1.	Preparation
<ol>
	<li>Prepare Xylazine-ketamine solution (Ketamine is a controlled substance. Detailed records should be kept and solutions stored in a safe, locked location).</li>
	<li>Clean and sterilize equipment.</li>
	<li>Prepare surgery area by wiping with aseptic agent and then covering with sterile paper toweling, and set up the micromanipulator.</li>
</ol>
2.	Preparation of Cells for Transplant
<ol>
<li>Cells should be resuspended to a concentration of 100,000 cells/&Eacute; l and, although only 250,000 cells are transplanted per mouse, an
excess of cells is required for syringe loading purposes (prepare at
least 300,000 cells per mouse receiving cells).</li>
<li>Wash cells to be transplanted 3 times in HBSS in a 50 ml conical vial. Count the cells before the final spin.</li>
<li>After the final spin down, decant HBSS and leave vial in upside down position to prevent droplets from reaching the pellet.</li>
<li>Dry inside walls with a UV-irradiated, sterile Kimwipe. Do not allow the pellet to dry.</li>
<li>Holding the tube upright, slowly and very delicately resuspend the cells in half the final desired volume of HBSS.</li>
<li>Measure the total volume by pipetting the majority of the suspension into a pipette tip and then adjusting the dial on the pipetter until the entire suspension is in the pipette tip. This will tell you how much more HBSS is needed for the desired concentration.</li>
<li>Bring volume to the desired amount by adding HBSS.</li>
<li>Place back on ice. Check viability of cells if they must be on ice for longer than 2 hrs.</li>
</ol>
3.	Preparation of mice for surgery and transplantation
<ol>
	<li>Anesthetize the mouse by intraperitoneal injection of ketamine (100mg/kg) and xylazine (10mg/kg) in ~100 &mu;l doses or an equivalent anesthetic. The entire procedure from surgical prep to suturing will take 30-40 minutes.</li> 
	<li>(Optional: apply numbered, colored tape to the tail of each mouse to ensure identification)</li>
	<li>Shave the dorsal area of the mouse from the lower back to the neck, and extending 2 cm bilateral from the midline, with electric clippers. The hair should be cut as close as possible (it may be necessary to go over the area several times).</li>
	<li>To remove the remaining hair, apply a thin layer of hair removal cream (Nair) with a gauze tipped applicator.</li>
	<li>After 1-2 minutes, wipe Nair off with gauze wetted lightly with soapy water. The prepared area should be clean bare skin without any stray pieces of hair that could get into the wound during the subsequent surgical procedure. 
Sterilize the prepared area with iodide solution.</li>
</ol>
4.	Laminectomy
<ol>
	<li>Frequently change and/or sterilize gloves throughout the procedure. Position the mouse dorsal side up with head pointing to the left (if you are right handed). Drape animal to ensure sterility and that only the shaved area is exposed. Make a vertical incision (&tilde;1.3cm) over the laminecomy site spanning from about thoracic vertebrae T8 to T12.</li>
	<li>With the graefe forceps held in the left hand, firmly secure the spinal column at T9 (Figure 1A) and lift the mouse up to exaggerate the spinal curvature.</li>
	<li>Use the scalpel to score the junction between T10 and T11, the space between the two spiny protrusions. Further expose the junction by carefully scrapping the muscle layer away to expose the bone (see Figure 1B,C).</li>
	<li>Use the scissors to further clear muscle away from the lamina and around the pedicle with small snips. This will open up a small space between the vertebrae. Slowly and delicately insert one blade of the scissors into this gap and snip the pedicle. Make sure the curvature of the scissors is always positioned laterally, away from the cord. Repeat on the other side. (see Figure 1D,E)</li>
	<li>Lift the lamina to expose the cord and carefully snip it off. Be sure not to leave any free or jagged bone fragments behind. (see Figure 1F)</li>
	<li>Prior to injection, clean any blood away with sterile cotton swabs.</li>
</ol>
5.	Injection of cells
<ol>
<li>Attach the needle with needle nut to the Hamilton syringe and clean them by flushing several times with water, then 70% ethanol, and finally HBSS. Insert plunger after each fill with water, 70% ethanol, or HBSS.</li>
<li>Prepare the Hamilton syringe for cell loading by removing the plunger and loosening the needle nut and pulling the needle away from the syringe to prevent backpressure. Ensure that careful handling of the needle and nut are done with sterile gloves.</li>
<li>Load 15&mu;l of cells into pipette tip and press the tip tightly into the back end of the syringe to load the cells into the syringe.</li>
<li>Insert the plunger about 5mm and then tighten the needle nut.</li>
<li>Depress plunger until some of the cell suspension is seen exiting the needle.</li>
<li>Make sure there are no bubbles in the syringe and lay the syringe down in the horizontal position to prevent the cells from making a gradient by gravity.</li>
<li>Grab hold of the laminectomized mouse by the spinalis dorsi muscle connecting the spines of T8 and T9 (Figure 2B, C).</li> 
<li>Clamp the hemostat to the left (vertical) micromanipulator arm so that the mouse&#x2019;s front paws are in the air and its rear paws lightly touching a platform of sterile paper towels as in Figures 2A and 2B.</li>
<li>Attach the syringe to the right micromanipulator arm (at a 70&deg; angle) and slide the syringe to the lowest position possible before clamping.</li>
<li>Stabilize the mouse by pinning its tail against the paper towels and slowly lower the syringe (Figure 2B).</li>
<li>Lower the needle towards the cord and insert the needle 1mm into the opposite hemisphere through the dorsal midline (Figure 2C). The tip of the needle should be in the gray matter close to the central canal.</li>
<li>Slowly inject 2.5&mu;l of cells. Inject at a rate of 1&mu;l / 5 seconds. After injecting the cells, wait a 10 seconds and retract the needle a tenth of a turn at a time every 10 seconds until the needle is out of the cord. Pay attention to possible efflux of cell suspension.</li>
<li>Quickly retract the syringe and detach it from the micromanipulator arm. Lay the syringe down horizontally.</li>
<li>Release the mouse and transfer to suturing table.</li>
<li>Repeat steps 5.7-5.14 for each mouse until the syringe is emptied. Sterilize tools (in the sterilizer) and the needle (by wiping with ethanol) between animals. Discard cells and reload if clumping is visible.</li>
<li>Clean the syringe as in step 5.1 between loads.</li>
</ol>
6.	Sutures and post-op care
<ol>
<li>Suture the incision. Suture needle is inserted into the superficial fascia on both sides of the incision. The thread is strung through, pulling superficial fascia together (Figure 3A), thereby covering the exposed spinal cord at the site of removed lamina. Do not suture the cutaneous muscle attached to the skin or the skeletal muscle of the back. Pull the entire thread through, leaving approximately 1/2". Using the needle holder, three knots are formed and thread is trimmed as close to the knot as possible.</li>
<li>Close the incision by applying two to three staples (depending on size of incision) to the skin (Figure 3B). Carefully pull skin away from the mouse to avoid stapling the underlying muscle.</li> 
<li>Using a 26G3/8 needle inject 0.5ml Lactated Ringers sub-cutaneously in the lower back away from the incision.</li>
<li>Place mouse back in its cage. To ensure it is able to breathe
comfortably while under anesthesia, the mouse should be placed on its
side in the cage, avoiding contact between the surgery site and cage
bedding. Cages should be placed on heating pads. </li>
<li>Mice are monitored after anesthesia wears off to ensure bleeding subsides, sutures remain closed, and that mice return to pre-surgery mobility.</li>
<li>Treat mice one time with the analgesic buprenorphine (0.05-0.1 mg/kg) following surgery.</li>
<li>Ensure impaired mice have sufficient access to food and water: water bottles are fitted with extended 3.5 inch spouts and mice that are unable to walk are hand fed water and/or high-calorie dietary supplement (Nutri-Cal, Tomlyn).</li>
</ol>
7. Representative Results:
Desired results will be identifiable by the lack of efflux of the cell suspension during the injection and by the intact appearance of the spinal cord following the procedure. To this end, it is vital to have bright and direct lighting on the mouse&#x2019;s spine during the laminectomy and during the injection. Optimal lighting is facilitated by fiber optic lighting (Figure 2A).
<img src="/files/ftp_upload/2834/2834fig1.jpg" alt="Figure 1"/> 
Figure 1- Laminectomy. (A) Once vertebra T9 is solidly held with the Graefe forceps, (B, C) score the spine with scalpel between T10 and T11 to facilitate entry of the micro scissors. (D) Carefully slide the micro scissors through the space between T10 and T11(arrows and inset, E) and cut the pedicles on each side (dashes, E) to free the dorsal lamina. (F) Flip the lamina up rostrally and cut it off.
<img src="/files/ftp_upload/2834/2834fig2.jpg" alt="Figure 2"/> 
Figure 2. Injection of NSCs. (A) The general setup of the micromanipulator with the hemostat holding the mouse attached to the left arm and the Hamilton syringe on the right arm at a 70 angle. (B) The hemostat hold the spinalis dorsi muscle connecting the spines of T8 and T9. (C) The needle is lowered through the midline and into the gray matter on the opposite hemisphere, proximal to the central canal.
<img src="/files/ftp_upload/2834/2834fig3.jpg" alt="Figure 3"/> 
Figure 3. Sutures and wound closure. (A) Sutures are applied to the superficial fascia on both sides of the incision. (B) The incision is closed with 2-3 staples as needed (one staple shown on a wound needing 3).

<ol>
	<li>Bergmann, C. C., Lane, T. E., Stohlman, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coronavirus+infection+of+the+central+nervous+system:+host-virus+stand-off.">Coronavirus infection of the central nervous system: host-virus stand-off.</a> Nat Rev Microbiol. 4, 121-132 (2006).</li><li>Weiner, H. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+challenge+of+multiple+sclerosis:+how+do+we+cure+a+chronic+heterogeneous+disease.">The challenge of multiple sclerosis: how do we cure a chronic heterogeneous disease.</a> Ann Neurol. 65, 239-248 (2009).</li><li>Fleming, J. O., Trousdale, M. D., Bradbury, J., Stohlman, S. A., Weiner, L. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental+demyelination+induced+by+coronavirus+JHM+(MHV-4):+molecular+identification+of+a+viral+determinant+of+paralytic+disease.">Experimental demyelination induced by coronavirus JHM (MHV-4): molecular identification of a viral determinant of paralytic disease.</a> Microb Pathog. 3, 9-20 (1987).</li><li>Totoiu, M. O., Nistor, G. I., Lane, T. E., Keirstead, H. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Remyelination,+axonal+sparing,+and+locomotor+recovery+following+transplantation+of+glial-committed+progenitor+cells+into+the+MHV+model+of+multiple+sclerosis.">Remyelination, axonal sparing, and locomotor recovery following transplantation of glial-committed progenitor cells into the MHV model of multiple sclerosis.</a> Exp Neurol. 187, 254-265 (2004).</li><li>Carbajal, K. S., Schaumburg, C., Strieter, R., Kane, J., Lane, T. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Migration+of+engrafted+neural+stem+cells+is+mediated+by+CXCL12+signaling+through+CXCR4+in+a+viral+model+of+multiple+sclerosis.">Migration of engrafted neural stem cells is mediated by CXCL12 signaling through CXCR4 in a viral model of multiple sclerosis.</a> Proc Natl Acad Sci U S A. 107, 11068-11073 (2010).</li><li>Hardison, J. L., Nistor, G., Gonzalez, R., Keirstead, H. S., Lane, T. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transplantation+of+glial-committed+progenitor+cells+into+a+viral+model+of+multiple+sclerosis+induces+remyelination+in+the+absence+of+an+attenuated+inflammatory+response.">Transplantation of glial-committed progenitor cells into a viral model of multiple sclerosis induces remyelination in the absence of an attenuated inflammatory response.</a> Exp Neurol. 197, 420-429 (2006).</li><li>Blakemore, W. F., Crang, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Transplantation of glial cells into areas of demyelination in the adult rat spinal cord. , (1992).</li><li>Liu, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Embryonic+stem+cells+differentiate+into+oligodendrocytes+and+myelinate+in+culture+and+after+spinal+cord+transplantation.">Embryonic stem cells differentiate into oligodendrocytes and myelinate in culture and after spinal cord transplantation.</a> Proc Natl Acad Sci U S A. 97, 6126-6131 (2000).</li><li>Keirstead, H. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+embryonic+stem+cell-derived+oligodendrocyte+progenitor+cell+transplants+remyelinate+and+restore+locomotion+after+spinal+cord+injury.">Human embryonic stem cell-derived oligodendrocyte progenitor cell transplants remyelinate and restore locomotion after spinal cord injury.</a> J Neurosci. 25, 4694-4705 (2005).</li><li>Mayor, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=First+patient+enters+trial+to+test+safety+of+stem+cells+in+spinal+injury.">First patient enters trial to test safety of stem cells in spinal injury.</a> BMJ. 341, c5724-c5724 (2010).</li></ol>

No conflicts of interest declared.

A well-executed transplant will hinge primarily on the careful laminectomy and injection of the cells. The primary pitfall to avoid during a laminectomy is the damaging of the spinal cord. This can occur during the procedure itself or by damage caused by sharp bone fragments left behind following the procedure. To avoid these, ensure that the points of the curved micro scissors are always facing away from the cord and carefully examine the laminectomized spine to ensure that all bone fragments are cleared and that the remaining vertebral structure does not have overtly protruding or jagged edges. 
As previously mentioned, the detection of efflux will be possible if the light is shining brightly and directly onto the exposed spinal cord during the injection. Efflux is most likely to happen with 30 gauge needles (versus 33 gauge) and if the injection is done too rapidly. Though this protocol has given us good results, others have reported longer waiting periods (up to 5 minutes) before retracting the needle following injection 8,9. Also, smaller gauge needles are preferable, but we have observed that some cells are too easily lysed when passed through 33 gauge needles.
To maximize efficiency, a transplant team manning the four different stations (mouse prep, laminectomy, injection, and sutures) is desirable. Furthermore, the timing for each procedure should be optimized to minimize the time the cells are waiting on ice. For example, we transplant our mice in groups of four (the number of doses in each load of the syringe), the person injecting the cells starts loading the syringe after the third mouse has been laminectomized and the person preparing the mice should anesthetize the following group after the second mouse of the previous group has been laminectomized. In this manner, we can transplant cells (or control media) into 40 mice in about 3 hrs even though each mouse will take approximately 30-40 min. 
Cell replacement therapies for the treatment of some CNS disorders are currently in clinical trials 10. There is no substitute for in vivo models of NSC transplantation and our protocol for the engraftment of NSCs into the spinal cords of mice with viral induced demyelination facilitates the use of an important model of MS and can also be easily adapted to other models.

<table border="1">
	<tbody>
 	<tr>
 	<th>Name of the reagent/equipment</th>
 <th>Company</th>
 <th>Catalogue number</th>
 <th>Comments (optional)</th>
 </tr>
 <tr>
 	<td>Ketaject</td>
 <td>Phoenix Parmaceuticals</td>
 <td>NDC 57319-542-02</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 	<td>Xylazine Hydrochloride</td>
 <td>MP Biomedicals</td>
 <td>158307</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 	<td>Nair</td>
 <td>Church &amp; Dwight Co.</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 	<td>10&#xF020;&mu;l Hamilton Syringe w/ Removable needle</td>
 <td>Hamilton Company</td>
 <td>7635-01</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 	<td>Hamilton Needles, 30G or 33 G, &frac12; inch, 30&deg; bevel </td>
 <td>Hamilton Company</td>
 <td>7803-077803-05</td>
 <td>Test the viability of your cells following passage through needles to identify the best gauge to use</td>
 </tr>
 <tr>
 	<td>Micro Scissors</td>
 <td>World Precision Instruments</td>
 <td>555500S</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 	<td>Small Graefe Forceps</td>
 <td>FST</td>
 <td>11053-10</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 	<td>Stereotaxic</td>
 <td>KOPF Instruments</td>
 <td>Model 1772 Universal Holder</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 	<td>Stereotaxic</td>
 <td>KOPF Instruments</td>
 <td>Model 1773 Electrode Holder</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 	<td>Stereotaxic</td>
 <td>KOPF Instruments</td>
 <td>Model 902 small animal stereotaxic</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 	<td>Stereotaxic</td>
 <td>KOPF Instruments</td>
 <td>Model 960 left electrode carrier</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 	<td>Sutures</td>
 <td>Ethicon</td>
 <td>95057-064</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 	<td>Lactated Ringers</td>
 <td>Hospira</td>
 <td>NDC 0409-7953-03</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 	<td>Staples</td>
 <td>Fine Science</td>
 <td>12032-07</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 	<td>Hemostat</td>
 <td>FST</td>
 <td>13010-12</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 	<td>Scalpels, sizes 10,11</td>
 <td>Fisher</td>
 <td>268878, 268879</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 	<td>Sutures</td>
 <td>ETHICON</td>
 <td>1676G</td>
 <td>size 5-0, 3/8" circle, 19mm needle, 45cm braided thread</td>
 </tr>
 <tr>
 	<td>Reflex 7 wound clip applicator</td>
 <td>FST</td>
 <td>12031-07</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 	<td>7mm Reflex wound clips</td>
 <td>FST</td>
 <td>12032-07</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 	<td>Olsen-Hegar needle holder</td>
 <td>FST</td>
 <td>12502-12</td>
 <td>&nbsp;</td>
 </tr>
 </tbody>
</table>

surgical transplantation of mouse neural stem cells into the spinal cords of mice infected with neurotropic mouse hepatitis virus

Mice infected with the neurotropic JHM strain of mouse hepatitis virus (MHV) develop pathological and clinical outcomes similar to patients with the demyelinating disease Multiple Sclerosis (MS). We have shown that transplantation of NSCs into the spinal cords of sick mice results in a significant improvement in both remyelination and in clinical outcome. Cell replacement therapies for the treatment of chronic neurologic diseases are now a reality and in vivo models are vital in understanding the interactions between the engrafted cells and host tissue microenvironment. This presentation provides an adapted method for transplanting cells into the spinal cord of JHMV-infected mice. In brief, we provide a procedure for i) preparation of NSCs prior to transplant, ii) pre-operative care of mice, iii) exposure of the spinal cord via laminectomy, iv) stereotactic injection of NSCs, and iv) post-operative care.

Watch this Scientific Journal Video about Surgical Transplantation of Mouse Neural Stem Cells into the Spinal Cords of Mice Infected with Neurotropic Mouse Hepatitis Virus at JoVE.com

Surgical Transplantation of Mouse Neural Stem Cells into the Spinal Cords of Mice Infected with Neurotropic Mouse Hepatitis Virus

The transplantation of mouse neural stem cells (NSCs) into the spinal cords of mice with established demyelination is detailed. The preparation of NSCs, the laminectomy of thoracic vertebra 9 (T9), and transplantation of NSCs is outlined along with the pre- and post-operative care of the mice.

Mice infected with the neurotropic JHM strain of mouse hepatitis virus (MHV) develop pathological and clinical outcomes similar to patients with the ...

surgical-transplantation-mouse-neural-stem-cells-into-spinal-cords

Research

JoVE Journal

Neuroscience

23.3K vistas.  University of California, Irvine. El trasplante de células madre neurales (NSC) en la médula espinal de ratones con desmielinización establecido se detalla. La preparación de los comités directivos nacionales, la laminectomía de vértebra torácica 9 (T9), y el trasplante de NSCs se perfila, junto con la atención pre y post-operatorio de los ratones.

Video: Surgical Transplantation of Mouse Neural Stem Cells into the Spinal Cords of Mice Infected with Neurotropic Mouse Hepatitis Virus

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 ...

One Mouse, Two Cultures: Isolation and Culture of Adult Neural Stem Cells from the Two Neurogenic Zones of Individual Mice

The neurosphere assay and the adherent monolayer culture system are valuable tools to determine the potential (proliferation or differentiation) of adult neural stem cells in vitro. These assays can be used to compare the precursor potential of cells isolated from genetically different or differentially treated animals&#160;to determine the effects of exogenous factors on neural precursor cell proliferation and differentiation and to generate neural precursor cell lines that can be assayed over continuous passages. The neurosphere assay is traditionally used for the post-hoc identification of stem cells, primarily due to the lack of definitive markers with which they can be isolated from primary tissue&#160;and has the major advantage of giving a quick estimate of precursor cell numbers in brain tissue derived from individual animals. Adherent monolayer cultures, in contrast, are not traditionally used to compare proliferation between individual animals, as each culture is generally initiated from the combined tissue of between 5-8 animals. However, they have the major advantage that, unlike neurospheres, they consist of a mostly homogeneous population of precursor cells and are useful for following the differentiation process in single cells. Here, we describe, in detail, the generation of neurosphere cultures and, for the first time, adherent cultures from individual animals. This has many important implications including paired analysis of proliferation and/or differentiation potential in both the subventricular zone (SVZ) and dentate gyrus (DG) of treated or genetically different mouse lines, as well as a significant reduction in animal usage.

Here we describe a detailed protocol for the simultaneous generation of neural precursor cell cultures, as either adherent monolayers or neurospheres, from the subventricular zone and dentate gyrus of individual adult mice.

The neurosphere assay and the adherent monolayer culture system are valuable tools to determine the potential (proliferation or differentiation) of adult ...

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 ...

Development of a Refined Protocol for Trans-scleral Subretinal Transplantation of Human Retinal Pigment Epithelial Cells into Rat Eyes

Degenerative retinal diseases such as age-related macular degeneration (AMD) are the leading cause of irreversible vision loss worldwide. AMD is characterized by the degeneration of retinal pigment epithelial (RPE) cells, which are a monolayer of cells functionally supporting and anatomically wrapping around the neural retina. Current pharmacological treatments for the non-neovascular AMD (dry AMD) only slow down the disease progression but cannot restore vision, necessitating studies aimed at identifying novel therapeutic strategies. Replacing the degenerative RPE cells with healthy cells holds promise to treat dry AMD in the future. Extensive preclinical studies of stem cell replacement therapies for AMD involve the transplantation of stem cell-derived RPE cells into the subretinal space of animal models, in which the subretinal injection technique is applied. The approach most frequently used in these preclinical animal studies is through the trans-scleral route, which is made difficult by the lack of direct visualization of the needle end and can often result in retinal damage. An alternative approach through the vitreous allows for direct observation of the needle end position, but it carries a high risk of surgical traumas as more eye tissues are disturbed. We have developed a less risky and reproducible modified trans-scleral injection method that uses defined needle angles and depths to successfully and consistently deliver RPE cells into the rat subretinal space and avoid excessive retinal damage. Cells delivered in this manner have been previously demonstrated to be efficacious in the Royal College of Surgeons (RCS) rat for at least 2 months. This technique can be used not only for cell transplantation but also for delivery of small molecules or gene therapies.

Subretinal injection has been widely applied in preclinical studies of stem cell replacement therapy for age-related macular degeneration. In this visualized article, we describe a less risky, reproducible and precisely modified subretinal injection technique via the trans-scleral approach to deliver cells into rat eyes.

Degenerative retinal diseases such as age-related macular degeneration (AMD) are the leading cause of irreversible vision loss worldwide. AMD is ...

Differentiation of Mouse Embryonic Stem Cells into Cortical Interneuron Precursors

GABAergic cortical interneurons are a heterogeneous population of cells that play critical roles in regulating the output of excitatory pyramidal neurons as well as synchronizing the outputs of pyramidal neuron ensembles. Deficits in interneuron function have been implicated in a variety of neuropsychiatric disorders, including schizophrenia, autism, and epilepsy. The derivation of cortical interneurons from embryonic stem cells not only allows for the study of their development and function, but provides insight into the molecular mechanisms underlying the pathogenesis of cortical interneuron-related disorders. Interneurons also have the remarkable capacity to survive, migrate, and integrate into host cortical circuitry post-transplantation, making them ideal candidates for use in cell-based therapies. Here, we present a scalable, highly efficient, modified embryoid body-to-monolayer method for the derivation of Nkx2.1-expressing interneuron progenitors and their progeny from mouse embryonic stem cells (mESCs). Using a Nkx2.1::mCherry:Lhx6::GFP dual reporter mESC line, Nkx2.1 progenitors or their Lhx6-expressing post-mitotic progeny can be isolated via fluorescence-activated cell sorting (FACS) and subsequently used in a number of downstream applications. We also provide methods to enrich for parvalbumin (PV) or somatostatin (SST) interneuron subgroups, which may be helpful for studying aspects of fate determination or for use in therapeutic applications that would benefit from interneuron subgroup-enriched transplantations.

This protocol describes a method for generating cortical interneuron progenitors and post-mitotic interneuron precursors from mouse embryonic stem cells using a modified embryoid body-to-monolayer method. These progenitors/precursors can be used in vitro or fluorescently sorted and transplanted into neonatal neocortex for studying fate determination, or used in therapeutic applications.

GABAergic cortical interneurons are a heterogeneous population of cells that play critical roles in regulating the output of excitatory pyramidal neurons ...

Homochronic Transplantation of Interneuron Precursors into Early Postnatal Mouse Brains

Neuronal fate determination and maturation requires an intricate interplay between genetic programs and environmental signals. However, disentangling the roles of intrinsic vs. extrinsic mechanisms that regulate this differentiation process is a conundrum for all developmental neurobiologists. This issue is magnified for GABAergic interneurons, an incredibly heterogeneous cell population that is born from transient embryonic structures and undergo a protracted migratory phase to disperse throughout the telencephalon. To explore how different brain environments affect interneuron fate and maturation, we developed a protocol for harvesting fluorescently labeled immature interneuron precursors from specific brain regions in newborn mice (P0-P2). At this age, interneuron migration is nearly complete and these cells are residing in their final resting environments with relatively little synaptic integration. Following collection of single cell solutions via flow cytometry, these interneuron precursors are transplanted into P0-P2 wildtype postnatal pups. By performing both homotopic (e.g., cortex-to-cortex) or heterotopic (e.g., cortex-to-hippocampus) transplantations, one can assess how challenging immature interneurons in new brain environments affects their fate, maturation, and circuit integration. Brains can be harvested in adult mice and assayed with a wide variety of posthoc analysis on grafted cells, including immunohistochemical, electrophysiological and transcriptional profiling. This general approach provides investigators with a strategy to assay how distinct brain environments can influence numerous aspects of neuron development and identify if specific neuronal characteristics are primarily driven by hardwired genetic programs or environmental cues.

Challenging young neurons in new brain regions can reveal important insights into how the environment sculpts neuronal fate and maturation. This protocol describes a procedure to harvest interneuron precursors from specific brain regions and transplant them either homotopically or heterotopically into the brain of postnatal pups.

Neuronal fate determination and maturation requires an intricate interplay between genetic programs and environmental signals. However, disentangling the ...

Isolation and Culture of Embryonic Mouse Neural Stem Cells

Neural stem cells (NSCs) are multipotent and can give rise to the three major cell types of the central nervous system (CNS). In vitro culture and expansion of NSCs provide a suitable source of cells for neuroscientists to study the function of neurons and glial cells along with their interactions. There are several reported techniques for the isolation of neural stem cells from adult or embryo mammalian brains. During the microsurgical operation to isolate NSCs from different regions of the embryonic CNS, it is very important to reduce the damage to the brain cells to obtain the highest ratio of live and expandable stem cells. A possible technique for stress reduction during isolation of these cells from the mouse embryo brain is the reduction of surgical time. Here, we demonstrate a developed technique for rapid isolation of these cells from the E13 mouse embryo ganglionic eminence. Surgical procedures include harvesting E13 mouse embryos from the uterus, cutting the frontal fontanelle of the embryo with a bent needle tip, extracting the brain from the skull, microdissection of the isolated brain to harvest the ganglionic eminence, dissociation of the harvested tissue in NSC medium to gain a single cell suspension, and finally plating cells in suspension culture to generate neurospheres.

Here, we introduce a novel microsurgical technique for the isolation of neural stem cells from the E13 mouse embryo ganglionic eminence.

Neural stem cells (NSCs) are multipotent and can give rise to the three major cell types of the central nervous system (CNS). In vitro culture and ...

Transplantation of Chemogenetically Engineered Cortical Interneuron Progenitors into Early Postnatal Mouse Brains

Neuronal development is regulated by a complex combination of environmental and genetic factors. Assessing the relative contribution of each component is a complicated task, which is particularly difficult in regards to the development of &#947;-aminobutyric acid (GABA)ergic cortical interneurons (CIs). CIs are the main inhibitory neurons in the cerebral cortex, and they play key roles in neuronal networks, by regulating both the activity of individual pyramidal neurons, as well as the oscillatory behavior of neuronal ensembles. They are generated in transient embryonic structures (medial and caudal ganglionic eminences -&#160;MGE and CGE) that are very difficult to efficiently target using in utero electroporation approaches. Interneuron progenitors migrate long distances during normal embryonic development, before they integrate in the cortical circuit. This remarkable ability to disperse and integrate into a developing network can be hijacked by transplanting embryonic interneuron precursors into early post-natal host cortices. Here, we present a protocol that allows genetic modification of embryonic interneuron progenitors using focal ex vivo electroporation. These engineered interneuron precursors are then transplanted into early post-natal host cortices, where they will mature into easily identifiable CIs. This protocol allows the use of multiple genetically encoded tools, or the ability to regulate the expression of specific genes in interneuron progenitors, in order to investigate the impact of either genetic or environmental variables on the maturation and integration of CIs.

Here we present a protocol, designed to use chemogenetic tools to manipulate the activity of cortical interneuron progenitors transplanted into the cortex of early postnatal mice.

Neuronal development is regulated by a complex combination of environmental and genetic factors. Assessing the relative contribution of each component is ...

The Detection of 5-Hydroxymethylcytosine in Neural Stem Cells and Brains of Mice

Multiple&#160;DNA modifications have been identified in the mammalian genome. Of that, 5-methylcytosine and 5-hydroxymethylcytosine-mediated epigenetic mechanisms have been intensively studied. 5-hydroxymethylcytosine displays dynamic features during embryonic and postnatal development of the brain, plays a regulatory function in gene expression, and is involved in multiple neurological disorders. Here, we describe the detailed methods including immunofluorescence staining and&#160;DNA dot-blot to detect 5-hydroxymethylcytosine in cultured cells and brain tissues of mouse.

Here, we present a protocol to detect 5-hydroxymethylcytosine in cells and brain tissues, utilizing immunofluorescence staining and DNA dot-blot methods.

Multiple&#160;DNA modifications have been identified in the mammalian genome. Of that, 5-methylcytosine and 5-hydroxymethylcytosine-mediated epigenetic ...

Intra-Arterial Delivery of Neural Stem Cells to the Rat and Mouse Brain: Application to Cerebral Ischemia

Neural stem cell (NSC) therapy is an emerging innovative treatment for stroke, traumatic brain injury and neurodegenerative disorders. As compared to intracranial delivery, intra-arterial administration of NSCs is less invasive and produces a more diffuse distribution of NSCs within the brain parenchyma. Further, intra-arterial delivery allows the first-pass effect in the brain circulation, lessening the potential for trapping of cells in peripheral organs, such as liver and spleen, a complication associated with peripheral injections. Here, we detail the methodology, in both mice and rats, for delivery of NSCs through the common carotid artery (mouse) or external carotid artery (rat) to the ipsilateral hemisphere after an ischemic stroke. Using GFP-labeled NSCs, we illustrate the widespread distribution achieved throughout the rodent ipsilateral hemisphere at 1 d, 1 week and 4 weeks after postischemic delivery, with a higher density in or near the ischemic injury site. In addition to long-term survival, we show evidence of differentiation of GFP-labeled cells at 4 weeks. The intra-arterial delivery approach described here for NSCs can also be used for administration of therapeutic compounds, and thus has broad applicability to varied CNS injury and disease models across multiple species.

A method for delivering neural stem cells, adaptable for injecting solutions or suspensions, through the common carotid artery (mouse) or external carotid artery (rat) after ischemic stroke is reported. Injected cells are distributed broadly throughout the brain parenchyma and can be detected up to 30 d after delivery.

Neural stem cell (NSC) therapy is an emerging innovative treatment for stroke, traumatic brain injury and neurodegenerative disorders. As compared to ...