migration assay of murine adipose-derived mesenchymal stromal cells

Coculturing AdMSCs and Glia to Reduce Prion-Induced Inflammation

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.

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.

Migration Assay of Murine Adipose-Derived Mesenchymal Stromal Cells

Treat passage two AdMSCs with media containing 10 nanograms per milliliter of TNF alpha for 24 hours or without TNF alpha for unstimulated cells. After washing and incubating the cells in serum-free media for four hours, add 25, 000 AdMSCs to each insert and incubate for 24 hours at 37 degrees Celsius. Aspirate the contents of both well and insert, then wash twice with PBS leaving one milliliter of PBS in each well.Using forceps, remove the inserts one by one and gently but thoroughly wipe the top chamber with a cotton swab to remove unmigrated cells. Aspirate the remaining PBS and pipette 500 microliters of crystal violet solution to the well and the top chamber of the insert. Aspirate the crystal violet solution from the well and top chamber of the insert after one hour incubation.After washing and wiping the top chamber, place the insert in a new 24 well plate containing one milliliter of PBS. Using an inverted microscope with a camera, image four random areas towards the insert's center under a 10 times objective. Upload the images to the preferred image processing software and adjust the background for enhanced contrast with purple stained cells.CLIA infected with 22L showed increased mRNA expression for inflammatory markers CCL2, CCL5, and IL1 beta, and the astrocyte marker S100 beta compared to those treated with NBH. Coculturing with AdMSCs decreased expression of the inflammatory cytokines CCL2, CCL5, and IL1 beta, but not TNF alpha for both MBH and 22L infected cells. BV2s cocultured with AdMSCs showed a decrease in mRNA for the inflammatory markers IL1 beta, IL-6, TNF alpha, and the complement protein C1qa in both NBH and RML treated cells.Additionally, AdMSCs decreased the M1 microglia gene CD16 and increased the M2 microglia marker ARG-1. The in vitro migration assay suggested that AdMSCs showed significant migration toward media containing 1%RML.

migration-assay-of-murine-adipose-derived-mesenchymal-stromal-cells

Research

JoVE Journal

Neuroscience

37 Views.  Colorado State University. Treat passage two AdMSCs with media containing 10 nanograms per milliliter of TNF alpha for 24 hours or without TNF alpha for unstimulated cells. After washing and incubating the cells in serum-free media for four hours, add 25, 000 AdMSCs to each insert and incubate for 24 hours at 37 degrees Celsius. Aspirate the contents of both well and insert, then wash twice with PBS leaving one milliliter of PBS in each well.Using forceps, remove the inserts one by one and gently but thoroughly ...

Video: Migration Assay of Murine Adipose-Derived Mesenchymal Stromal Cells

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.

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 ...

Isolating and Infecting Primary Cortical Mixed Glia with Prions

To begin, take the brain isolated from the euthanized mouse pups and place it in a three-centimeter Petri dish containing cold MEM with EBSS and 2X PSN. Under a dissecting scope, separate brain hemispheres, remove and discard the midbrain, hippocampus, and meninges. Place both cortical hemispheres in a 50-milliliter conical tube containing five milliliters of MEM EBSS with 2X PSN, and keep on ice.In a tissue culture hood, aspirate the media from the 50-milliliter conical tube using a pipette, leaving the cortical tissue pieces at the bottom. Add 10 milliliters of pre-warmed dissociation media with a coated glass pipette, and triturate the tissue 10 to 20 times. Transfer the suspension to a 50-milliliter beaker containing a small stir bar and place it on a stir plate.Then remove the beaker, set it at a 30-degree angle for three minutes to allow the tissue to settle, and transfer the cell suspension to a new 50-milliliter conical tube on ice. Centrifuge the 50-milliliter conical tube at 1000G for 10 minutes at four degrees Celsius. Replace the supernatant with glial growth medium and count the cells using a hemocytometer.Plate the cells at a density of one million in 10-centimeter cell culture-treated dishes and incubate for 24 hours. Then replace the media with fresh glial media and allow the cells to reach 100%confluence within two weeks before plating them for experimentation. Plate 100, 000 glial cells per well in six-well plates and allow them to reach 80%to 100%confluence for in vitro prion infection.Expose 20%diluted brain homogenates and PBS to ultraviolet light for 30 minutes. Aspirate the media from the Glia to be infected and add 1.5 to 2 milliliters of glial growth media with 0.1%normal or prion brain homogenate per well. After 72 hours, remove the media, wash the cells with PBS and add fresh glial growth media.Carefully aspirate the adipose tissue in approximately one milliliter of HBSS in 0.25%trypsin solution, using a serological pipette. Transfer the tissue to a four-centimeter Petri dish containing two milliliters of DMEM F-12 media with Dnase-1, collagenase and Dispase mixture. Then cut the adipose tissue into small pieces using scissors and incubate the mixture at 37 degrees Celsius for 1.5 hours.Transfer the tissue to a 50-milliliter conical tube, triturate the tissue, and pellet the stromal vascular fraction, which will appear red. After washing the pellet with sterile PBS and centrifuging for three minutes, resuspend the pellet in one milliliter of ADMSC media. Afterward, pipette the cell suspension through a 40-micrometer strainer into a sterile 50-milliliter conical tube, to remove non-dissociated tissue.Add nine milliliters of ADMSC media to a 10-centimeter cell culture-treated dish. Pipette the strained cell suspension into it and incubate at 37 degrees Celsius with 5%carbon dioxide. Change the media the next day and passage the cells once they attain 80%to 90%confluency.After the cells have undergone two to three passages, resuspend them in three to 10 milliliters of media and count using a hemocytometer. Plate 100, 000 cells per well in six-well plates containing ADMSC media and incubate overnight, as shown earlier. The next day, prepare the ADMSC media for cytokine-stimulated cells with either 10 nanograms per milliliter of TNF-alpha or 200 nanograms per milliliter of IFN-gamma.Create ADMSC media with a final concentration of 0.1%prion-infected or normal brain homogenate for control samples. Aspirate the old media, add 1.5 milliliters of cytokine or brain homogenate-containing media into the corresponding wells in triplicate, and return plates to the incubator. Then wash the cells with PBS and isolate RNA by adding 350 microliters of lysis buffer with 1%beta-mercaptoethanol to each well.Remove cell lysates using a cell lifter and reverse transcribe 25 nanograms of RNA per sample and amplify complementary DNA. Plate BV2 microglia at 50, 000 cells per well or primary mixed glia at 100, 000 cells per well in a six-well plate. Treat BV2 cells with media containing 0.1%prion-infected or normal brain homogenate and incubate for 72 hours.Following incubation, wash the cells two times with PBS to remove the remaining brain homogenate. Add fresh media to the cells and return the plate to the incubator. To stimulate ADMCs, treat them with 10 nanograms per milliliter of TNF-alpha for 24 hours before co-culturing with BV2 or mixed glia.Wash the stimulated ADMCs with PBS three times and spin the ADMSC suspension as demonstrated earlier. Replace the media on BV2 cells or mixed glia with two milliliters per well of ADMSC media, and place inserts for six-well plates with a pore size of 0.4 micrometers into half of the wells. Add two milliliters of ADMSC media to each insert and add an additional two milliliters of media to wells that do not receive inserts.Upon pelleting, resuspend and counting the ADMCs, add 50, 000 to 100, 000 cells to each insert and incubate them. At the end of the incubation, remove and discard the inserts or replace them in a new six-well plate and wash the inserts twice with PBS. Add the buffer provided by the RNA isolation kit to the mixed glia or BV2 cells and scrape the wells.