Name: dissection of hippocampal dentate gyrus from adult mouse
Uploaded: 2009-11-17T20:30:00
Duration: 7 min 42 s
Description: Watch this Scientific Journal Video about Dissection of Hippocampal Dentate Gyrus from Adult Mouse at JoVE.com

We thank Dr. Yoko Nabeshima at Kyoto University for her instruction on the dissection technique and Ms. Aki Miyakawa at Fujita Health University for her support to film. This work was supported by the Program for Promotion of Fundamental Studies in Health Sciences of the National Institute of Biomedical Innovation, a Grant-in-Aid for Scientific Research on Priority Areas -Integrative Brain Research (Shien)- from MEXT in Japan, and by a Grant-in-Aid from CREST of the Japan Science and Technology Agency.

Dissection of hippocampal dentate gyrus
<ol>
 <li>In a deeply anesthetized mouse, carefully dissect the brain out from the skull and place it into ice-cold phosphate-buffered saline (PBS).</li>
 <li>In a Petri dish containing ice-cold PBS, cut the brain along the longitudinal fissure of the cerebrum using a surgical knife, and cut off the regions posterior to lambda (midbrain, hindbrain, and cerebellum).</li>
 <li>Place the cerebral hemisphere medial side up and, using forceps, carefully remove the diencephalon (thalamus and hypothalamus) under a dissection microscope. This will expose the medial side of the hippocampus, allowing for visualization of the dentate gyrus. The dentate gyrus is distinguishable from Ammon's horn by the gaps between them. Injury to the hippocampus or surrounding area will make it more difficult to isolate the dentate gyrus.</li>
 <li>Insert a sharp needle-tip (e.g., 27-gauge needle) into each side of the dentate gyrus (boundaries of the dentate gyrus and Ammon's horn; Figure 1), and slide the needles superficially along the septo-temporal axis of hippocampus to isolate the dentate gyrus.</li>
 <li>Pick up the isolated dentate gyrus using a needle or forceps and place it in a sample tube. The thus-obtained dentate gyrus tissue sample can be used immediately for any assay or stored in a deep-freezer for later use.</li>
 <li>Isolate the dentate gyrus from the other cerebral hemisphere using the same method.</li>
</ol>
Quantitative real-time PCR
The dentate gyrus was isolated using the above-mentioned method and the remaining hippocampus was dissected out as the Ammon's horn sample from wild-type mice. Real-time PCR of beta-actin, TDO2, Dsp, Mrg1b and Tyro3 were performed with the dentate gyrus and the Ammon's horn samples as described previously1. Primers 5'-CTGGCGAGATCACGATGACG and 5'-AAGCTACGCTGTTGTCTAACC were used for Mrg1b, and GCCTCCAAATTGCCCGTCA and 5'-CCAGCACTGGTACATGAGATCA for Tyro3. 
Microarray analysis
Microarray experiments were performed with male wild-type mice and mice heterozygous for the alpha-isoform of calcium/calmodulin-dependent protein kinase II (alpha-CaMKII+/- mice) as described previously1. Briefly, RNA isolated from the whole hippocampus or dentate gyrus of wild-type and mutant mice was hybridized with a Mouse Genome 430 2.0 Array (Affymetrix, Santa Clara, CA), and each GeneChip was scanned by an Affymetrix GeneChip Scanner 3000 (GCS3000). GeneChip analysis was performed with Microarray Analysis Suite version 5.0.

<ol>
	<li>Yamasaki, N., Maekawa, M., Kobayashi, K., Kajii, Y., Maeda, J., Soma, M., Takao, K., Tanda, K., Ohira, K., Toyama, K., Kanzaki, K., Fukunaga, K., Sudo, Y., Ichinose, H., Ikeda, M., Iwata, N., Ozaki, N., Suzuki, H., Higuchi, M., Suhara, T., Yuasa, S., Miyakawa, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alpha-CaMKII+deficiency+causes+immature+dentate+gyrus,+a+novel+candidate+endophenotype+of+psychiatric+disorders.">Alpha-CaMKII deficiency causes immature dentate gyrus, a novel candidate endophenotype of psychiatric disorders.</a> Mol. Brain. 1, 6-6 (2008).</li><li>Insausti, A. M., Megias, M., Crespo, D., Cruz-Orive, L. M., Dierssen, M., Vallina, I. F., Insausti, R., Florez, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hippocampal+volume+and+neuronal+number+in+Ts65Dn+mice:+a+murine+model+of+Down+syndrome.">Hippocampal volume and neuronal number in Ts65Dn mice: a murine model of Down syndrome.</a> Neurosci. Lett. 253, 175-175 (1998).</li><li>Redwine, J. M., Kosofsky, B., Jacobs, R. E., Games, D., Reilly, J. F., Morrison, J. H., Young, W. G., Bloom, F. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dentate+gyrus+volume+is+reduced+before+onset+of+plaque+formation+in+PDAPP+mice:+a+magnetic+resonance+microscopy+and+stereologic+analysis.">Dentate gyrus volume is reduced before onset of plaque formation in PDAPP mice: a magnetic resonance microscopy and stereologic analysis.</a> Proc. Natl. Acad. Sci. U.S.A. 100, 1381-1381 (2003).</li><li>Lein, E. S., Zhao, X., Gage, F. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Defining+a+molecular+atlas+of+the+hippocampus+using+DNA+microarrays+and+high-throughput+in+situ+hybridization.">Defining a molecular atlas of the hippocampus using DNA microarrays and high-throughput in situ hybridization.</a> J. Neurosci. 24, 3879-3879 (2004).</li><li>Matsuo, N., Yamasaki, N., Ohira, K., Takao, K., Toyama, K., Eguchi, M., Yamaguchi, S., Miyakawa, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neural+activity+changes+underlying+the+working+memory+deficit+in+alpha-CaMKII+heterozygous+knockout+mice.">Neural activity changes underlying the working memory deficit in alpha-CaMKII heterozygous knockout mice.</a> Front. Behav. Neurosci. 3, 20-20 (2009).</li><li>Franklin, K. B. J., Paxinos, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Mouse Brain in Stereotaxic Coordinates. , (1997).</li></ol>

Animal experiments were performed in accordance with the National Institute of Health Guide for Care and Use of Laboratory Animals, and approved by the animal care and use committee at Fujita Health University.

The dentate gyrus occupies approximately 25% to 30% of the volume of the hippocampal formation2,3. It has a unique cell composition and plays crucial roles in various brain functions. Therefore, techniques to isolate the dentate gyrus are useful for analyzing the events that occur specifically in this region.
Here, we demonstrated a procedure to efficiently dissect the dentate gyrus from adult mouse hippocampus and confirmed the precision of the technique. First, histologic study revealed that the dentate gyrus was separated without contamination by other regions (Figure 1), indicating that a pure dentate gyrus sample can be prepared.
Second, we confirmed that the dissected tissue is dentate gyrus by conducting real-time PCR of dentate gyrus-specific genes, TDO2 and Dsp, and Ammon's horn enriched genes, Mrg1b and Tyro34 (Figure 2). The mRNA expressions of TDO2 (p=0.000023; n's=4 and 4, respectively) and Dsp (p=0.0000030; n's=4 and 4, respectively) in the dentate gyrus samples were detected at obviously higher levels, whereas Mrg1b (p=0.000080; n's=4 and 4, respectively) and Tyro3 (p=0.00017; n's=4 and 4, respectively) were lower levels, than those in the Ammon's horn samples. Beta-actin expression levels did not differ in these samples (p=0.11; n's=4 and 4, respectively). Thus, we could check whether or not the dentate gyrus was accurately dissected out by conducting such simple real-time PCR experiments.
Third, to assess the usefulness of this dissection method, we compared the mRNA expression level of whole hippocampus with that of dentate gyrus. Whole hippocampus and dentate gyrus obtained from wild-type (n's=9 and 4, respectively) and alpha-CaMKII+/- mice (n's=18 and 4, respectively) were processed for microarray analysis, and for all genes scored, the fold-change was calculated by dividing the mutant value by the wild-type value. The results indicated that the changes in mRNA expression, especially of dentate gyrus-specific molecules such as Dsp and TDO2, were detected with up to a 5-fold increase in sensitivity in dentate gyrus samples compared to whole hippocampal samples (Table 1). We previously demonstrated that alpha-CaMKII+/- mice exhibit behaviors related to human psychiatric disorders such as working memory deficits and an exaggerated infradian rhythm1,5. Furthermore, morphologic and electrophysiologic features of the dentate gyrus neurons in mutant mice are strikingly similar to those of immature dentate gyrus neurons in normal rodents, indicating that the neurons in these mutant mice fail to develop to maturity1. The immature dentate gyrus and down-regulated expression of Dsp and TDO2 mRNA in alpha-CaMKII+/- mice are consistent with the finding that Dsp and TDO2 can be used as markers of mature granule cells in the dentate gyrus (Ohira et al., unpublished data).
Taken together, this convenient and accurate dissection technique can be reliably used for studies focused on the dentate gyrus. Dentate gyrus tissue obtained using this method is applicable to other types of analyses as well, including proteomic and cell biology analyses.
<img src="/files/ftp_upload/1543/1543fig1.jpg" alt="figure 1" /> Figure 1. Verification of the isolated dentate gyrus by histologic study. A coronal section of the brain after isolating dentate gyrus was processed for Nissl staining (left panel), and a schematic diagram adapted from the mouse brain atlas6 represents the approximately the same level of the section shown in the left panel (right panel). Arrows indicate the directions of the needle-tip insertion. Scale bar, 1 mm.
<img src="/files/ftp_upload/1543/1543fig2.jpg" alt="figure 2" /> Figure 2. Verification of the isolated dentate gyrus by real-time PCR. The dentate gyrus and the Ammon s horn obtained from four wild-type mice were processed for real-time PCR of beta-actin, TDO2, Dsp, Mrg1b and Tyro3. Results are presented as means &plusmn; SEM. For statistical analysis, Student s t test was employed, and p values are followed: beta-actin, p=0.11; TDO2, p=0.000023 (**1); Dsp, p=0.0000030 (**2); Mrg1b, p=0.000080 (**3); and Tyro3, p=0.00017 (**4).
Table 1. Microarray analysis of whole hippocampus and dentate gyrus. Genes differentially expressed in dentate gyrus and whole hippocampus of alpha-CaMKII+/- mice were determined by calculating the fold-change from that detected in wild-type mice. Data were analyzed for statistical significance using the Student s t test between wild-type and alpha-CaMKII+/- mice. Among the genes whose expression exhibited p&lt;0.05 in the dentate gyrus of alpha-CaMKII+/- mice compared to that of wild-type mice, the top 50 genes are listed. Note that the numbers of samples for dentate gyrus are much less than those for whole hippocampus. AffyID, Affymetrix probe identifier; CKII, alpha-CaMKII+/- mice; WT, wild-type mice. 
<table border="1">
 <tbody>
 <tr>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td colspan="2"> Dentate gyrus (p &lt;0.05) WT: n=4, CKII+/-: n=4</td>
 <td>&nbsp;</td>
 <td colspan="2"> Whole hippocampus WT: n=9, CKII+/-: n=18</td>
 </tr>
 <tr>
 <td>Gene Title</td>
 <td>Genebank</td>
 <td>AffyID</td>
 <td>&nbsp;</td>
 <td>Fold change</td>
 <td>p value</td>
 <td>&nbsp;</td>
 <td>Fold change</td>
 <td>p value</td>
 </tr>
 <tr>
 <td>desmoplakin</td>
 <td>AV297961</td>
 <td>1435494_s_at</td>
 <td>&nbsp;</td>
 <td>0.011018913</td>
 <td>7.02694E-06</td>
 <td>&nbsp;</td>
 <td>0.037021003</td>
 <td>1.86126E-13</td>
 </tr>
 <tr>
 <td>desmoplakin</td>
 <td>AV297961</td>
 <td>1435493_at</td>
 <td>&nbsp;</td>
 <td>0.014369734</td>
 <td>7.86747E-06</td>
 <td>&nbsp;</td>
 <td>0.04232106</td>
 <td>1.00579E-12</td>
 </tr>
 <tr>
 <td>tryptophan 2,3-dioxygenase</td>
 <td>AI098840</td>
 <td>1419093_at</td>
 <td>&nbsp;</td>
 <td>0.020986484</td>
 <td>5.23546E-09</td>
 <td>&nbsp;</td>
 <td>0.101037776</td>
 <td>4.14823E-13</td>
 </tr>
 <tr>
 <td>nephronectin</td>
 <td>AA223007</td>
 <td>1452106_at</td>
 <td>&nbsp;</td>
 <td>0.075479901</td>
 <td>1.05191E-08</td>
 <td>&nbsp;</td>
 <td>0.234001154</td>
 <td>1.66301E-15</td>
 </tr>
 <tr>
 <td>nephronectin</td>
 <td>AA223007</td>
 <td>1452107_s_at</td>
 <td>&nbsp;</td>
 <td>0.079457767</td>
 <td>1.40433E-07</td>
 <td>&nbsp;</td>
 <td>0.177974715</td>
 <td>3.9758E-12</td>
 </tr>
 <tr>
 <td>thyrotropin releasing hormone receptor</td>
 <td>M59811</td>
 <td>1449571_at</td>
 <td>&nbsp;</td>
 <td>0.103105815</td>
 <td>0.003093796</td>
 <td>&nbsp;</td>
 <td>0.801412732</td>
 <td>0.283994361</td>
 </tr>
 <tr>
 <td>ryanodine receptor 1, skeletal muscle</td>
 <td>X83932</td>
 <td>1427306_at</td>
 <td>&nbsp;</td>
 <td>0.104825517</td>
 <td>3.38513E-07</td>
 <td>&nbsp;</td>
 <td>0.650685017</td>
 <td>0.000308462</td>
 </tr>
 <tr>
 <td>nescient helix loop helix 1</td>
 <td>NM_010916</td>
 <td>1419533_at</td>
 <td>&nbsp;</td>
 <td>9.431896</td>
 <td>6.7979E-06</td>
 <td>&nbsp;</td>
 <td>4.078815314</td>
 <td>5.27E-11</td>
 </tr>
 <tr>
 <td>copine family member IX</td>
 <td>BB274531</td>
 <td>1454653_at</td>
 <td>&nbsp;</td>
 <td>9.159157</td>
 <td>7.99492E-06</td>
 <td>&nbsp;</td>
 <td>1.797304153</td>
 <td>0.000296375</td>
 </tr>
 <tr>
 <td>doublecortin-like kinase 3</td>
 <td>BB326709</td>
 <td>1436532_at</td>
 <td>&nbsp;</td>
 <td>0.109336662</td>
 <td>1.95278E-07</td>
 <td>&nbsp;</td>
 <td>0.56697229</td>
 <td>2.62633E-08</td>
 </tr>
 <tr>
 <td>calpain 3</td>
 <td>AF127766</td>
 <td>1426043_a_at</td>
 <td>&nbsp;</td>
 <td>0.111269769</td>
 <td>8.07053E-06</td>
 <td>&nbsp;</td>
 <td>0.370956608</td>
 <td>2.04421E-14</td>
 </tr>
 <tr>
 <td>Adult male corpus striatum cDNA, RIKEN full-length enriched library, clone:C030023B07 product:unclassifiable, full insert sequence</td>
 <td>BB357628</td>
 <td>1460043_at</td>
 <td>&nbsp;</td>
 <td>0.118712341</td>
 <td>6.16926E-07</td>
 <td>&nbsp;</td>
 <td>0.682339204</td>
 <td>2.33001E-06</td>
 </tr>
 <tr>
 <td>collagen and calcium binding EGF domains 1</td>
 <td>AV264768</td>
 <td>1437385_at</td>
 <td>&nbsp;</td>
 <td>0.124043978</td>
 <td>3.65669E-05</td>
 <td>&nbsp;</td>
 <td>0.488394112</td>
 <td>4.05538E-06</td>
 </tr>
 <tr>
 <td>amyloid beta (A4) precursor protein-binding, family A, member 2 binding protein</td>
 <td>AK013520</td>
 <td>1431946_a_at</td>
 <td>&nbsp;</td>
 <td>7.7986307</td>
 <td>1.2098E-06</td>
 <td>&nbsp;</td>
 <td>2.099164713</td>
 <td>1.67047E-06</td>
 </tr>
 <tr>
 <td>calbindin-28K</td>
 <td>BB177770</td>
 <td>1456934_at</td>
 <td>&nbsp;</td>
 <td>0.130255444</td>
 <td>3.32186E-06</td>
 <td>&nbsp;</td>
 <td>0.572605751</td>
 <td>1.99157E-10</td>
 </tr>
 <tr>
 <td>Transcribed locus</td>
 <td>AV328597</td>
 <td>1443322_at</td>
 <td>&nbsp;</td>
 <td>0.133290835</td>
 <td>5.43583E-06</td>
 <td>&nbsp;</td>
 <td>0.562767164</td>
 <td>7.56544E-06</td>
 </tr>
 <tr>
 <td>neuropeptide Y receptor Y2</td>
 <td>NM_008731</td>
 <td>1417489_at</td>
 <td>&nbsp;</td>
 <td>0.135319609</td>
 <td>0.000113407</td>
 <td>&nbsp;</td>
 <td>0.781498474</td>
 <td>0.00394504</td>
 </tr>
 <tr>
 <td>ras responsive element binding protein 1</td>
 <td>BE197381</td>
 <td>1428657_at</td>
 <td>&nbsp;</td>
 <td>0.138235114</td>
 <td>7.93691E-07</td>
 <td>&nbsp;</td>
 <td>0.651220705</td>
 <td>2.94209E-05</td>
 </tr>
 <tr>
 <td>glial cell line derived neurotrophic factor family receptor alpha 2</td>
 <td>BB284482</td>
 <td>1433716_x_at</td>
 <td>&nbsp;</td>
 <td>0.139062563</td>
 <td>2.35371E-06</td>
 <td>&nbsp;</td>
 <td>0.669544709</td>
 <td>0.000214146</td>
 </tr>
 <tr>
 <td>preproenkephalin 1</td>
 <td>M13227</td>
 <td>1427038_at</td>
 <td>&nbsp;</td>
 <td>6.9850435</td>
 <td>2.39074E-08</td>
 <td>&nbsp;</td>
 <td>1.766018828</td>
 <td>0.000250501</td>
 </tr>
 <tr>
 <td>RIKEN cDNA 1810010H24 gene</td>
 <td>BI729991</td>
 <td>1428809_at</td>
 <td>&nbsp;</td>
 <td>6.8658915</td>
 <td>1.88516E-05</td>
 <td>&nbsp;</td>
 <td>2.77573142</td>
 <td>6.81865E-09</td>
 </tr>
 <tr>
 <td>ryanodine receptor 1, skeletal muscle</td>
 <td>BG793713</td>
 <td>1457347_at</td>
 <td>&nbsp;</td>
 <td>0.151364292</td>
 <td>3.35612E-05</td>
 <td>&nbsp;</td>
 <td>0.503144617</td>
 <td>4.32907E-05</td>
 </tr>
 <tr>
 <td>protocadherin 21</td>
 <td>NM_130878</td>
 <td>1418304_at</td>
 <td>&nbsp;</td>
 <td>0.152671849</td>
 <td>8.57783E-06</td>
 <td>&nbsp;</td>
 <td>0.670714726</td>
 <td>1.56309E-05</td>
 </tr>
 <tr>
 <td>cornichon homolog 3 (Drosophila)</td>
 <td>NM_028408</td>
 <td>1419517_at</td>
 <td>&nbsp;</td>
 <td>0.153724144</td>
 <td>8.90755E-06</td>
 <td>&nbsp;</td>
 <td>0.95780695</td>
 <td>0.661055608</td>
 </tr>
 <tr>
 <td>harakiri, BCL2 interacting protein (contains only BH3 domain)</td>
 <td>BQ175572</td>
 <td>1439854_at</td>
 <td>&nbsp;</td>
 <td>0.154284407</td>
 <td>2.0118E-05</td>
 <td>&nbsp;</td>
 <td>0.56516812</td>
 <td>4.86925E-09</td>
 </tr>
 <tr>
 <td>carbohydrate (N-acetylgalactosamine 4-0) sulfotransferase 9</td>
 <td>AK017407</td>
 <td>1431897_at</td>
 <td>&nbsp;</td>
 <td>0.155238951</td>
 <td>5.37423E-06</td>
 <td>&nbsp;</td>
 <td>1.14910007</td>
 <td>0.215637733</td>
 </tr>
 <tr>
 <td>calpain 3</td>
 <td>AI323605</td>
 <td>1433681_x_at</td>
 <td>&nbsp;</td>
 <td>0.160871988</td>
 <td>1.07655E-05</td>
 <td>&nbsp;</td>
 <td>0.477164757</td>
 <td>1.33753E-11</td>
 </tr>
 <tr>
 <td>zinc finger, CCHC domain containing 5</td>
 <td>BQ126004</td>
 <td>1437355_at</td>
 <td>&nbsp;</td>
 <td>0.161812078</td>
 <td>3.08262E-06</td>
 <td>&nbsp;</td>
 <td>0.421252632</td>
 <td>0.01152969</td>
 </tr>
 <tr>
 <td>loricrin</td>
 <td>NM_008508</td>
 <td>1448745_s_at</td>
 <td>&nbsp;</td>
 <td>0.165129967</td>
 <td>1.86362E-05</td>
 <td>&nbsp;</td>
 <td>0.639733409</td>
 <td>0.000729772</td>
 </tr>
 <tr>
 <td>spondin 1, (f-spondin) extracellular matrix protein</td>
 <td>BC020531</td>
 <td>1451342_at</td>
 <td>&nbsp;</td>
 <td>0.168035879</td>
 <td>6.67867E-07</td>
 <td>&nbsp;</td>
 <td>0.821042412</td>
 <td>0.023650765</td>
 </tr>
 <tr>
 <td>RIKEN cDNA A930035E12 gene</td>
 <td>AV348640</td>
 <td>1429906_at</td>
 <td>&nbsp;</td>
 <td>5.9086795</td>
 <td>1.747E-07</td>
 <td>&nbsp;</td>
 <td>1.470383201</td>
 <td>0.104085454</td>
 </tr>
 </tbody>
 <tbody>
 <tr>
 <td>BB247294 RIKEN full-length enriched, 7 days neonate cerebellum Mus musculus cDNA clone A730018G18 3', mRNA sequence.</td>
 <td>BB247294</td>
 <td>1447907_x_at</td>
 <td>&nbsp;</td>
 <td>5.9047494</td>
 <td>1.04931E-05</td>
 <td>&nbsp;</td>
 <td>1.968147585</td>
 <td>0.00010636</td>
 </tr>
 <tr>
 <td>FERM domain containing 3</td>
 <td>BB099015</td>
 <td>1437075_at</td>
 <td>&nbsp;</td>
 <td>5.860216</td>
 <td>0.000345581</td>
 <td>&nbsp;</td>
 <td>2.780297178</td>
 <td>1.83072E-06</td>
 </tr>
 <tr>
 <td>neuronal pentraxin 2 /// hypothetical protein LOC100044234</td>
 <td>NM_016789</td>
 <td>1420720_at</td>
 <td>&nbsp;</td>
 <td>5.7568517</td>
 <td>1.34227E-06</td>
 <td>&nbsp;</td>
 <td>2.652516957</td>
 <td>0.000206279</td>
 </tr>
 <tr>
 <td>Transcribed sequences</td>
 <td>BG076361</td>
 <td>1460101_at</td>
 <td>&nbsp;</td>
 <td>5.657735</td>
 <td>2.5015E-06</td>
 <td>&nbsp;</td>
 <td>1.296248831</td>
 <td>0.22870031</td>
 </tr>
 <tr>
 <td>spondin 1, (f-spondin) extracellular matrix protein</td>
 <td>BC020531</td>
 <td>1424415_s_at</td>
 <td>&nbsp;</td>
 <td>0.17783576</td>
 <td>1.01658E-06</td>
 <td>&nbsp;</td>
 <td>0.836181248</td>
 <td>0.001380141</td>
 </tr>
 <tr>
 <td>calbindin-28K</td>
 <td>BB246032</td>
 <td>1448738_at</td>
 <td>&nbsp;</td>
 <td>0.180317904</td>
 <td>1.35961E-05</td>
 <td>&nbsp;</td>
 <td>0.647334052</td>
 <td>4.12268E-09</td>
 </tr>
 <tr>
 <td>MARCKS-like 1</td>
 <td>AV110584</td>
 <td>1437226_x_at</td>
 <td>&nbsp;</td>
 <td>0.186235935</td>
 <td>1.47067E-06</td>
 <td>&nbsp;</td>
 <td>0.499291387</td>
 <td>2.34984E-08</td>
 </tr>
 <tr>
 <td>matrilin 2</td>
 <td>BB338441</td>
 <td>1455978_a_at</td>
 <td>&nbsp;</td>
 <td>0.187783528</td>
 <td>6.19122E-05</td>
 <td>&nbsp;</td>
 <td>0.8967688</td>
 <td>0.282337853</td>
 </tr>
 <tr>
 <td>matrilin 2</td>
 <td>BC005429</td>
 <td>1419442_at</td>
 <td>&nbsp;</td>
 <td>0.188195795</td>
 <td>0.000105295</td>
 <td>&nbsp;</td>
 <td>0.915528892</td>
 <td>0.35282097</td>
 </tr>
 <tr>
 <td>spondin 1, (f-spondin) extracellular matrix protein</td>
 <td>BQ175871</td>
 <td>1442613_at</td>
 <td>&nbsp;</td>
 <td>0.189956563</td>
 <td>9.41195E-06</td>
 <td>&nbsp;</td>
 <td>0.861033222</td>
 <td>0.1394266</td>
 </tr>
 <tr>
 <td>arrestin 3, retinal</td>
 <td>NM_133205</td>
 <td>1450329_a_at</td>
 <td>&nbsp;</td>
 <td>5.2130346</td>
 <td>2.90599E-05</td>
 <td>&nbsp;</td>
 <td>3.944218329</td>
 <td>1.07437E-07</td>
 </tr>
 <tr>
 <td>RIKEN cDNA A330050F15 gene</td>
 <td>AV325555</td>
 <td>1457558_at</td>
 <td>&nbsp;</td>
 <td>0.19186781</td>
 <td>0.000119342</td>
 <td>&nbsp;</td>
 <td>0.660282035</td>
 <td>2.47342E-05</td>
 </tr>
 <tr>
 <td>contactin 3</td>
 <td>BB559510</td>
 <td>1438628_x_at</td>
 <td>&nbsp;</td>
 <td>0.194404608</td>
 <td>4.08641E-07</td>
 <td>&nbsp;</td>
 <td>0.918742591</td>
 <td>0.022545297</td>
 </tr>
 <tr>
 <td>calbindin-28K</td>
 <td>BB246032</td>
 <td>1417504_at</td>
 <td>&nbsp;</td>
 <td>0.196381321</td>
 <td>2.24182E-05</td>
 <td>&nbsp;</td>
 <td>0.619305124</td>
 <td>3.6222E-06</td>
 </tr>
 <tr>
 <td>gastrin releasing peptide</td>
 <td>BC024515</td>
 <td>1424525_at</td>
 <td>&nbsp;</td>
 <td>4.9436426</td>
 <td>3.00588E-05</td>
 <td>&nbsp;</td>
 <td>2.752845903</td>
 <td>5.72954E-07</td>
 </tr>
 <tr>
 <td>sortilin-related VPS10 domain containing receptor 3</td>
 <td>AK018111</td>
 <td>1425111_at</td>
 <td>&nbsp;</td>
 <td>4.885766</td>
 <td>1.03645E-05</td>
 <td>&nbsp;</td>
 <td>1.29051599</td>
 <td>0.029733649</td>
 </tr>
 <tr>
 <td>dopamine receptor D1A</td>
 <td>BE957273</td>
 <td>1455629_at</td>
 <td>&nbsp;</td>
 <td>4.869493</td>
 <td>3.77525E-05</td>
 <td>&nbsp;</td>
 <td>1.815881979</td>
 <td>0.000516498</td>
 </tr>
 <tr>
 <td>proprotein convertase subtilisin/kexin type 5</td>
 <td>BB241731</td>
 <td>1437339_s_at</td>
 <td>&nbsp;</td>
 <td>0.210528027</td>
 <td>7.83039E-05</td>
 <td>&nbsp;</td>
 <td>0.574126078</td>
 <td>9.15496E-05</td>
 </tr>
 </tbody>
 <tbody>
 <tr>
 <td>interleukin 1 receptor, type I</td>
 <td>NM_008362</td>
 <td>1448950_at</td>
 <td>&nbsp;</td>
 <td>0.210572243</td>
 <td>9.64524E-06</td>
 <td>&nbsp;</td>
 <td>0.241135352</td>
 <td>2.79816E-08</td>
 </tr>
 </tbody>
</table>

dissection of hippocampal dentate gyrus from adult mouse

The hippocampus is one of the most widely studied areas in the brain because of its important functional role in memory processing and learning, its remarkable neuronal cell plasticity, and its involvement in epilepsy, neurodegenerative diseases, and psychiatric disorders. The hippocampus is composed of distinct regions; the dentate gyrus, which comprises mainly granule neurons, and Ammon's horn, which comprises mainly pyramidal neurons, and the two regions are connected by both anatomic and functional circuits. Many different mRNAs and proteins are selectively expressed in the dentate gyrus, and the dentate gyrus is a site of adult neurogenesis; that is, new neurons are continually generated in the adult dentate gyrus. To investigate mRNA and protein expression specific to the dentate gyrus, laser capture microdissection is often used. This method has some limitations, however, such as the need for special apparatuses and complicated handling procedures. In this video-recorded protocol, we demonstrate a dissection technique for removing the dentate gyrus from adult mouse under a stereomicroscope. Dentate gyrus samples prepared using this technique are suitable for any assay, including transcriptomic, proteomic, and cell biology analyses. We confirmed that the dissected tissue is dentate gyrus by conducting real-time PCR of dentate gyrus-specific genes, tryptophan 2,3-dioxygenase (TDO2) and desmoplakin (Dsp), and Ammon's horn enriched genes, Meis-related gene 1b (Mrg1b) and TYRO3 protein tyrosine kinase 3 (Tyro3). The mRNA expressions of TDO2 and Dsp in the dentate gyrus samples were detected at obviously higher levels, whereas Mrg1b and Tyro3 were lower levels, than those in the Ammon's horn samples. To demonstrate the advantage of this method, we performed DNA microarray analysis using samples of whole hippocampus and dentate gyrus. The mRNA expression of TDO2 and Dsp, which are expressed selectively in the dentate gyrus, in the whole hippocampus of alpha-CaMKII+/- mice, exhibited 0.037 and 0.10-fold changes compared to that of wild-type mice, respectively. In the isolated dentate gyrus, however, these expressions exhibited 0.011 and 0.021-fold changes compared to that of wild-type mice, demonstrating that gene expression changes in dentate gyrus can be detected with greater sensitivity. Taken together, this convenient and accurate dissection technique can be reliably used for studies focused on the dentate gyrus.

Watch this Scientific Journal Video about Dissection of Hippocampal Dentate Gyrus from Adult Mouse at JoVE.com

Dissection of Hippocampal Dentate Gyrus from Adult Mouse

A dissection technique for removal of the dentate gyrus from adult mouse under a stereomicroscope was demonstrated in this video-recorded protocol.

The hippocampus is one of the most widely studied areas in the brain because of its important functional role in memory processing and learning, its ...

dissection-of-hippocampal-dentate-gyrus-from-adult-mouse

Research

JoVE Journal

Biology

Dissection of 6.5 dpc Mouse Embryos

Analysis of gene expression patterns during early stages of mammalian embryonic development can provide important clues about gene function, cell-cell interaction and signaling mechanisms that guide embryonic patterning. However, dissection of the mouse embryo from the decidua shortly after implantation can be a challenging procedure, and detailed step-by-step documentation of this process is lacking.

Here we demonstrate how post-implantation (6.5 dpc) embryos are isolated by first dissecting the uterus of a pregnant mouse (detection of the vaginal plug was designated day 0.5 poist coitum) and subsequently dissecting the embryo from maternal decidua. The dissection of Reichert's membrane is described as well as the removal of the ectoplacental cone.

Isolation of postimplantation-stage embryos allows one to study gene patterning and analyze cell-lineage decision making processes during embryonic development, but proper dissection of the early embryo can be challenging. This protocol describes a method for isolating early primitive-streak-stage embryos (~6.5 days post coitum [dpc]).

Analysis of gene expression patterns during early stages of mammalian embryonic development can provide important clues about gene function, cell-cell ...

Dissection of Organs from the Adult Zebrafish

Over the last 20 years, the zebrafish has become a powerful model organism for understanding vertebrate development and disease.  Although experimental analysis of the embryo and larva is extensive and the morphology has been well documented, descriptions of adult zebrafish anatomy and studies of development of the adult structures and organs, together with techniques for working with adults are lacking. The organs of the larva undergo significant changes in their overall structure, morphology, and anatomical location during the larval to adult transition.  Externally, the transparent larva develops its characteristic adult striped pigment pattern and paired pelvic fins, while internally, the organs undergo massive growth and remodeling.  In addition, the bipotential gonad primordium develops into either testis or ovary.  This protocol identifies many of the organs of the adult and demonstrates methods for dissection of the brain, gonads, gastrointestinal system, heart, and kidney of the adult zebrafish. The dissected organs can be used for in situ hybridization, immunohistochemistry, histology, RNA extraction, protein analysis, and other molecular techniques.  This protocol will assist in the broadening of studies in the zebrafish to include the remodeling of larval organs, the morphogenesis of organs specific to the adult and other investigations of the adult organ systems.

This protocol describes a procedure for identifying and dissecting organs from the adult zebrafish.

Over the last 20 years, the zebrafish has become a powerful model organism for understanding vertebrate development and disease.  Although experimental ...

Dissection of Oenocytes from Adult Drosophila melanogaster

In Drosophila melanogaster, as in other insects, a waxy layer on the outer surface of the cuticle, composed primarily of hydrocarbon compounds, provides protection against desiccation and other environmental challenges. Several of these cuticular hydrocarbon (CHC) compounds also function as semiochemical signals, and as such mediate pheromonal communications between members of the same species, or in some instances between different species, and influence behavior. Specialized cells referred to as oenocytes are regarded as the primary site for CHC synthesis. However, relatively little is known regarding the involvement of the oenocytes in the regulation of the biosynthetic, transport, and deposition pathways contributing to CHC output. Given the significant role that CHCs play in several aspects of insect biology, including chemical communication, desiccation resistance, and immunity, it is important to gain a greater understanding of the molecular and genetic regulation of CHC production within these specialized cells. The adult oenocytes of D. melanogaster are located within the abdominal integument, and are metamerically arrayed in ribbon-like clusters radiating along the inner cuticular surface of each abdominal segment. In this video article we demonstrate a dissection technique used for the preparation of oenocytes from adult D. melanogaster. Specifically, we provide a detailed step-by-step demonstration of (1) how to fillet prepare an adult Drosophila abdomen, (2) how to identify the oenocytes and discern them from other tissues, and (3) how to remove intact oenocyte clusters from the abdominal integument. A brief experimental illustration of how this preparation can be used to examine the expression of genes involved in hydrocarbon synthesis is included. The dissected preparation demonstrated herein will allow for the detailed molecular and genetic analysis of oenocyte function in the adult fruit fly.

Dissection of Oenocytes from Adult Drosophila melanogaster

In insects, the oenocytes produce cuticular hydrocarbon compounds. These compounds protect against desiccation and facilitate chemical communication. Here we demonstrate a dissection technique used to isolate the oenocytes from adult Drosophila melanogaster, and illustrate how this preparation can be utilized to study genes involved in hydrocarbon synthesis.

In Drosophila melanogaster, as in other insects, a waxy layer on the outer surface of the cuticle, composed primarily of hydrocarbon compounds, provides ...

Dissection of the Adult Zebrafish Kidney

Researchers working in the burgeoning field of adult stem cell biology seek to understand the signals that regulate the behavior and function of stem cells during normal homeostasis and disease states. The understanding of adult stem cells has broad reaching implications for the future of regenerative medicine1. For example, better knowledge about adult stem cell biology can facilitate the design of therapeutic strategies in which organs are triggered to heal themselves or even the creation of methods for growing organs in vitro that can be transplanted into humans1. The zebrafish has become a powerful animal model for the study of vertebrate cell biology2. There has been extensive documentation and analysis of embryonic development in the zebrafish3. Only recently have scientists sought to document adult anatomy and surgical dissection techniques4, as there has been a progressive movement within the zebrafish community to broaden the applications of this research organism to adult studies. For example, there are expanding interests in using zebrafish to investigate the biology of adult stem cell populations and make sophisticated adult models of diseases such as cancer5. Historically, isolation of the zebrafish adult kidney has been instrumental for studying hematopoiesis, as the kidney is the anatomical location of blood cell production in fish6,7. The kidney is composed of nephron functional units found in arborized arrangements, surrounded by hematopoietic tissue that is dispersed throughout the intervening spaces. The hematopoietic component consists of hematopoietic stem cells (HSCs) and their progeny that inhabit the kidney until they terminally differentiate8. In addition, it is now appreciated that a group of renal stem/progenitor cells (RPCs) also inhabit the zebrafish kidney organ and enable both kidney regeneration and growth, as observed in other fish species9-11. In light of this new discovery, the zebrafish kidney is one organ that houses the location of two exciting opportunities for adult stem cell biology studies. It is clear that many outstanding questions could be well served with this experimental system. To encourage expansion of this field, it is beneficial to document detailed methods of visualizing and then isolating the adult zebrafish kidney organ. This protocol details our procedure for dissection of the adult kidney from both unfixed and fixed animals.
Dissection of the kidney organ can be used to isolate and characterize hematopoietic and renal stem cells and their offspring using established techniques such as histology, fluorescence activated cell sorting (FACS)11,12, expression profiling13,14, and transplantation11,15. 
We hope that dissemination of this protocol will provide researchers with the knowledge to implement broader use of zebrafish studies that ultimately can be translated for human application.

The zebrafish kidney is home to both renal and hematopoietic adult stem/progenitor cells, and represents an outstanding opportunity to study these cell types and their progeny in a vertebrate model organism. Here, we demonstrate a detailed dissection procedure that enables the researcher to identify and surgically remove the adult zebrafish kidney, which can be used for applications such as cell isolation, transplantation, and expression studies of kidney and/or blood cell populations.

Researchers working in the burgeoning field of adult stem cell biology seek to understand the signals that regulate the behavior and function of stem ...

Heart Dissection in Larval, Juvenile and Adult Zebrafish, Danio rerio

Zebrafish have become a beneficial and practical model organism for the study of embryonic heart development (see recent reviews1-6), however, work examining post-embryonic through adult cardiac development has been limited7-10. Examining the changing morphology of the maturing and aging heart are restricted by the lack of techniques available for staging and isolating juvenile and adult hearts. In order to analyze heart development over the fish's lifespan, we dissect zebrafish hearts at numerous stages and photograph them for further analysis11. The morphological features of the heart can easily be quantified and individual hearts can be further analyzed by a host of standard methods. Zebrafish grow at variable rates and maturation correlates better with fish size than age, thus, post-fixation, we photograph and measure fish length as a gauge of fish maturation. This protocol explains two distinct, size dependent dissection techniques for zebrafish, ranging from larvae 3.5mm standard length (SL) with hearts of 100&mu;m ventricle length (VL), to adults, with SL of 30mm and VL 1mm or larger. Larval and adult fish have quite distinct body and organ morphology. Larvae are not only significantly smaller, they have less pigment and each organ is visually very difficult to identify. For this reason, we use distinct dissection techniques.
We used pre-dissection fixation procedures, as we discovered that hearts dissected directly after euthanization have a more variable morphology, with very loose and balloon like atria compared with hearts removed following fixation. The fish fixed prior to dissection, retain in vivo morphology and chamber position (data not shown). In addition, for demonstration purposes, we take advantage of the heart (myocardial) specific GFP transgenic Tg(myl7:GFP)twu34 (12), which allows us to visualize the entire heart and is particularly useful at early stages in development when the cardiac morphology is less distinct from surrounding tissues. Dissection of the heart makes further analysis of the cell and molecular biology underlying heart development and maturation using in situ hybridization, immunohistochemistry, RNA extraction or other analytical methods easier in post-embryonic zebrafish. This protocol will provide a valuable technique for the study of cardiac development maturation and aging.

Heart Dissection in Larval, Juvenile and Adult Zebrafish, Danio rerio

A clear, standardized method for dissection and isolation of the zebrafish heart at multiple developmental stages are described. Annotation and quantification techniques are also discussed.

Zebrafish have become a beneficial and practical model organism for the study of embryonic heart development (see recent reviews1-6), however, work ...

Generation of Mice Derived from Induced Pluripotent Stem Cells

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also produce a source of patient-matched cells for regenerative therapies. iPSCs may be generated using multiple protocols and derived from multiple cell sources. Once generated, iPSCs are tested using a variety of assays including immunostaining for pluripotency markers, generation of three germ layers in embryoid bodies and teratomas, comparisons of gene expression with embryonic stem cells (ESCs) and production of chimeric mice with or without germline contribution2. Importantly, iPSC lines that pass these tests still vary in their capacity to produce different differentiated cell types2. This has made it difficult to establish which iPSC derivation protocols, donor cell sources or selection methods are most useful for different applications.
The most stringent test of whether a stem cell line has sufficient developmental potential to generate all tissues required for survival of an organism (termed full pluripotency) is tetraploid embryo complementation (TEC)3-5. Technically, TEC involves electrofusion of two-cell embryos to generate tetraploid (4n) one-cell embryos that can be cultured in vitro to the blastocyst stage6. Diploid (2n) pluripotent stem cells (e.g. ESCs or iPSCs) are then injected into the blastocoel cavity of the tetraploid blastocyst and transferred to a recipient female for gestation (see Figure 1). The tetraploid component of the complemented embryo contributes almost exclusively to the extraembryonic tissues (placenta, yolk sac), whereas the diploid cells constitute the embryo proper, resulting in a fetus derived entirely from the injected stem cell line.
Recently, we reported the derivation of iPSC lines that reproducibly generate adult mice via TEC1. These iPSC lines give rise to viable pups with efficiencies of 5-13%, which is comparable to ESCs3,4,7 and higher than that reported for most other iPSC lines8-12. These reports show that direct reprogramming can produce fully pluripotent iPSCs that match ESCs in their developmental potential and efficiency of generating pups in TEC tests. At present, it is not clear what distinguishes between fully pluripotent iPSCs and less potent lines13-15. Nor is it clear which reprogramming methods will produce these lines with the highest efficiency. Here we describe one method that produces fully pluripotent iPSCs and "all- iPSC" mice, which may be helpful for investigators wishing to compare the pluripotency of iPSC lines or establish the equivalence of different reprogramming methods.

Generating induced pluripotent stem cell (iPSC) lines produces lines of differing developmental potential even when they pass standard tests for pluripotency. Here we describe a protocol to produce mice derived entirely from iPSCs, which defines the iPSC lines as possessing full pluripotency1.

The production of induced pluripotent stem cells (iPSCs) from somatic cells provides a means to create valuable tools for basic research and may also ...

Isolation and Culture of Dental Epithelial Stem Cells from the Adult Mouse Incisor

Understanding the cellular and molecular mechanisms that underlie tooth regeneration and renewal has become a topic of great interest1-4, and the mouse incisor provides a model for these processes. This remarkable organ grows continuously throughout the animal&#39;s life and generates all the necessary cell types from active pools of adult stem cells housed in the labial (toward the lip) and lingual (toward the tongue) cervical loop (CL) regions. Only the dental stem cells from the labial CL give rise to ameloblasts that generate enamel, the outer covering of teeth, on the labial surface. This asymmetric enamel formation allows abrasion at the incisor tip, and progenitors and stem cells in the proximal incisor ensure that the dental tissues are constantly replenished. The ability to isolate and grow these progenitor or stem cells in vitro allows their expansion and opens doors to numerous experiments not achievable in vivo, such as high throughput testing of potential stem cell regulatory factors. Here, we describe and demonstrate a reliable and consistent method to culture cells from the labial CL of the mouse incisor.

The continuously growing mouse incisor provides a model for studying renewal of dental tissues from dental epithelial stem cells (DESCs). A robust system for consistently and reliably obtaining these cells from the incisor and expanding them in vitro is reported here.

Understanding the cellular and molecular mechanisms that underlie tooth regeneration and renewal has become a topic of great interest1-4, and the mouse ...

Isolation and Culture of Primary Mouse Keratinocytes from Neonatal and Adult Mouse Skin

The keratinocyte (KC) is the predominant cell type in the epidermis, the outermost layer of the skin. Epidermal KCs play a critical role in providing skin defense by forming an intact skin barrier against environmental insults, such as UVB irradiation or pathogens, and also by initiating an inflammatory response upon those insults. Here we describe methods to isolate KCs from neonatal mouse skin and from adult mouse tail skin. We also describe culturing conditions using defined growth supplements (dGS) in comparison to chelexed fetal bovine serum (cFBS). Functionally, we show that both neonatal and adult KCs are highly responsive to high calcium-induced terminal differentiation, tight junction formation and stratification. Additionally, cultured adult KCs are susceptible to UVB-triggered cell death and can release large amounts of TNF upon UVB irradiation. Together, the methods described here will be useful to researchers for the setup of in vitro models to study epidermal biology in the neonatal mouse and/or the adult mouse.

Epidermal keratinocytes form a functional skin barrier and are positioned at the front line of host defense against external environmental insults. Here we describe methods for isolation and primary culture of epidermal keratinocytes from neonatal and adult mouse skin, and induction of terminal differentiation and UVB-triggered inflammatory response from keratinocytes.

The keratinocyte (KC) is the predominant cell type in the epidermis, the outermost layer of the skin. Epidermal KCs play a critical role in providing skin ...

Isolation of Enteric Glial Cells from the Submucosa and Lamina Propria of the Adult Mouse

The enteric nervous system (ENS) consists of neurons and enteric glial cells (EGCs) that reside within the smooth muscle wall, submucosa and lamina propria. EGCs play important roles in gut homeostasis through the release of various trophic factors and contribute to the integrity of the epithelial barrier. Most studies of primary enteric glial cultures use cells isolated from the myenteric plexus after enzymatic dissociation. Here, a non-enzymatic method to isolate and culture EGCs from the intestinal submucosa and lamina propria is described. After manual removal of the longitudinal muscle layer, EGCs were liberated from the lamina propria and submucosa using sequential HEPES-buffered EDTA incubations followed by incubation in commercially available non-enzymatic cell recovery solution. The EDTA incubations were sufficient to strip most of the epithelial mucosa from the lamina propria, allowing the cell recovery solution to liberate the submucosal EGCs. Any residual lamina propria and smooth muscle was discarded along with the myenteric glia. EGCs were easily identified by their ability to express glial fibrillary acidic protein (GFAP). Only about 50% of the cell suspension contained GFAP+ cells after completing tissue incubations and prior to plating on the poly-D-lysine/laminin substrate. However, after 3 days of culturing the cells in glial cell-derived neurotrophic factor (GDNF)-containing culture media, the cell population adhering to the substrate-coated plates comprised of &#62;95% enteric glia. We created a hybrid mouse line by breeding a hGFAP-Cre mouse to the ROSA-tdTomato reporter line to track the percentage of GFAP+ cells using endogenous cell fluorescence. Thus, non-myenteric enteric glia can be isolated by non-enzymatic methods and cultured for at least 5 days.

Here, we describe the isolation of enteric-glial cells from the intestinal-submucosa using sequential EDTA incubations to chelate divalent cations and then incubation in non-enzymatic cell recovery solution. Plating the resultant cell suspension on poly-D-lysine and laminin results in a highly enriched culture of submucosal glial cells for functional analysis.

The enteric nervous system (ENS) consists of neurons and enteric glial cells (EGCs) that reside within the smooth muscle wall, submucosa and lamina ...

Isolation, Characterization, and Differentiation of Cardiac Stem Cells from the Adult Mouse Heart

Myocardial infarction (MI) is a leading cause of morbidity and mortality around the world. A major goal of regenerative medicine is to replenish the dead myocardium after MI. Although several strategies have been used to regenerate myocardium, stem cell therapy remains a major approach to replenish the dead myocardium of an MI heart. Accumulating evidence suggests the presence of resident cardiac stem cells (CSCs) in the adult heart and their endocrine and/or paracrine effects on cardiac regeneration. However, CSC isolation and their characterization and differentiation toward myocardial cells, especially cardiomyocytes, remains a technical challenge. In the present study, we provided a simple method for the isolation, characterization, and differentiation of CSCs from the adult mouse heart. Here, we describe a density gradient method for the isolation of CSCs, where the heart is digested by a 0.2% collagenase II solution. To characterize the isolated CSCs, we evaluated the expression of CSCs/cardiac markers Sca-1, NKX2-5, and GATA4, and pluripotency/stemness markers OCT4, SOX2, and Nanog. We also determined the proliferation potential of isolated CSCs by culturing them in a Petri dish and assessing the expression of the proliferation marker Ki-67. For evaluating the differentiation potential of CSCs, we selected seven- to ten-days cultured CSCs. We transferred them to a new plate with a cardiomyocyte differentiation medium. They are incubated in a cell culture incubator for 12 days, while the differentiation medium is changed every three days. The differentiated CSCs express cardiomyocyte-specific markers: actinin and troponin I. Thus, CSCs isolated with this protocol have stemness and cardiac markers, and they have a potential for proliferation and differentiation toward cardiomyocyte lineage.

The overall goal of this article is to standardize the protocol for the isolation, characterization, and differentiation of cardiac stem cells (CSCs) from the adult mouse heart. Here, we describe a density gradient centrifugation method to isolate murine CSCs and elaborated methods for CSC culture, proliferation, and differentiation into cardiomyocytes.

Myocardial infarction (MI) is a leading cause of morbidity and mortality around the world. A major goal of regenerative medicine is to replenish the dead ...