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 (PrP^C) misfolds to the disease-associated prion protein (PrP^Sc), 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

Mice were bred and maintained at Colorado State'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

1. To isolate primary mixed glia containing both astrocytes and microglia, obtain C57Bl/6 mouse pups aged zero to two days old.
   NOTE: This protocol is adapted from previous protocols ^15,16.
2. Euthanize pups one at a time by decapitation and extract their brain, separating and discarding the cerebellum and brain stem.
   1. Place the brain in a 3 cm Petri dish containing cold MEM/EBSS with 2x Penicillin/streptomycin/neomycin (PSN). Under a dissecting scope, separate brain hemispheres and remove and discard the midbrain. Remove and discard the hippocampus and meninges from the cortex.
   2. Place both cortical hemispheres in a 50 mL conical tube containing 5 mL of MEM/EBSS + 2x PSN and place on ice.
3. In a tissue culture hood, remove the media from the 50 mL conical tube by gently aspirating with a pipet, leaving the cortical tissue pieces at the bottom of the tube. With a coated glass pipet, add 10 mL of prewarmed dissociation media and triturate the tissue with the glass pipet 10-20x.
4. Transfer the suspension to a 50 mL beaker containing a small stir bar and place on a stir plate at the lowest setting, approximately 30 rpm, for 10 min. Remove the beaker from the stir plate and set it at a 30° angle for 3 min to allow the tissue to settle at the bottom. Remove the cell suspension (supernatant) and transfer to a new 50 mL conical tube on ice.
5. Add DNase-I (4,000 U/mL) to 10 mL of dissociation media and resuspend the tissue and stir for an additional 10 min. Repeat by adding fresh dissociation media (without DNase-I) 2-4 additional times (one time for every two mouse pups used), and transferring the cell suspension to the 50 mL conical tube on ice, until only fibrous tissue remains in the bottom of the beaker.
6. Centrifuge the cell suspension in the conical tube for 10 min at 1,000 × g at 4 °C. Aspirate the supernatant and replace with glial growth medium. Count the cells with a hemocytometer and plate the cells at a density of 10⁶ in 10 cm cell-culture treated dishes. Place in an incubator at 37 °C with 5% CO₂.
7. After 24 h, remove the media and replace it with fresh glial media. Let the cells reach 100% confluence within 2 weeks; then, split and plate them for experimentation.
8. For in vitro prion infection, plate glia at 100,000 cells per well in 6-well plates and allow to reach 80%-100% confluence. Expose brain homogenates, both prion and normal, diluted to 20% in PBS to UV light for 30 min prior to adding to cell culture. Aspirate media off glia to be infected, and to each well, add 1.5-2 mL of glial growth media containing a final volume of 0.1% normal or prion brain homogenate.
9. After 72 h, aspirate off the media and wash the cells with PBS to remove any residual brain homogenate. Replace with fresh glial growth media (containing no brain homogenates).
10. To isolate AdMSCs cells, use a serological pipet and gently aspirate the adipose tissue along with ~1 mL of HBSS/Trypsin solution. Transfer to a 4 cm Petri dish containing 2 mL of DMEM/F12 media with 200 U/mL DNase-I and 400 U/mL collagenase/Dispase mixture. With small scissors, cut the chunks of adipose tissue into small pieces (less than 5 mm in size) and incubate at 37 °C for 1.5 h.
11. Transfer the contents of the Petri dish to a 50 mL conical tube with a serological pipet and triturate the mixture ~10x with the pipet. Make sure the tissue breaks apart easily and forms a relatively homogeneous mixture. Centrifuge the mixture at 4 °C for 5 min at 1,000 × g to pellet the stromal vascular fraction, which will appear red.
12. Aspirate the supernatant carefully and wash the pellet with 5 mL of prewarmed sterile PBS and centrifuge at 4 °C at 1,000 × g for 3 min. Aspirate the supernatant and resuspend the pellet in 1 mL of AdMSC media (low glucose DMEM containing L-glutamine and supplemented with 15% heat-inactivated fetal bovine serum (hiFBS), 1% amino acids, 1% non-essential amino acids, and 1% PSN).
13. Place a 40 µm cell strainer on top of a fresh sterile 50 mL conical tube and pipet the cell suspension through the strainer to remove any non-dissociated tissue. Add 9 mL of AdMSC media to a 10 cm cell culture-treated dish and pipet the strained cell suspension into the dish. Place in an incubator at 37 °C with 5% CO₂.
14. Remove and replace with fresh AdMSC media the following day. Wait for the cells to become 80-90% confluent when they will be ready to be passaged between 72 h and 96 h.
15. Isolate AdMSCs as described above and passage twice to obtain a consistent homogeneous population ¹⁴. Wash cells twice with sterile PBS and pipet 2 mL of prewarmed trypsin (0.25%) onto each plate. Incubate cells at 37 °C for 5 min or until they become fully dislodged from the plate. Add 8 mL of prewarmed AdMSC media to each plate, pipet to mix the cell suspension, transfer to the conical tube, and centrifuge at 1,000 × g at 4 °C to pellet.
16. Resuspend AdMSCs in 3-10 mL of media (depending on the number of plates used) and count cells using a hemocytometer. Plate 100,000 cells/well in 6-well plates containing AdMSC media. Place in an incubator at 37 °C with 5% CO₂ overnight.
17. The following day, stimulate cells with either cytokines or brain homogenate.
    1. For cytokine-stimulated cells, make AdMSC media containing either 10 ng/mL TNFα or 200 ng/mL IFNγ. Use fresh media for control wells.
    2. For prion-infected brain homogenate treatments, obtain 20% brain homogenate (in PBS) from terminally infected prion mice (22L or RML strains). Make AdMSC mediawith a final concentration of 0.1% prion-infected or normal brain homogenate for controls.
    3. Aspirate media off the wells and pipet 1.5 mL of cytokine- or brain homogenate-containing media to corresponding wells. Perform in triplicate. Return plates to the incubator.
18. Remove the media, wash cells 2x with prewarmed PBS, isolate RNA by adding 350 mL of lysis buffer containing 1% βME to each well, and use a cell lifter to remove cell lysates. Perform RNA isolation following the mini kit manufacturer's protocol, including a DNase digestion step. For RT-qPCR, reverse-transcribe 25 ng of RNA per sample and amplify cDNA with SYBR Green and primers for each gene at 10 mM. Analyze mRNA expression using the 2^-ΔΔCT method and normalize to the expression of reference gene β-actin ¹⁷.
    NOTE: All RT-PCR was done following MIQE guidelines.
19. Plate BV2 microglia at 50,000 cells per well, or primary mixed glia at 100,000 cells per well in a 6-well plate. Treat BV2 cells the following day with media containing 0.1% prion-infected or normal brain homogenate. For mixed glia, wait until cells are 80-90% confluent to infect with brain homogenate.
20. Incubate cells in media containing brain homogenate for 72 h. Wash cells 2x with PBS to remove remaining brain homogenate and add fresh media and return to incubator.
21. Use AdMSCs at passage 3 for co-culture. If stimulating AdMSCs, use media containing 10 ng/mL TNFα and treat for 24 h prior to co-culturing with BV2 or mixed glia. Wash stimulated AdMSCs 3x with PBS to remove any remaining TNFα.
22. Trypsinize AdMSCs as described in step 1.15 and resuspend in AdMSC media.
23. Spin AdMSC suspension at 1,000 × g at 4 °C for 5 min. During this time, replace media on BV2 cells or mixed glia with 2 mL per well of AdMSC media and place inserts for 6-well plates with a pore size of 0.4 micrometers into half of the wells. Add 2 mL of AdMSC media to each insert and an additional 2 mL of media to wells that do not receive inserts.
24. When finished pelleting AdMSCs, resuspend the pellet in AdMSC media. Count AdMSCs with a hemocytometer and add 50,000-100,000 cells to each insert.
25. For BV2 cells, incubate co-cultures for 24 h. For mixed glia, incubate for as few as 24 h and as many as 7 days.
26. After incubation with AdMSCs for the desired time, remove the inserts and discard, or place them in a new 6-well plate, wash inserts 2x with PBS, add buffer provided by RNA isolation kit to the mixed glia or BV2 cells, and scrape inserts to analyze AdMSC RNA.
27. Treat AdMSCs at passage 2 with media containing 10 ng/mL TNFα. The following day, wash cells with prewarmed sterile PBS 3x and incubate in serum-free AdMSC media for 4 h.
28. To perform migratory cell assay, add 25,000 AdMSCs per insert and incubate cells for 24 h at 37 °C. During this time, migratory cells will move from the top chamber of the insert and adhere to the underside of the insert. Carefully aspirate the contents of both the well and inserts and wash 2x with PBS, leaving 1 mL of PBS in each well to ensure that the inserts remain damp on each side.
29. One at a time, using forceps, remove the inserts and gently but thoroughly wipe the top chamber with a cotton swab to remove any cells that have not migrated. Aspirate the remaining PBS and pipet 500 μL of crystal violet solution to both the well and top chamber of the insert, ensuring that the insert membrane is fully submerged.
30. Incubate inserts for 1 h in crystal violet solution at room temperature. To best retain color, perform the next steps quickly, with only one insert at a time.
    1. Aspirate crystal violet solution from the well and top chamber of the insert and wash 3x with PBS. Wipe the top chamber again with a cotton swab.
    2. Place the insert in a well in a new 24-well plate containing 1 mL of PBS. Place the plate above an inverted microscope with a camera attached. Using the 10x objective, take images of four random areas toward the center of the insert, focusing only on the bottom side of the insert.
31. After performing this with each well, upload images to the image processing software of choice, and adjust the background to enhance the contrast with the purple-stained cells. Count the cells manually with a cell counter.

Results

Stimulating AdMSCs with TNFα or interferon-gamma (IFNγ) for 24 h induces changes in the expression of anti-inflammatory molecules and growth factors. Treating AdMSCs with TNFα or interferon-gamma (IFNγ) increases TNF-stimulated gene 6 (TSG-6) mRNA, whereas TNFα, but not IFNγ, causes an increase in transforming growth factor beta-1 (TGFβ-1) mRNA. Stimulation with TNFα or IFNγ induces an increase in vascular endothelial growth factor (VEGF)mRNA, but no change in the expression of fibroblast growth factor ...

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...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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