Name: Author Spotlight: Collecting Neural Stem and Progenitor Cells from Live Animals Using a Novel Brain Milking Protocol
Uploaded: 2024-02-09T12:30:00
Description: Watch this Scientific Journal Video about Collecting Neural Stem and Progenitor Cells from Live Animals Using a Novel Brain Milking Protocol at JoVE.com

This work was supported by an Action Medical Research (UK) grant (GN2291) to R.J.M.F. and I.K. The research work was also partly supported (animal costs and support to D.D) by the Hellenic Foundation for Research and Innovation (H.F.R.I.) under the &#34;First Call for H.F.R.I. Research Projects to support Faculty members and Researchers and the procurement of high-cost research equipment grant&#34; (Project Number: 3395).

Animal breeding, maintenance, and experimental procedures were conducted in accordance with the UK Animals (Scientific Procedures) Act 1986, authorized by the Home Office, and with the Presidential Decree 56/2013 of the Hellenic Republic, scrutinized by the Animal Welfare and Ethical Review Bodies of the Universities of Cambridge and Patras, as well as approved and scrutinized by the local Prefectural Animal Care and Use Committee (Protocol number: 5675/39/18-01-2021). Male and female Sprague-Dawley, Wistar, and Long-Eva...

Rat "Brain Milking" for Neural Stem and Oligodendrocyte Progenitor Cell Isolation

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Stem and progenitor cells are relatively sparse in mammalian brain tissue. In addition, NSCs are located in areas inaccessible for easy and safe biopsies (ventricular walls, hippocampus). Therefore, the only way to work experimentally with such cells, so far, has been their postmortem isolation. A method allowing the single or repeated collection of NSCs and OPCs from live rats, named milking, is described here step by step. The method is based on two key features: i) NSCs or OPCs are separated by the ependymal cell mono...

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.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Release cocktail</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>&#946;1-integrin-blocking antibody</td>
			<td>BD Biosciences</td>
			<td>#555002</td>
			<td>purified NA/LE Hamster Anti-Rat CD29 Clone Ha2/5, 1 mg/mL. Any abntibody with blocking activity should be appropriate.</td>
		</tr>
		<tr>
			<td>Neuraminidase from Clostridium perfringens (Clostridium welchii)</td>
			<td>Sigma-Aldrich</td>
			<td>#N2876</td>
			<td>Neuraminidases fromother sources (e.g., from Vibrio cholerae) have not been tested.</td>
		</tr>
		<tr>
			<td>Recombinant Human FGF-basic (154 a.a.)</td>
			<td>Peprotech</td>
			<td>#100-18B</td>
			<td>kept as a 1 &#956;g/&#956;L stock, diluted in sterile water at -20 &#176;C</td>
		</tr>
		<tr>
			<td>Surgical procedures</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>10 &#181;L Syringe</td>
			<td>Hamilton</td>
			<td>#80330</td>
			<td>Model 701 RN, Small Removable Needle, 26s gauge, 2 in., point style 2</td>
		</tr>
		<tr>
			<td>BD Micro-fine 1 mL insulin syringes</td>
			<td>BD biosciences</td>
			<td>04085-00</td>
			<td>29 G x 12.7 mm</td>
		</tr>
		<tr>
			<td>BETADINE CUT.SOL 10% FLx30ML</td>
			<td>LAVIPHARM-CASTALIA</td>
			<td>SKU: 5201048131168</td>
			<td></td>
		</tr>
		<tr>
			<td>Bupaq</td>
			<td>RICHTERPHARMA</td>
			<td>1021854AF</td>
			<td>10 mL (buprenophine 0.3 mg/mL)</td>
		</tr>
		<tr>
			<td>Digital New Standard Stereotaxic, Rat and Mouse</td>
			<td>Stoelting</td>
			<td>51500D</td>
			<td></td>
		</tr>
		<tr>
			<td>Homeothermic Monitoring System</td>
			<td>Harvard Apparatus</td>
			<td>55-7020</td>
			<td></td>
		</tr>
		<tr>
			<td>ISOFLURIN 1,000 mg/g inhalation vapour, liquid</td>
			<td>Vetpharma Animal Health</td>
			<td>32509/4031</td>
			<td></td>
		</tr>
		<tr>
			<td>Ketamidor</td>
			<td>RICHTER PHARMA</td>
			<td>SKU: 9004114002531</td>
			<td>Ketamine 100 mg/mL</td>
		</tr>
		<tr>
			<td>Nylon suture, Ethilon</td>
			<td>Ethicon</td>
			<td>D9635</td>
			<td>Clear , size 5-0</td>
		</tr>
		<tr>
			<td>Rechargeable Cordless Surgical Trimmers</td>
			<td>Stoelting</td>
			<td>Item:51472</td>
			<td></td>
		</tr>
		<tr>
			<td>Scalpel blades, sterile</td>
			<td>Swann Morton</td>
			<td>AW050</td>
			<td></td>
		</tr>
		<tr>
			<td>Scopettes Jr.&#160; 8-inch Swabs</td>
			<td>Birchwood Laboratories</td>
			<td>34-7021-12P</td>
			<td></td>
		</tr>
		<tr>
			<td>Stereotaxic High Speed Drill</td>
			<td>Foredom</td>
			<td>1474w/o1464</td>
			<td></td>
		</tr>
		<tr>
			<td>Stoelting&#8217;s Stereotaxic Instrument Kit</td>
			<td>Stoelting</td>
			<td>Item: 52189</td>
			<td></td>
		</tr>
		<tr>
			<td>Xylan 2%</td>
			<td>Chanelle Pharmaceuticals</td>
			<td>13764/03/19-5-2004</td>
			<td>Xylazine, 25 mL</td>
		</tr>
		<tr>
			<td>Tissue and cells handling and immunostainings</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>96-well plates appropriate for microscopy</td>
			<td>Greiner</td>
			<td>#655866</td>
			<td>Screen star microplate</td>
		</tr>
		<tr>
			<td>B27 supplement</td>
			<td>ThermoFisher Scientific</td>
			<td>A1486701</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin (BSA)</td>
			<td>Merck</td>
			<td>P06-1391100</td>
			<td>Fraction V, heat shock</td>
		</tr>
		<tr>
			<td>Citrate</td>
			<td>Merck</td>
			<td>71497</td>
			<td>Sodium citrate monobasic</td>
		</tr>
		<tr>
			<td>Cryostat</td>
			<td>Leica</td>
			<td>CM1510S</td>
			<td></td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>Merck, Calbiochem</td>
			<td>28718-90-3</td>
			<td>Nuclear staining, Dilution: 1/1,000</td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>ThermoFisher Scientific</td>
			<td>11995065</td>
			<td>High glucose, pyruvate</td>
		</tr>
		<tr>
			<td>donkey anti-goat</td>
			<td>Biotium</td>
			<td>20016 or 20106 or 20048</td>
			<td>Dilution: 1/1,000</td>
		</tr>
		<tr>
			<td>donkey anti-mouse</td>
			<td>Biotium</td>
			<td>20014 or 20105 or 20046</td>
			<td>Dilution: 1/1,000</td>
		</tr>
		<tr>
			<td>donkey anti-rabbit</td>
			<td>Biotium</td>
			<td>20015 or 20098 or 20047</td>
			<td>Dilution: 1/1,000</td>
		</tr>
		<tr>
			<td>EGF</td>
			<td>Peprotech</td>
			<td>315-09</td>
			<td></td>
		</tr>
		<tr>
			<td>FGF-2 (or bFGF)</td>
			<td>Peprotech</td>
			<td>100-18B</td>
			<td></td>
		</tr>
		<tr>
			<td>goat anti-GFAP</td>
			<td>Abcam</td>
			<td>ab53554</td>
			<td>Dilution: 1/500</td>
		</tr>
		<tr>
			<td>goat anti-SOX2</td>
			<td>Santa Cruz Biotecnology</td>
			<td>sc-17320</td>
			<td>Dilution: 1/200</td>
		</tr>
		<tr>
			<td>mouse anti-ID3</td>
			<td>Santa Cruz Biotecnology</td>
			<td>sc-56712</td>
			<td>Dilution: 1/200</td>
		</tr>
		<tr>
			<td>mouse anti-S100&#946;</td>
			<td>Sigma</td>
			<td>S2532</td>
			<td>Dilution: 1/200</td>
		</tr>
		<tr>
			<td>Mowiol</td>
			<td>Merck, Calbiochem</td>
			<td>475904</td>
			<td>Mounting medium</td>
		</tr>
		<tr>
			<td>N2 supplement</td>
			<td>ThermoFisher Scientific</td>
			<td>17502048</td>
			<td></td>
		</tr>
		<tr>
			<td>Parafolmadehyde</td>
			<td>Merck</td>
			<td>158127</td>
			<td></td>
		</tr>
		<tr>
			<td>Poly-D-Lysine</td>
			<td>Merck, Millipore</td>
			<td>A-003-E</td>
			<td>Solution, 1.0 mg/mL</td>
		</tr>
		<tr>
			<td>rabbit anti-Doublecortin (DCX)</td>
			<td>Abcam</td>
			<td>ab18723</td>
			<td>Dilution: 1/500</td>
		</tr>
		<tr>
			<td>rabbit anti-PDGFR&#945;</td>
			<td>Abcam</td>
			<td>ab51875</td>
			<td>Dilution: 1/200</td>
		</tr>
		<tr>
			<td>rabbit anti-&#946;- catenin</td>
			<td>Abcam</td>
			<td>ab16051</td>
			<td>Dilution: 1/500</td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Merck</td>
			<td>X100</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscopy and image analysis</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Confocal microscope</td>
			<td>Leica</td>
			<td>SP6 and SP8</td>
			<td></td>
		</tr>
		<tr>
			<td>Image analysis</td>
			<td>NIH, USA</td>
			<td>ImageJ</td>
			<td></td>
		</tr>
		<tr>
			<td>Image analysis</td>
			<td>Leica</td>
			<td>LasX</td>
			<td></td>
		</tr>
	</tbody>
</table>

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 neurons and glia. One such niche is the subependymal zone (SEZ; also called the ventricular-subventricular zone), which is located across the lateral walls of the lateral ventricles, adjacent to the ependymal cell layer. Oligodendrocyte progenitor cells (OPCs) are abundantly distributed throughout the central nervous system, constituting a pool of proliferative progenitor cells that can generate oligodendrocytes.
Both NSCs and OPCs exhibit self-renewal potential and quiescence/activation cycles. Due to their location, the isolation and experimental investigation of these cells is performed postmortem. Here, we describe in detail &#34;brain milking&#34;, a method for the isolation of NSCs and OPCs, amongst other cells, from live animals. This is a two-step protocol designed for use in rodents and tested in rats. First, cells are &#34;released&#34; from the tissue via stereotaxic intracerebroventricular (i.c.v.) injection of a &#34;release cocktail&#34;. The main components are neuraminidase, which targets ependymal cells and induces ventricular wall denudation, an integrin-&#946;1-blocking antibody, and fibroblast growth factor-2. At a second &#34;collection&#34; step, liquid biopsies of cerebrospinal fluid are performed from the cisterna magna, in anesthetized rats without the need of an incision.
Results presented here show that isolated cells retain their endogenous profile and that NSCs of the SEZ preserve their quiescence. The denudation of the ependymal layer is restricted to the anatomical level of injection and the protocol (release and collection) is tolerated well&#160; by the animals. This novel approach paves the way for performing longitudinal studies of endogenous neurogenesis and gliogenesis in experimental animals.

Author Spotlight: Collecting Neural Stem and Progenitor Cells from Live Animals Using a Novel Brain Milking Protocol 

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

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.

Release and collection of NSCs 
NSCs of the SEZ are separated from the CSF only by the monolayer of ependymal cells, albeit they remain in direct contact with the ventricular content via intercalating mono-ciliated processes8,9. Neuraminidase acts specifically on ependymal cells via cleavage of sialic acid residues and can induce denudation of the ventricular wall. This leads to neuroblast clustering on the surface of the v...

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A method for the isolation of neural stem cells and oligodendrocyte progenitor cells from the brains of live rats is presented here in experimental detail. It allows multiple collections of these cells from the same animals without compromising their well-being.

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