Name: Innsamling av murine nevrale stamme og oligodendrocytt stamceller av cerebrospinalvæske flytende biopsi
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

Rotte "hjernemelking" for isolering av nevrale stamceller og oligodendrocyttceller

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.

Forfatter i søkelyset: Innsamling av nevrale stam- og stamceller fra levende dyr ved hjelp av en ny hjernemelkeprotokoll 

"Brain Milking" -metoden for isolering av nevrale stamceller og oligodendrocyttprogenitorceller fra levende rotter

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

Innsamling av murine nevrale stamme og oligodendrocytt stamceller av cerebrospinalvæske flytende biopsi

For å begynne, administrer analgesi subkutant til den bedøvede rotten som tidligere ble injisert intracerebroventrikulært med frigjøringscocktailen. Monter rotta på den stereotaksiske rammen. Fest hodet med ørestenger, slik at rotasjonsbevegelsen mot forsiden og baksiden ikke hindres.Stabiliser hodet nedover i en 40-graders vinkel for å forlenge baksiden av nakken riktig. Barber nakkepelsen med en barberhøvel og desinfiser huden med tre alternerende runder med 10% povidonjod og alkohol ved hjelp av sterile bomullspinner, gni i forskjellige retninger i tre til fem sekunder hver gang. Med fingertuppen, identifiser det rundformede depressible området mellom occipital fremspring og ryggraden i atlaset.Merk dette punktet med en markør. Fest en milliliter sprøyte til den stereotaktiske rammen. Plasser nålen for å komme i kontakt med huden ved merket.Bruk tang til å løfte huden mens sprøyten føres inn gjennom hudlagene. Trekk stempelet forsiktig tilbake for å skape undertrykk inne i sprøyten. Sett kanylen gradvis inn til cerebralt spinalvæske er synlig i sprøyten.Hold nålen stødig i denne posisjonen og tillat den langsomme strømmen av cerebral spinalvæske. Fjern langsomt det cerebrale spinalvæsken opp til 120 mikroliter. Hent sprøyten forsiktig.Administrer en milliliter normalt serum intraperitonealt for å støtte det eksperimentelle dyrets væskepåfylling. Kombiner flytende biopsi med 400 mikroliter nevrale stamcellemedium og lagre mikrosentrifugerøret ved fire grader Celsius for senere bruk. Overfør deretter dyret til overvåkingsområdet etter operasjonen.Melkebiopsier resulterte i nevrale stamcellekulturer med et gjennomsnitt på 3,17 passasjepotensial, og nådde til og med ni passasjer. Innsamlede celler ble belagt på poly-D-lysinbelagte brønner hvor de vokste som både adherente monolag og sjeldnere som nevrosfærer. Nylig isolerte celler som var GFAP-positive var også immunpositive for hvilemarkøren ID3 og hadde karakteristikken NSE bipolar morfologi.Immunocytokjemisk sammenligning av biopsi-deriverte og postmortem-deriverte celler viste at profilen til de innsamlede cellene var lik endogene subependymale soneceller. Vekstfaktoren resulterte i en samtidig og signifikant økning i SOX2-celler og i reduserte dobbeltkortin-positive neuroblaster i begge prøvene.

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 ...