltreat | 229210 | |
| 15 mL conical tubes | Celltreat | 667015B | |
| 50 mL conical tubes | Celltreat | 667050B | |
| BV2 microglia cell line | AcceGen Biotech | ABC-TC212S | |
| Cell lifter | Biologix Research Company | 70-2180 | |
| Crystal violet | Electron Microscopy Sciences | 12785 | |
| Dispase | Thermo Scientific | 17105041 | |
| DMEM/F12 | Caisson Labs | DFL14-500ML | |
| DNase-I | Sigma Aldrich | 11284932001 | |
| Essential amino acids | Thermo Scientific | 11130051 | |
| Ethanol (100%) | EMD Millipore | EX0276-1 | |
| Fetal bovine serum (heat inactivated) | Peak Serum | PS-FB4 | Can be purchased as heat inactivated or inactivated in the laboratory |
| Formaldehyde | EMD Millipore | 1.04003.1000 | |
| Glass 10 mL serological pipet | Corning | 7077-10N | |
| Hank’s Balances Salt Solution | Sigma Aldrich | H8264-500ML | |
| Hemocytometer/Neubauer Chamber | Daigger | HU-3100 | |
| High Glucose DMEM | Cytiva | SH30022.01 | |
| low glucose DMEM containing L-glutamine | Cytiva | SH30021.01 | |
| MEM/EBSS | Cytiva | SH30024.FS | |
| non-essential amino acids | Sigma-Aldrich | M7145-100M | |
| Paraformaldehyde (16%) | MP Biomedicals | 219998320 | |
| Penicillin/streptomycin/neomycin | Sigma-Aldrich | P4083-100ML | |
| Phosphate buffered saline | Cytiva | SH30256.01 | |
| Recombinant Mouse IFN-gamma Protein | R&D Systems | 485-MI | |
| Recombinant Mouse TNF-alpha (aa 80-235) Protein, CF | R&D Systems | 410-MT | |
| RNeasy mini kit | Qiagen | 74104 | |
| Sigmacote | Sigma Aldrich | SL2-100ML | 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. |
| Stemxyme | Worthington Biochemical Corporation | LS004106 | Collagenase/Dispase mixture |
| Sterile, individually wrapped cotton swab | Puritan Medical | 25-8061WC | |
| Thincert Tissue Culture Inserts, 24 well, Pore Size=8 µm | Greiner Bio-One | 662638 | |
| Thincert Tissue Culture Inserts, 6 well, Pore Size=0.4 µm | Greiner Bio-One | 657641 |
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