migration assay of murine adipose-derived mesenchymal stromal cells

Samdyrking av AdMSC og glia for å redusere prionindusert betennelse

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.

Forfatter i søkelyset: Innsikt i fremskritt innen forskning på prionsykdommer

Fettavledede mesenkymale stromaceller samdyrket med primær blandet glia for å redusere prionindusert betennelse

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.

Migrasjonsanalyse av murine adipose-derived mesenkymale stromale celler

Behandle passasje to AdMSC med medier som inneholder 10 nanogram per milliliter TNF alfa i 24 timer eller uten TNF alfa for ustimulerte celler. Etter å ha vasket og inkubert cellene i serumfrie medier i fire timer, tilsett 25 000 AdMSCs til hver innsats og inkuber i 24 timer ved 37 grader Celsius. Aspirer innholdet i begge godt og sett inn, vask deretter to ganger med PBS og la en milliliter PBS ligge i hver brønn.Bruk tang, fjern innsatsene en etter en og tørk forsiktig men grundig av det øverste kammeret med en bomullspinne for å fjerne umigrerte celler. Aspirer de resterende PBS og pipetten 500 mikroliter krystallfiolett løsning til brønnen og det øverste kammeret på innsatsen. Aspirer den krystallfiolette løsningen fra brønnen og toppkammeret på innsatsen etter en times inkubasjon.Etter vask og tørk av toppkammeret, plasser innsatsen i en ny 24-brønnplate som inneholder en milliliter PBS. Ved hjelp av et omvendt mikroskop med et kamera, bilde fire tilfeldige områder mot innsatsen sentrum under en 10 ganger mål. Last opp bildene til den foretrukne bildebehandlingsprogramvaren og juster bakgrunnen for forbedret kontrast med lilla fargede celler.CLIA infisert med 22L viste økt mRNA-uttrykk for betennelsesmarkørene CCL2, CCL5 og IL1 beta, og astrocyttmarkøren S100 beta sammenlignet med de som ble behandlet med NBH. Kokulering med AdMSC reduserte ekspresjonen av de inflammatoriske cytokinene CCL2, CCL5 og IL1 beta, men ikke TNF alfa for både MBH og 22L infiserte celler. BV2s dyrket sammen med AdMSC viste en reduksjon i mRNA for de inflammatoriske markørene IL1 beta, IL-6, TNF alfa og komplementproteinet C1qa i både NBH- og RML-behandlede celler.I tillegg reduserte AdMSCs M1 microglia-genet CD16 og økte M2 microglia-markøren ARG-1. In vitro-migrasjonsanalysen antydet at AdMSC-er viste signifikant migrasjon mot medier som inneholdt 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 og infisering av primær kortikal blandet glia med prioner