migration assay of murine adipose-derived mesenchymal stromal cells

Cocoltura di ADMSC e glia per ridurre l'infiammazione indotta da prioni

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.

Autore in primo piano: Approfondimento sui progressi nella ricerca sulle malattie da prioni

Cellule stromali mesenchimali di derivazione adiposa co-coltivate con glia mista primaria per ridurre l'infiammazione indotta da prioni

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.

Saggio di migrazione di cellule stromali mesenchimali di derivazione tesa murina

Trattare le AdMSC di passaggio due con terreni contenenti 10 nanogrammi per millilitro di TNF alfa per 24 ore o senza TNF alfa per le cellule non stimolate. Dopo aver lavato e incubato le cellule in terreni privi di siero per quattro ore, aggiungere 25.000 AdMSC a ciascun inserto e incubare per 24 ore a 37 gradi Celsius. Aspirare il contenuto di entrambi i pozzetti e inserirli, quindi lavare due volte con PBS lasciando un millilitro di PBS in ciascun pozzetto.Usando una pinza, rimuovere gli inserti uno per uno e pulire delicatamente ma accuratamente la camera superiore con un batuffolo di cotone per rimuovere le cellule non migrate. Aspirare il PBS rimanente e pipettare 500 microlitri di soluzione di cristallo violetto nel pozzetto e nella camera superiore dell'inserto. Aspirare la soluzione di cristallo viola dal pozzetto e dalla camera superiore dell'inserto dopo un'ora di incubazione.Dopo aver lavato e pulito la camera superiore, posizionare l'inserto in una nuova piastra da 24 pozzetti contenente un millilitro di PBS. Utilizzando un microscopio invertito con una fotocamera, è possibile visualizzare quattro aree casuali verso il centro dell'inserto sotto un obiettivo 10 volte. Carica le immagini sul software di elaborazione delle immagini preferito e regola lo sfondo per migliorare il contrasto con le cellule colorate di viola.CLIA infettata con 22L ha mostrato un aumento dell'espressione di mRNA per i marcatori infiammatori CCL2, CCL5 e IL1 beta e il marcatore astrocitario S100 beta rispetto a quelli trattati con NBH. La cocoltura con AdMSC ha ridotto l'espressione delle citochine infiammatorie CCL2, CCL5 e IL1 beta, ma non il TNF alfa sia per le cellule infettate da MBH che per quelle infettate da 22L. Le BV2 cocoltivate con AdMSC hanno mostrato una diminuzione dell'mRNA per i marcatori infiammatori IL1 beta, IL-6, TNF alfa e la proteina del complemento C1qa in entrambe le cellule trattate con NBH e RML.Inoltre, le AdMSC hanno diminuito il gene della microglia M1 CD16 e aumentato il marcatore della microglia M2 ARG-1. Il test di migrazione in vitro ha suggerito che le AdMSC hanno mostrato una migrazione significativa verso terreni contenenti l'1% di 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 ...

Ricerca

Neuroscienze

Isolating and Infecting Primary Cortical Mixed Glia with Prions

Isolamento e infezione della glia mista corticale primaria con prioni