migration assay of murine adipose-derived mesenchymal stromal cells

Cocultureing AdMSCs och glia för att minska prioninducerad 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.

Författare i fokus: Inblick i framsteg inom forskning om prionsjukdomar

Mesenkymala stromaceller från fett odlade tillsammans med primära blandade glia för att minska prioninducerad 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.

Migrationsanalys av musfetthärledda mesenkymala stromaceller

Behandla passage två AdMSC med media som innehåller 10 nanogram per milliliter TNF-alfa i 24 timmar eller utan TNF-alfa för ostimulerade celler. Efter tvättning och inkubation av cellerna i serumfritt medium i fyra timmar, tillsätt 25 000 AdMSC till varje insats och inkubera i 24 timmar vid 37 grader Celsius. Aspirera innehållet i både brunnen och insatsen, tvätta sedan två gånger med PBS och lämna en milliliter PBS i varje brunn.Använd en pincett, ta bort insatserna en efter en och torka försiktigt men noggrant av den övre kammaren med en bomullspinne för att ta bort omigrerade celler. Sug upp den återstående PBS:en och pipettera 500 mikroliter kristallviolett lösning till brunnen och den övre kammaren på insatsen. Aspirera den kristallvioletta lösningen från insatsens brunn och övre kammare efter en timmes inkubation.Efter att ha tvättat och torkat av den övre kammaren, placera insatsen i en ny 24-hålsplatta som innehåller en milliliter PBS. Använd ett inverterat mikroskop med en kamera och avbilda fyra slumpmässiga områden mot insatsens centrum under ett 10 gånger objektiv. Ladda upp bilderna till önskat bildbehandlingsprogram och justera bakgrunden för förbättrad kontrast med lila färgade celler.CLIA infekterad med 22L visade ökat mRNA-uttryck för inflammatoriska markörer CCL2, CCL5 och IL1 beta samt astrocytmarkören S100 beta jämfört med de som behandlades med NBH. Samodling med AdMSCs minskade uttrycket av de inflammatoriska cytokinerna CCL2, CCL5 och IL1 beta, men inte TNF alfa för både MBH- och 22L-infekterade celler. BV2 samodlade med AdMSCs visade en minskning av mRNA för de inflammatoriska markörerna IL1 beta, IL-6, TNF alfa och komplementproteinet C1qa i både NBH- och RML-behandlade celler.Dessutom minskade AdMSCs M1 microglia-genen CD16 och ökade M2-mikrogliamarkören ARG-1. In vitro-migrationsanalysen tydde på att AdMSC visade signifikant migration mot media som innehöll 1 % RML.

Research

JoVE Journal

Neuroscience

Migration Assay of Murine Adipose-Derived Mesenchymal Stromal Cells

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 ...

Isolating and Infecting Primary Cortical Mixed Glia with Prions

Isolering och infektion av primära kortikala blandade glia med prioner