Name: 뇌척수액 액체 생검에 의한 쥐 신경줄기 및 희소돌기아교세포 전구세포 수집
Uploaded: 2024-02-09T12:30:00
Description: Watch this Scientific Journal Video about Collecting Murine Neural Stem and Oligodendrocyte Progenitor Cells by Cerebrospinal Fluid Liquid Biopsy at JoVE.com

collecting murine neural stem and oligodendrocyte progenitor cells by cerebrospinal fluid liquid biopsy

신경 줄기 및 희소돌기아교세포 전구 세포 분리를 위한 쥐 "뇌 착유"

Tissue-specific stem cells are partially committed cells that can give rise to all cell populations that constitute the respective tissues. Apart from being multipotent, they are self-renewing cells and crucial for maintaining the homeostasis and the regenerative capacity of tissues1. Some tissue-specific stem cells remain in an active, strongly proliferative state, such as intestinal or hematopoietic stem cells. Others, such as brain stem cells, remain largely quiescent or dormant2. In the adult brain, neural stem cells (NSCs) can be found in specialized areas, often called niches. Two such well described areas exist in the subependymal zone (SEZ) of the lateral ventricles and in the dentate gyrus of the hippocampus. The SEZ niche generates the highest numbers of cells, primarily neuroblasts that migrate toward the olfactory bulbs and contribute to the local interneuron population; in contrast, generated oligodendroblasts migrate to the adjacent corpus callosum (CC)3. Oligodendrocyte progenitor cells (OPCs) are mitotically active cells, widely distributed throughout the central nervous system, that: i) are committed to the oligodendroglial lineage, ii) can migrate to sites of demyelination, and iii) can differentiate into myelinating oligodendrocytes. OPCs also exhibit self-renewal potential and quiescence4.
Until now, the isolation and study of NSCs and OPCs required postmortem dissociation of the dissected brain and spinal cord tissue. To circumvent this experimental limitation, we established a method that allows, for the first time, the isolation of brain NSCs and OPCs from live animals. We call this method &#34;milking&#34;, because it enables multiple collections of cells as their pools are not depleted. The protocol was developed in rats, due to their large brain size, targeting mainly the SEZ, or the CC, and includes two major steps. First, NSCs or OPCs are &#34;removed&#34; from the tissue via i.c.v. injection of a &#34;release cocktail&#34; containing neuraminidase, a toxin that induces ventricular wall denudation, an integrin-&#946;1-blocking antibody, and fibroblast growth factor 2 (FGF2). The cocktail is stereotaxically injected bilaterally within the lateral ventricles. If the intended use is the isolation of NSCs, rostral areas of the lateral ventricles are targeted. If the aim is to isolate OPCs more purely, the cocktail is injected caudally in the area of the hippocampal fimbria. At a second &#34;collection&#34; step, liquid biopsies of cerebrospinal fluid (CSF) are performed from the cisterna magna of anesthetized rats, without the need of an incision. The liquid biopsy is mixed with NSC culture medium and can be kept at 4 &#176;C until plating.

저자 스포트라이트: Collecting Neural Stem and Progenitor Cells from Live Animals Using a Novel Brain Milking Protocol (새로운 뇌 착유 프로토콜을 사용하여 살아있는 동물에서 신경 줄기 및 전구 세포 수집) 

살아있는 쥐에서 신경 줄기 세포와 희소돌기아교세포 전구 세포를 분리하기 위한 "뇌 착유" 방법

We are working with brain neural stem cells, very sparse cells that cluster within specialized microenvironments called niches. Although they contribute to olfaction, memory, and learning, they are extremely inefficient in driving regeneration. Thus, we investigate their properties and ways to manipulate them to increase the healing of the nervous tissue.We have shown that the contribution of brain neural stem cells to regeneration is limited by endogenous and endogenous factors, such as their restricted differentiation potential, and their dependence on microenvironment factors. Equally, we have identified key properties that can be used to devise strategies to modulate their capacity. The milking protocol allows the collection of brain neural stem and oligodendrocyte progenitor cells from live experimental animals.Thus, it allows a more uninterrupted analysis of their properties and the performance of longitudinal studies. Currently, there are no alternative techniques for the isolation of brain murine stem and progenitor cells from live experimental animals. The present standard of comparison is the postmortem isolation of such cells, which introduces various experimental uncertainties and biological bias.We are now investigating how brain neural stem cells become activated by existing quiescence. We are also comparing the properties of different progenitor populations of the brain. For example, neurogenic versus oligodendrogenic, in order to define their differential properties and their adaptations to varied microenvironment.

Watch this Scientific Journal Video about Collecting Murine Neural Stem and Oligodendrocyte Progenitor Cells by Cerebrospinal Fluid Liquid Biopsy at JoVE.com

뇌척수액 액체 생검에 의한 쥐 신경줄기 및 희소돌기아교세포 전구세포 수집

먼저, 이전에 방출 칵테일과 함께 뇌내 주사를 맞은 마취된 쥐에게 진통제를 피하 투여합니다. 입체 프레임에 쥐를 장착하십시오. 이어 바를 사용하여 헤드를 고정하고 앞뒤로 회전 운동이 방해받지 않도록 합니다.머리를 40도 각도로 아래쪽으로 고정하여 목 뒤쪽을 적절하게 확장합니다. 면도기를 사용하여 목털을 면도하고 멸균 면봉을 사용하여 10% 포비돈 요오드와 알코올을 번갈아 가며 3-5초 동안 다른 방향으로 문질러 피부를 소독합니다. 손가락 끝으로 후두부 돌기와 아틀라스의 척추 사이의 둥근 모양의 함몰 영역을 식별합니다.마커를 사용하여 이 점을 표시합니다. 1밀리리터 주사기를 입체 프레임에 고정합니다. 바늘이 표시에서 피부에 닿도록 배치합니다.집게를 사용하여 피부층을 통해 주사기를 삽입하면서 피부를 들어 올립니다. 플런저를 부드럽게 뒤로 당겨 주사기 내부에 음압을 생성합니다. 주사기에 뇌척수액이 보일 때까지 바늘을 서서히 삽입합니다.이 위치에서 바늘을 안정적으로 잡고 뇌척수액이 천천히 흐르도록 합니다. 최대 120마이크로리터의 뇌척수액을 천천히 제거합니다. 주사기를 조심스럽게 회수합니다.실험 동물의 체액 보충을 지원하기 위해 1ml의 정상 혈청을 복강 내로 투여합니다. 액체 생검을 400마이크로리터의 신경 줄기 세포 배지와 결합하고 나중에 사용할 수 있도록 마이크로 원심분리 튜브를 섭씨 4도에서 보관합니다. 그런 다음 동물을 수술 후 모니터링 구역으로 옮깁니다.생검을 착유한 결과 평균 3.17개의 통과 가능성을 가진 신경 줄기 세포 배양이 이루어졌으며 심지어 9개의 통로에 도달했습니다. 수집된 세포는 Poly-D-Lysine으로 코팅된 웰에 도금되어 부착성 단층으로 성장하고 드물게는 신경구로 성장했습니다. GFAP 양성인 갓 분리된 세포는 정지 마커 ID3에 대해서도 면역 양성이었고 NSE 양극성 형태 특성을 가졌습니다.생검 유래 세포와 사후 유래 세포의 면역세포화학적 비교는 수집된 세포의 프로파일이 내인성 뇌막하 영역 세포와 유사하다는 것을 보여주었습니다. 성장 인자는 SOX2 세포의 수반적이고 중요한 증가와 두 샘플 모두에서 이중 코르틴 양성 신경 아세포를 감소시켰습니다.

Research

JoVE Journal

Neuroscience

Collecting Murine Neural Stem and Oligodendrocyte Progenitor Cells by Cerebrospinal Fluid Liquid Biopsy

The "Brain Milking" Method for the Isolation of Neural Stem Cells and Oligodendrocyte Progenitor Cells from Live Rats

Tissue-specific neural stem cells (NSCs) remain active in the mammalian postnatal brain. They reside in specialized niches, where they generate new ...