Name: Recolección de células madre neurales murinas y células progenitoras de oligodendrocitos mediante biopsia líquida de líquido cefalorraquídeo
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

"Ordeño cerebral" de ratas para el aislamiento de células progenitoras de oligodendrocitos y tronco neural

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.

Autor destacado: Recolección de células madre neurales y progenitoras de animales vivos utilizando un novedoso protocolo de ordeño cerebral 

El método de "ordeño cerebral" para el aislamiento de células madre neurales y células progenitoras de oligodendrocitos de ratas vivas

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

Recolección de células madre neurales murinas y células progenitoras de oligodendrocitos mediante biopsia líquida de líquido cefalorraquídeo

Para empezar, administrar analgesia por vía subcutánea a la rata anestesiada a la que previamente se le inyectó intracerebroventricularmente con el cóctel de liberación. Monta la rata en el marco estereotáxico. Asegure la cabeza con barras para los oídos, asegurándose de que el movimiento de rotación hacia adelante y hacia atrás no esté obstruido.Estabilice la cabeza hacia abajo en un ángulo de 40 grados para extender correctamente la parte posterior del cuello. Afeitar el pelaje del cuello con una navaja de afeitar y desinfectar la piel con tres rondas alternas de povidona yodada al 10% y alcohol con hisopos de algodón estériles, frotando en diferentes direcciones durante tres a cinco segundos cada vez. Con la yema del dedo, identifique el área depresible de forma redonda entre la protuberancia occipital y la columna vertebral del atlas.Marca este punto con un rotulador. Fije una jeringa de un mililitro al marco estereotáxico. Coloque la aguja para que entre en contacto con la piel en la marca.Con unas pinzas, levante la piel mientras inserta la jeringa a través de las capas de piel. Tire suavemente del émbolo hacia atrás para crear una presión negativa dentro de la jeringa. Inserte gradualmente la aguja hasta que el líquido cefalorraquídeo sea visible en la jeringa.Sostenga la aguja firmemente en esta posición y permita el flujo lento del líquido cefalorraquídeo. Retire lentamente el líquido cefalorraquídeo hasta 120 microlitros. Recupere con cuidado la jeringa.Administrar un mililitro de suero normal por vía intraperitoneal para apoyar la reposición de líquidos del animal de experimentación. Combine la biopsia líquida con 400 microlitros de medio de células madre neurales y almacene el tubo de microcentrífuga a cuatro grados centígrados para su uso posterior. A continuación, traslade al animal a la zona de seguimiento postoperatorio.Las biopsias de ordeño dieron como resultado cultivos de células madre neurales con un potencial de paso promedio de 3,17, alcanzando incluso nueve pasajes. Las células recolectadas se sembraron en pocillos recubiertos de poli-D-lisina, donde crecieron como monocapas adherentes y, más raramente, como neuroesferas. Las células recién aisladas que eran positivas para GFAP también eran inmunopositivas para el marcador quiescente ID3 y tenían la característica de morfología bipolar NSE.La comparación inmunocitoquímica de las células derivadas de la biopsia y las derivadas de la autopsia mostró que el perfil de las células recolectadas era similar al de las células endógenas de la zona subependimaria. El factor de crecimiento dio lugar a un aumento concomitante y significativo de las células SOX2 y a una disminución de los neuroblastos positivos para doble cortina en ambas muestras.

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 ...