migration assay of murine adipose-derived mesenchymal stromal cells

프리온 유발 염증을 줄이기 위한 AdMSCs 및 신경교세포 공동 배양

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.

저자 스포트라이트: 프리온 질환 연구의 발전에 대한 통찰력

프리온 유발 염증을 줄이기 위해 원발성 혼합 신경교세포와 공동 배양된 지방 유래 중간엽 기질 세포

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.

Murine Adipose-Derived Mesenchymal Stromal Cells의 이동 분석

TNF 알파 밀리리터당 10나노그램을 함유한 배지로 24시간 동안 또는 자극되지 않은 세포의 경우 TNF 알파 없이 2개의 AdMSC를 처리합니다. 4 시간 동안 혈청 자유로운 매체에 있는 세포를 세척하고 부양 한 후에, 각 삽입에 25, 000의 AdMSCs를 추가하고 섭씨 37 온도에 24 시간 동안 부화한다. 두 웰의 내용물을 흡인하고 삽입한 다음 PBS로 두 번 세척하여 각 웰에 1밀리리터의 PBS를 남깁니다.집게를 사용하여 삽입물을 하나씩 제거하고 면봉으로 상단 챔버를 부드럽지만 철저하게 닦아 이동하지 않은 세포를 제거합니다. 나머지 PBS와 피펫 500 마이크로리터의 결정 보라색 용액을 웰과 인서트의 상단 챔버에 흡입합니다. 1시간 배양 후 삽입물의 웰과 상단 챔버에서 결정 보라색 용액을 흡입합니다.상단 챔버를 세척하고 닦은 후 PBS 1mL가 들어 있는 새 24웰 플레이트에 삽입물을 넣습니다. 카메라가 장착된 도립 현미경을 사용하여 10배 대물렌즈 아래에서 인서트 중심을 향한 4개의 무작위 영역을 이미지화합니다. 이미지를 선호하는 이미지 처리 소프트웨어에 업로드하고 보라색 염색 세포와의 대비를 향상시키기 위해 배경을 조정합니다.22L에 감염된 CLIA는 NBH로 치료받은 환자에 비해 염증 마커 CCL2, CCL5, IL1 베타와 성상세포 마커 S100 베타에 대한 mRNA 발현이 증가한 것으로 나타났다. AdMSC를 사용한 공동 배양은 염증성 사이토카인 CCL2, CCL5 및 IL1 베타의 발현을 감소시켰지만 MBH 및 22L 감염 세포 모두에 대한 TNF 알파는 감소시키지 않았습니다. AdMSC와 병용배양한 BV2는 NBH 및 RML 처리 세포 모두에서 염증 마커 IL1 베타, IL-6, TNF 알파 및 보체 단백질 C1qa에 대한 mRNA의 감소를 보여주었습니다.또한 AdMSC는 M1 미세아교세포 유전자 CD16을 감소시키고 M2 미세아교세포 마커 ARG-1을 증가시켰습니다. in vitro 이동 분석은 AdMSC가 1% RML을 포함하는 배지로 상당한 이동을 보였음을 시사했습니다.

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 ...

연구

신경과학

Isolating and Infecting Primary Cortical Mixed Glia with Prions

Isolating and infecting Primary Cortical Mixed Glia with Prions(원발성 피질 혼합 신경교세포와 프리온 분리 및 감염)