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-Evans rats, with ages varying between 2 and 4 months and with body weights between 150 g and 250 g, were used. The protocol is graphically summarized in Figure 1.
1. Release cocktail preparation
NOTE: Prepare fresh on the day of the procedure and keep on ice. The quantities are given per 2 &#181;L to be i.c.v. injected in each lateral ventricle. Prepare an additional 1 &#181;L per intended injection.
<ol>
	<li>Prepare 0.5 &#181;L of 500 mU neuraminidase from Clostridium perfringens (Clostridium welchii). 
	NOTE: Store this as a 1 U/&#181;L stock diluted in sterile water at -20 &#176;C. Other types of neuraminidases have not been tested.</li>
	<li>Prepare 1 &#181;L containing 1 &#181;g of integrin-&#946;1-blocking antibody. 
	NOTE: Store it at 4 &#176;C.</li>
	<li>Prepare 0.5 &#181;L containing 0.5 &#181;g of basic fibroblast growth factor (recombinant human FGF-basic). 
	&#8203;NOTE: Store it as a 1 &#181;g/&#181;L stock diluted in sterile water at -20 &#176;C.</li>
</ol>
2. Injection of release cocktail
NOTE: The whole process can be performed within 20 min. Take care to perform the surgery in aseptic conditions. Clean all surfaces with antiseptic (e.g., 3% or 6% hydrogen peroxide). Use autoclaved or readily sterile tools, gloves, gowns, and drapes.
<ol>
	<li>Anesthetize the experimental animal by isoflurane inhalation (2.5% for induction and 2% for maintenance). Confirm the depth of anesthesia by checking the corneal reflex, the reaction to stimuli (hind limb and tail pinch test), and the mode of breathing. 
	NOTE: Surgical procedures can be performed under general anesthesia induced by intraperitoneally injectable anesthesia (e.g., ketamine [40 mg/kg] and xylazine [10 mg/kg]). Deep anesthesia was confirmed by checking the corneal reflex, the reaction to stimuli (hind limb and tail pinch test), and the mode of breathing.</li>
	<li>Administer analgesia subcutaneously (e.g., 0.3 mg/mL buprenorphine) upon the induction of anesthesia.</li>
	<li>Mount the rat on the stereotaxic frame.</li>
	<li>Shave the fur of the head with a razor and clean the skin with antiseptic, such as 10% povidone-iodine solution and then alcohol. Apply the antiseptic three times&#160;using sterile cotton swabs. Rub in a circular motion for 3-5 s each time. Apply eye ointment to prevent dryness.</li>
	<li>Make a 2 cm incision in the head&#39;s skin along the middle line using a sterile scalpel.</li>
	<li>Meticulously clear the skull using sterile swabs and sterile saline.</li>
	<li>Identify the bregma using the edge of the needle of a 10 &#181;L Hamilton syringe mounted on the stereotaxic device. Set the bregma as &#34;point 0,0&#34;. 
	NOTE: The bregma is identified as the point of intersection between the sagittal and the coronal sutures. More details can be found in the rat brain atlas of Paxinos and Watson5.</li>
	<li>Move the device to the coordinates anterioposterior (AP) = 0.3 mm, lateral (L) = +1.2 mm (to target the SEZ), or AP = 1.5 mm, L = +2.0 mm (to target the CC).</li>
	<li>Drill a 1 mm burr hole using a dental drill. 
	NOTE: When drilling by hand, take care not to injure the cortical surface. The hemorrhage is not expected to be considerable. Clear any blood with saline and apply pressure to stop more persistent bleeding.</li>
	<li>Load 4 &#181;L of the release cocktail in the 10 &#181;L Hamilton syringe.</li>
	<li>Lower the needle (preferably blunt or conical edge) of the Hamilton syringe in order to get in contact with the dura.</li>
	<li>Insert the needle at the desired depth (D = 3.5 mm). 
	NOTE: Pour saline on the surface of the dura to keep the tissue hydrated.</li>
	<li>Infuse 2 &#181;L of the release cocktail at a rate of 1 &#181;L/min.</li>
	<li>Leave the needle in place for another 2 min before slow retrieval to avoid surfacing of the release cocktail.</li>
	<li>Repeat steps 2.8-2.14 for the other hemisphere at the coordinates AP = 0.3 mm, L = -1.2 mm (to target the SEZ), or AP = 1.5 mm, L = -2.0 mm (to target the CC).</li>
	<li>Suture the incision with 5-0 nylon (diameter 0.1 mm) and clean the sutured area with antiseptic.</li>
	<li>Remove the mask supplying the anesthetic and transfer the animal to the post operation monitoring area. 
	NOTE: Recovery from anesthesia and maintenance of recumbency should occur within 5-10 min, and full recovery (normal behavior) within 25 min.
	<ol>
		<li>Closely monitor the recovery (vivid and uninterrupted movement, frequent access to water) in a well heated (24-25 &#176;C), quiet space without small-particle bedding, which can block the airways. Return the animal to the company of its cage mates (not to new animals) only after confirming full recovery.</li>
		<li>Monitor the animal at the maintenance facility for at least 48 h after surgery. Address signs of pain (hiding of head, abnormal head or body posture, hypersensitivity, and hyperexcitability to handling) by administering analgesia (e.g., 0.3 mg/mL buprenorphine, subcutaneously). Refer animals with discolored or erected fur to the responsible vet.</li>
	</ol>
	</li>
</ol>
3. Cerebrospinal fluid (CSF) liquid biopsy
NOTE: The whole process can be performed within 10 min. The liquid biopsy described here is performed 3 days post injection of the release cocktail but can be performed in exactly the same way whenever required. Take care to perform the surgery in aseptic conditions. Clean all the surfaces with antiseptic (e.g., 3% or 6% hydrogen peroxide). Use autoclaved or readily sterile tools, gloves, gowns, and drapes.
<ol>
	<li>Anesthetize the animal by isoflurane inhalation (2.5% for induction and 2% for maintenance). Confirm the depth of anesthesia by checking the corneal 
	reflex, the reaction to stimuli (hind limb and tail pinch test), and the mode of breathing. 
	NOTE: The surgical procedures can be performed under general anesthesia induced by intraperitoneally injectable anesthesia (e.g., ketamine [40 mg/kg] and xylazine [10 mg/kg]). However, this is not advisable as the procedure is short, especially if biopsies will be performed multiple times on consecutive days.</li>
	<li>Administer analgesia subcutaneously (e.g., 0.3 mg/mL buprenorphine) upon the induction of anesthesia.</li>
	<li>Mount the experimental animal on the stereotaxic frame. Use ear bars to fix the head without obstructing rotational movement toward the front and the back.</li>
	<li>Stabilize the head at a downward 40&#176; angle so that a good extension of the back of the neck can be achieved. 
	NOTE: One way to do this is by using the mouth bar of the device6.</li>
	<li>Shave the fur in the neck area using a razor and clean the skin with antiseptic, such as 10% povidone-iodine solution and then alcohol. Apply the antiseptic three times&#160;using sterile cotton swabs. Rub in a circular motion for 3-5 s each time.</li>
	<li>With the fingertip, find the rhomb-shaped depressible area positioned in between the occipital protuberance and the spine of the atlas6.</li>
	<li>Draw a point-mark (e.g., using a marker).</li>
	<li>Fix a 1 mL or 0.5 mL syringe on the stereotaxic frame. 
	NOTE: It is advisable to use syringes with non-detachable needles as they produce better suction and have less dead volume.</li>
	<li>Bring the needle at the point of contact with the skin at the point-mark.</li>
	<li>With a pair of forceps, lift the skin while lowering the syringe through the skin layers.</li>
	<li>Retrieve the plunger slightly to generate negative pressure in the syringe.</li>
	<li>Restart to dip the needle very slowly until liquid (CSF) appears in the syringe.</li>
	<li>Settle the needle at this position and allow the slow flow of CSF. 
	NOTE: The needle can be lowered or elevated slightly if CSF flow is very slow.</li>
	<li>Start removing the CSF slowly (in steps of 40 &#181;L/min) up to 120 &#181;L.</li>
	<li>Slowly retrieve the syringe.</li>
	<li>Intraperitoneally administer 1 mL of normal serum to support the experimental animal&#39;s replenishment of fluids.</li>
	<li>Mix the liquid biopsy (CSF) with 400 &#181;L of NSC medium and keep the microcentrifuge tube at 4 &#176;C until further use. 
	NOTE: Liquid biopsies should be processed within 3 h if not frozen.</li>
	<li>Remove the mask that supplies the anesthetic and transfer the animal to the post operation monitoring area. 
	NOTE: Recovery from anesthesia and maintenance of recumbency should occur within 5-10 min, and full recovery (normal behavior) within 25 min.
	<ol>
		<li>Closely monitor the recovery (vivid and uninterrupted movement, frequent access to water) in a well heated (24-25 &#176;C), quiet space without small-particle bedding, which can block the airways. Return the animal to the company of its cage mates (not to new animals) only after full recovery has been confirmed.</li>
		<li>Monitor the animal at the maintenance facility for at least 48 h after surgery. If signs of pain (hiding of head, abnormal head or body posture, hypersensitivity, and hyperexcitability to handling) are observed, administer analgesia (e.g., 0.3 mg/mL buprenorphine subcutaneously). Refer animals with discolored or erected fur to the responsible vet.</li>
	</ol>
	</li>
</ol>
4. Processing of tissue for immunofluorescence
<ol>
	<li>Euthanize the animal by intracardial infusion of 20 mL of ice-cold saline, followed by 50 mL of ice-cold 4% paraformaldehyde (PFA; in phosphate-buffered saline [PBS] of pH = 7.4).</li>
	<li>Dissect the brain tissue.
	<ol>
		<li>Separate the head from the body using scissors to make a cut in the cervical area. Use the scissors to remove the skin and then cut the skull&#39;s bone along the sagittal midline, with the tips of the scissors pointing upward in order not to damage the brain tissue. Aim to continue cutting as much as possible, passing the coronal midline.</li>
		<li>Use forceps to remove the bones, starting from the supraoccipital and the parietal bones and finally the frontal bones.</li>
		<li>Once the brain tissue is revealed, use the forceps or a spatula to lift it out of the skull. To facilitate this, cut the nerves at the base of the brain and the olfactory bulbs, if not wanted.</li>
	</ol>
	</li>
	<li>Post-fix the tissue overnight in 2% PFA at 4 &#176;C.</li>
	<li>Cryo-protect the tissue in 30% sucrose (in PBS) for at least 48 h at 4 &#176;C until the tissue sinks to the bottom.</li>
	<li>Freeze the tissue at -80 &#176;C.</li>
	<li>Cut the brain tissue into 14 &#956;m thick coronal sections with a cryostat.</li>
	<li>Perform immunofluorescence staining using standard protocols 3,7
	<ol>
		<li>Incubate the sections with blocking buffer (3% bovine serum albumin [BSA], 0.1% Triton X-100, in PBS) for 2 h at room temperature.</li>
		<li>Perform antigen retrieval (boiling for 15 min in 10 mM citrate buffer, pH = 6.0, using glass jars). 
		&#8203;NOTE: This step is optional, but necessary for nuclear antigens.</li>
		<li>Bring to room temperature and incubate with primary antibodies against glial fibrillary acidic protein (GFAP), doublecortin (Dcx), S100&#946;, and &#946;-catenin (see the Table of Materials) in blocking buffer overnight at 4 &#176;C.</li>
		<li>Incubate for 2 h with secondary antibodies in PBS with 4&#39;,6-diamidino-2-phenylindole (DAPI) for nuclear counterstaining at room temperature (see the Table of Materials).</li>
		<li>Mount the coverslips.</li>
	</ol>
	</li>
</ol>
5. Processing isolated cells for immunofluorescence
<ol>
	<li>Plate the cells into 96-well plates compatible for microscopy, coated with poly-D-lysine (100 &#181;g/mL).</li>
	<li>Fix the cells in ice-cold 2% PFA for 10 min and wash 3x with PBS.</li>
	<li>Perform immunofluorescence using standard procedures3,7for PDGFR&#945;, GFAP, Dcx, SOX2, and ID3 (see the Table of Materials).</li>
	<li>Keep the cells in PBS + NaN3 (0.1%) at 4 &#176;C in the dark.</li>
</ol>
6. Microscopy and image analysis
<ol>
	<li>Take images using epifluorescence or confocal microscopy and perform cell counts using standard image analysis software tools, such as cell counters.</li>
</ol>

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

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The authors have no competing financial interests or other conflicts of interest to declare.

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><tbody><tr><td>&lt;strong&gt;Release cocktail&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>&amp;beta;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 &lt;em&gt;Clostridium perfringens&lt;/em&gt; (&lt;em&gt;Clostridium welchii&lt;/em&gt;)</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 &amp;mu;g/&amp;mu;L stock, diluted in sterile water at -20 &amp;deg;C</td></tr><tr><td>&lt;strong&gt;Surgical procedures&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>10 &amp;micro;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.&amp;nbsp; 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&amp;rsquo;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>&lt;strong&gt;Tissue and cells handling and immunostainings&lt;/strong&gt;</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&amp;beta;</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&amp;alpha;</td><td>Abcam</td><td>ab51875</td><td>Dilution: 1/200</td></tr><tr><td>rabbit anti-&amp;beta;- 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>&lt;strong&gt;Microscopy and image analysis&lt;/strong&gt;</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.

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 ven...

Watch this Scientific Journal Video about Collecting Neural Stem and Progenitor Cells from Live Animals Using a Novel Brain Milking Protocol at JoVE.com

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

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.

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 ...

the-brain-milking-method-for-isolation-neural-stem-cells

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Neuroscience

1.9K Views.  University of Patras. We werken met neurale stamcellen in de hersenen, zeer schaarse cellen die clusteren in gespecialiseerde micro-omgevingen die niches worden genoemd. Hoewel ze bijdragen aan reukzin, geheugen en leren, zijn ze uiterst inefficiënt in het stimuleren van regeneratie. Daarom onderzoeken we hun eigenschappen en manieren om ze te manipuleren om de genezing van het zenuwweefsel te vergroten.We hebben aangetoond dat de bijdrage van neurale stamcellen in de hersenen aan regeneratie wordt beperkt...

Video: Auteur in de schijnwerpers: neurale stam- en voorlopercellen verzamelen van levende dieren met behulp van een nieuw hersenmelkprotocol 

Isolation and Expansion of the Adult Mouse Neural Stem Cells Using the Neurosphere Assay

Isolation and expansion of the putative neural stem cells (NSCs) from the adult murine brain was first described by Reynolds and Weiss in 1992 employing a chemically defined serum-free culture system known as the neurosphere assay (NSA). In this assay, the majority of differentiated cell types die within a few days of culture but a small population of growth factor responsive precursor cells undergo active proliferation in the presence of epidermal growth factor (EGF) and/ basic fibroblastic growth factor (bFGF). These cells form colonies of undifferentiated cells called neurospheres, which in turn can be subcultured to expand the pool of neural stem cells. Moreover, the cells can be induced to differentiate, generating the three major cell types of the CNS i.e. neurons, astrocytes, and oligodendrocytes. This assay provides an invaluable tool to supply a consistent, renewable source of undifferentiated CNS precursors, which could be used for in vitro studies and also for therapeutic purposes.
This video demonstrates the NSA method to generate and expand NSCs from the adult mouse periventricular region, and provides technical insights to ensure one can achieve reproducible neurosphere cultures. The procedure includes harvesting the brain from the adult mouse, micro-dissection of the periventricular region, tissue preparation and culture in the NSA. The harvested tissue is first chemically digested using trypsin-EDTA and then mechanically dissociated in NSC medium to achieve a single cell suspension and finally plated in the NSA. After 7-10 days in culture, the resulting primary neurospheres are ready for subculture to reach the amount of cells required for future experiments.

This video protocol demonstrates the neurosphere assay method to generate and expand neural stem cells from the adult mouse periventricular region, and provides technical insights to ensure one can achieve reproducible neurosphere cultures.

Isolation and expansion of the putative neural stem cells (NSCs) from the adult murine brain was first described by Reynolds and Weiss in 1992 employing a ...

Time-lapse Live Imaging of Clonally Related Neural Progenitor Cells in the Developing Zebrafish Forebrain

Precise patterns of division, migration and differentiation of neural progenitor cells are crucial for proper brain development and function1,2. To understand the behavior of neural progenitor cells in the complex in vivo environment, time-lapse live imaging of neural progenitor cells in an intact brain is critically required. In this video, we exploit the unique features of zebrafish embryos to visualize the development of forebrain neural progenitor cells in vivo. We use electroporation to genetically and sparsely label individual neural progenitor cells. Briefly, DNA constructs coding for fluorescent markers were injected into the forebrain ventricle of 22 hours post fertilization (hpf) zebrafish embryos and electric pulses were delivered immediately. Six hours later, the electroporated zebrafish embryos were mounted with low melting point agarose in glass bottom culture dishes. Fluorescently labeled neural progenitor cells were then imaged for 36hours with fixed intervals under a confocal microscope using water dipping objective lens. The present method provides a way to gain insights into the in vivo development of forebrain neural progenitor cells and can be applied to other parts of the central nervous system of the zebrafish embryo.

The present video demonstrates a method which takes advantage of the combination of electroporation and confocal microscopy to perform live imaging on individual neural progenitor cells in the developing zebrafish forebrain. In vivo analysis of the development of forebrain neural progenitor cells at a clonal level can be achieved in this way.

Precise patterns of division, migration and differentiation of neural progenitor cells are crucial for proper brain development and function1,2. To ...

Neural-Colony Forming Cell Assay: An Assay To Discriminate Bona Fide Neural Stem Cells from Neural Progenitor Cells

The neurosphere assay (NSA) is one of the most frequently used methods to isolate, expand and also calculate the frequency of neural stem cells (NSCs). Furthermore, this serum-free culture system has also been employed to expand stem cells and determine their frequency from a variety of tumors and normal tissues. It has been shown recently that a one-to-one relationship does not exist between neurosphere formation and NSCs. This suggests that the NSA as currently applied, overestimates the frequency of NSCs in a mixed population of neural precursor cells isolated from both the embryonic and adult mammalian brain. This video practically demonstrates a novel collagen based semi- solid assay, the neural-colony forming cell assay (N-CFCA), which has the ability to discriminate stem from progenitor cells based on their long-term proliferative potential, and thus provides a method to enumerate NSC frequency. In the N-CFCA, colonies &ge;2 mm in diameter are derived from cells that meet all the functional criteria of a NSC, while colonies &lt; 2mm are derived from progenitors. The N-CFCA procedure can be used for cells prepared from different sources including primary and cultured adult or embryonic mouse CNS cells. Here we use cells prepared from passage one neurospheres generated from embryonic day 14 mice brain to perform N-CFCA. The cultures are replenished with proliferation medium every seven days for three weeks to allow the plated cells to exhibit their full proliferative potential and then the frequency of neural progenitor and bona fide neural stem cells is calculated respectively by counting the number of colonies that are &lt; 2mm and the ones that are &ge;2mm in reference to the number of cells that were initially plated.

This video protocol demonstrates how to discriminate and enumerate bona fide neural stem cells in a mixed population of neural precursor cells using the neural colony-forming cell assay.

The neurosphere assay (NSA) is one of the most frequently used methods to isolate, expand and also calculate the frequency of neural stem cells (NSCs). ...

Live-cell Imaging of Sensory Organ Precursor Cells in Intact Drosophila Pupae

Since the discovery of Green Fluorescent Protein (GFP), there has been a revolutionary change in the use of live-cell imaging as a tool for understanding fundamental biological mechanisms. Striking progress has been particularly evident in Drosophila, whose extensive toolkit of mutants and transgenic lines provides a convenient model to study evolutionarily-conserved developmental and cell biological mechanisms. We are interested in understanding the mechanisms that control cell fate specification in the adult peripheral nervous system (PNS) in Drosophila. Bristles that cover the head, thorax, abdomen, legs and wings of the adult fly are individual mechanosensory organs, and have been studied as a model system for understanding mechanisms of Notch-dependent cell fate decisions. Sensory organ precursor (SOP) cells of the microchaetes (or small bristles), are distributed throughout the epithelium of the pupal thorax, and are specified during the first 12 hours after the onset of pupariation. After specification, the SOP cells begin to divide, segregating the cell fate determinant Numb to one daughter cell during mitosis. Numb functions as a cell-autonomous inhibitor of the Notch signaling pathway.
Here, we show a method to follow protein dynamics in SOP cell and its progeny within the intact pupal thorax using a combination of tissue-specific Gal4 drivers and GFP-tagged fusion proteins 1,2.This technique has the advantage over fixed tissue or cultured explants because it allows us to follow the entire development of an organ from specification of the neural precursor to growth and terminal differentiation of the organ. We can therefore directly correlate changes in cell behavior to changes in terminal differentiation. Moreover, we can combine the live imaging technique with mosaic analysis with a repressible cell marker (MARCM) system to assess the dynamics of tagged proteins in mitotic SOPs under mutant or wildtype conditions. Using this technique, we and others have revealed novel insights into regulation of asymmetric cell division and the control of Notch signaling activation in SOP cells (examples include references 1-6,7 ,8).

Live-cell Imaging of Sensory Organ Precursor Cells in Intact Drosophila Pupae

In this video, we describe a method for live cell imaging of asymmetrically dividing sensory organ progenitor cells and epidermal cells in intact Drosophila pupae

Since the discovery of Green Fluorescent Protein (GFP), there has been a revolutionary change in the use of live-cell imaging as a tool for understanding ...

The Neuroblast Assay: An Assay for the Generation and Enrichment of Neuronal Progenitor Cells from Differentiating Neural Stem Cell Progeny Using Flow Cytometry

Neural stem cells (NSCs) can be isolated and expanded in large-scale, using the neurosphere assay and differentiated into the three major cell types of the central nervous system (CNS); namely, astrocytes, oligodendrocytes and neurons. These characteristics make neural stem and progenitor cells an invaluable renewable source of cells for in vitro studies such as drug screening, neurotoxicology and electrophysiology and also for cell replacement therapy in many neurological diseases. In practice, however, heterogeneity of NSC progeny, low production of neurons and oligodendrocytes, and predominance of astrocytes following differentiation limit their clinical applications. Here, we describe a novel methodology for the generation and subsequent purification of immature neurons from murine NSC progeny using fluorescence activated cell sorting (FACS) technology. Using this methodology, a highly enriched neuronal progenitor cell population can be achieved without any noticeable astrocyte and bona fide NSC contamination. The procedure includes differentiation of NSC progeny isolated and expanded from E14 mouse ganglionic eminences using the neurosphere assay, followed by isolation and enrichment of immature neuronal cells based on their physical (size and internal complexity) and fluorescent properties using flow cytometry technology. Overall, it takes 5-7 days to generate neurospheres and 6-8 days to differentiate NSC progeny and isolate highly purified immature neuronal cells.

This video protocol demonstrates a novel method for the generation and subsequent purification of neuronal progenitor cells from a renewable source of neural stem cells (NSCs) based on their physical (size and internal granularity) and fluorescent properties using flow cytometry technology.

Neural stem cells (NSCs) can be isolated and expanded in large-scale, using the neurosphere assay and differentiated into the three major cell types of ...

Isolation and Culture of Neural Crest Cells from Embryonic Murine Neural Tube

The embryonic neural crest (NC) is a multipotent progenitor population that originates at the dorsal aspect of the neural tube, undergoes an epithelial to mesenchymal transition (EMT) and migrates throughout the embryo, giving rise to diverse cell types 1-3. NC also has the unique ability to influence the differentiation and maturation of target organs4-6. When explanted in vitro, NC progenitors undergo self-renewal, migrate and differentiate into a variety of tissue types including neurons, glia, smooth muscle cells, cartilage and bone. 
NC multipotency was first described from explants of the avian neural tube7-9. In vitro isolation of NC cells facilitates the study of NC dynamics including proliferation, migration, and multipotency. Further work in the avian and rat systems demonstrated that explanted NC cells retain their NC potential when transplanted back into the embryo10-13. Because these inherent cellular properties are preserved in explanted NC progenitors, the neural tube explant assay provides an attractive option for studying the NC in vitro. 
To attain a better understanding of the mammalian NC, many methods have been employed to isolate NC populations. NC-derived progenitors can be cultured from post-migratory locations in both the embryo and adult to study the dynamics of post-migratory NC progenitors11,14-20, however isolation of NC progenitors as they emigrate from the neural tube provides optimal preservation of NC cell potential and migratory properties13,21,22. Some protocols employ fluorescence activated cell sorting (FACS) to isolate a NC population enriched for particular progenitors11,13,14,17. However, when starting with early stage embryos, cell numbers adequate for analyses are difficult to obtain with FACS, complicating the isolation of early NC populations from individual embryos. Here, we describe an approach that does not rely on FACS and results in an approximately 96% pure NC population based on a Wnt1-Cre activated lineage reporter23. 
The method presented here is adapted from protocols optimized for the culture of rat NC11,13. The advantages of this protocol compared to previous methods are that 1) the cells are not grown on a feeder layer, 2) FACS is not required to obtain a relatively pure NC population, 3) premigratory NC cells are isolated and 4) results are easily quantified. Furthermore, this protocol can be used for isolation of NC from any mutant mouse model, facilitating the study of NC characteristics with different genetic manipulations. The limitation of this approach is that the NC is removed from the context of the embryo, which is known to influence the survival, migration and differentiation of the NC2,24-28.

Isolation of embryonic neural crest from the neural tube facilitates the use of in vitro methods for studying migration, self-renewal, and multipotency of neural crest.

The embryonic neural crest (NC) is a multipotent progenitor population that originates at the dorsal aspect of the neural tube, undergoes an epithelial to ...

A cGMP-applicable Expansion Method for Aggregates of Human Neural Stem and Progenitor Cells Derived From Pluripotent Stem Cells or Fetal Brain Tissue

A cell expansion technique to amass large numbers of cells from a single specimen for research experiments and clinical trials would greatly benefit the stem cell community. Many current expansion methods are laborious and costly, and those involving complete dissociation may cause several stem and progenitor cell types to undergo differentiation or early senescence. To overcome these problems, we have developed an automated mechanical passaging method referred to as &#8220;chopping&#8221; that is simple and inexpensive. This technique avoids chemical or enzymatic dissociation into single cells and instead allows for the large-scale expansion of suspended, spheroid cultures that maintain constant cell/cell contact. The chopping method has primarily been used for fetal brain-derived neural progenitor cells or neurospheres, and has recently been published for use with neural stem cells derived from embryonic and induced pluripotent stem cells. The procedure involves seeding neurospheres onto a tissue culture Petri dish and subsequently passing a sharp, sterile blade through the cells effectively automating the tedious process of manually mechanically dissociating each sphere. Suspending cells in culture provides a favorable surface area-to-volume ratio; as over 500,000 cells can be grown within a single neurosphere of less than 0.5 mm in diameter. In one T175 flask, over 50 million cells can grow in suspension cultures compared to only 15 million in adherent cultures. Importantly, the chopping procedure has been used under current good manufacturing practice (cGMP), permitting mass quantity production of clinical-grade cell products.

This protocol describes a novel mechanical chopping method that allows the expansion of spherical neural stem and progenitor cell aggregates without dissociation to a single cell suspension.&#160; Maintaining cell/cell contact allows rapid and stable growth for over 40 passages.

A cell expansion technique to amass large numbers of cells from a single specimen for research experiments and clinical trials would greatly benefit the ...

Feeder-free Derivation of Neural Crest Progenitor Cells from Human Pluripotent Stem Cells

Human pluripotent stem cells (hPSCs) have great potential for studying human embryonic development, for modeling human diseases in the dish and as a source of transplantable cells for regenerative applications after disease or accidents. Neural crest (NC) cells are the precursors for a large variety of adult somatic cells, such as cells from the peripheral nervous system and glia, melanocytes and mesenchymal cells. They are a valuable source of cells to study aspects of human embryonic development, including cell fate specification and migration. Further differentiation of NC progenitor cells into terminally differentiated cell types offers the possibility to model human diseases in vitro, investigate disease mechanisms and generate cells for regenerative medicine. This article presents the adaptation of a currently available in vitro differentiation protocol for the derivation of NC cells from hPSCs. This new protocol requires 18 days of differentiation, is feeder-free, easily scalable and highly reproducible among human embryonic stem cell (hESC) lines as well as human induced pluripotent stem cell (hiPSC) lines. Both old and new protocols yield NC cells of equal identity.

Neural crest (NC) cells derived from human pluripotent stem cells (hPSC) have great potential for modeling human development and disease and for cell replacement therapies. Here, a feeder-free adaptation of the currently widely used in vitro differentiation protocol for the derivation of NC cells from hPSCs is presented.

Human pluripotent stem cells (hPSCs) have great potential for studying human embryonic development, for modeling human diseases in the dish and as a ...

Cell Sorting of Neural Stem and Progenitor Cells from the Adult Mouse Subventricular Zone and Live-imaging of their Cell Cycle Dynamics

Neural stem cells (NSCs) in the subventricular zone of the lateral ventricles (SVZ) sustain olfactory neurogenesis throughout life in the mammalian brain. They successively generate transit amplifying cells (TACs) and neuroblasts that differentiate into neurons once they integrate the olfactory bulbs. Emerging fluorescent activated cell sorting (FACS) techniques have allowed the isolation of NSCs as well as their progeny and have started to shed light on gene regulatory networks in adult neurogenic niches. We report here a cell sorting technique that allows to follow and distinguish the cell cycle dynamics of the above-mentioned cell populations from the adult SVZ with a LeX/EGFR/CD24 triple staining. Isolated cells are then plated as adherent cells to explore in details their cell cycle progression by time-lapse video microscopy. To this end, we use transgenic Fluorescence Ubiquitination Cell Cycle Indicator (FUCCI) mice in which cells are red-fluorescent during G1 phase due to a G1 specific red-Cdt1 reporter. This method has recently revealed that proliferating NSCs progressively lengthen their G1 phase during aging, leading to neurogenesis impairment. This method is easily transposable to other systems and could be of great interest for the study of the cell cycle dynamics of brain cells in the context of brain pathologies.

We report a Fluorescent Activated Cell Sorting (FACS)-based method to isolate neural stem cells (NSCs) and their progeny from the subventricular zone (SVZ) of the adult mouse brain. Applied to Fluorescence Ubiquitination Cell Cycle Indicator (FUCCI) transgenic mice, it allows the study of cell cycle progression by live imaging.

Neural stem cells (NSCs) in the subventricular zone of the lateral ventricles (SVZ) sustain olfactory neurogenesis throughout life in the mammalian brain. ...

Isolation and Culture of Adult Neural Stem Cells from the Mouse Subcallosal Zone

Adult neural stem cells (aNSCs) can be used for the regeneration of damaged brain tissue. NSCs have the potential for differentiation and proliferation into three types of cells: neurons, astrocytes, and oligodendrocytes. Identifying aNSC-derived regions and characterizing the aNSC properties are critical for the potential use of aNSCs and for the elucidation of their role in neural regeneration. The subcallosal zone (SCZ), located between white matter and the hippocampus, has recently been reported to contain aNSCs and continuously give rise to neuroblasts. A low percentage of aNSCs from the SCZ is differentiated into neurons; most cells are differentiated into glial cells, such as oligodendrocytes and astrocytes. These cells are suggested to have a therapeutic potential for traumatic cortical injury. This protocol describes in detail the process to generate SCZ-aNSCs from an adult mouse brain. A brain matrix with intervals of 1 mm is used to obtain the SCZ-containing coronal slices and to precisely dissect the SCZ from the whole brain. The SCZ sections are initially subjected to a neurosphere culture. A well-developed culture system allows for the verification of their characteristics and can increase research on NSCs. A neurosphere culture system provides a useful tool for determining proliferation and collecting the genuine NSCs. A monolayer culture is also an in vitro system to assay proliferation and differentiation. Significantly, this culture system provides a more homogenous environment for NSCs than the neurosphere culture system. Thus, using a discrete brain region, these culture systems will be helpful for expanding our knowledge about aNSCs and their applications for therapeutic uses.

Establishing culture systems for the expansion of adult neural stem cells (aNSCs) allows for the examination and application of aNSCs for therapy. The subcallosal zone (SCZ) has recently been recognized as a novel neuroblast-forming region in adult mice. Here, methods for the isolation, expansion, and differentiation of SCZ-aNSCs are described.

Adult neural stem cells (aNSCs) can be used for the regeneration of damaged brain tissue. NSCs have the potential for differentiation and proliferation ...

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

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