This study was supported by the Beijing Natural Science Foundation (No. 7202119) and the National Natural Science Foundation of China (No. 82274380).

The experiments were performed in the Laboratory of Physiology at the Beijing University of Chinese Medicine, China. All study methods were performed in accordance with the relevant guidelines and regulations of Beijing University.
1. Cell culture
<ol>
	<li>Add 10% fetal bovine serum and 1% penicillin/streptomycin to Dulbecco&#39;s modified Eagle medium (DMEM) basic medium (with 4.5 g/L D-glucose, 4.g.g/L L-glutamine, and 110 mg/L sodium pyruvate; see Table of Materials) to prepare DMEM complete medium.</li>
	<li>Thaw the liquid nitrogen-frozen H9c2 myocardial cells (see Table of Materials) in warm water at 37 &#176;C with a quick and uniform stir until the ice melts.</li>
	<li>Transfer the cells to a centrifuge tube and add four times the volume of the DMEM complete medium. Centrifuge at 358 x g for 5 min at room temperature and discard the supernatant using a pipette.</li>
	<li>Dilute the obtained cell suspension with culture medium, blow gently, and inoculate the cells in a culture flask. Culture in an incubator at 37 &#176;C with 5% CO2.</li>
</ol>
2. Determination of cell viability
<ol>
	<li>Dissolve crocetin (see Table of Materials) in dimethyl sulfoxide (DMSO) to concentrations of 0.05 mM, 0.1 mM, 0.5 mM, 1 mM, 5 mM, 10 mM, 50 mM, 100 mM, and 200 mM.</li>
	<li>Dissociate the H9c2 myocardial cells by trypsin, then neutralize the mixture with DMEM complete medium.</li>
	<li>Transfer the cells to a centrifuge tube and centrifuge at 179 x g for 5 min at room temperature. Discard the supernatant and mix the cells in DMEM complete medium by gently blowing.</li>
	<li>Count the cells using a blood cell counting plate22 (see Table of Materials) and dilute them to 5 &#215; 104 cells/mL with DMEM complete medium.</li>
	<li>Divide the cells into nine equal portions. Add DMSO (diluted in a 1:1,000 ratio with DMEM complete medium) to the control group and add different concentrations of crocetin to the remaining groups at a ratio of 1:1,000.</li>
	<li>Seed the cells in 96-well plates at 100 &#181;L per well. Discard the supernatant after incubation for 24 h and wash the cells three times with PBS.</li>
	<li>After adding 100 &#181;L of DMEM basal medium, incubate the cells for 4 h and add 20 &#181;L of MTS (see Table of Materials).</li>
	<li>Incubate the cells for another 2 h, measure the absorbance at a wavelength of 490 nm, and calculate the cell viability. 
	&#8203;NOTE: Cell viability = (OD treated - OD blank) / (OD control - OD blank) &#215; 100.</li>
</ol>
3. Determination of lactate dehydrogenase (LDH), creatine kinase (CK), malondialdehyde (MDA), superoxide dismutase (SOD), glutathione peroxidase (GSH Px), and catalase (CAT)
<ol>
	<li>Seed the H9c2 cells in a 6-well plate at a density of 1 &#215; 105 cells. Collect the supernatant after the intervention and detect LDH and SOD levels, according to the manufacturer&#39;s instructions (see Table of Materials).</li>
	<li>Collect the supernatant and wash it with phosphate-buffered saline (PBS) once. Add the cell lysate to the culture dish and allow it to sit on ice for 20 min. Collect the liquid from the culture dish into a centrifuge tube.</li>
	<li>Detect CK, MDA, GSH-Px, and CAT levels according to the manufacturer&#39;s instructions23 (see Table of Materials and Supplementary File 1).</li>
	<li>Detect the total protein concentration of the lysis by the bicinchoninic acid (BCA) method to correct the concentration of CK, MDA, GSH-Px, and CAT24(see Table of Materials).</li>
</ol>
4. Determination of ROS
<ol>
	<li>Seed the H9c2 cells in a 48-well plate at a density of 5 &#215; 103 cells. Dilute DCFH-DA (see Table of Materials) at a 1:1,000 ratio with the serum-free medium. Discard the cell supernatant and wash the cell twice with a serum-free medium.</li>
	<li>Add 150 &#181;L of diluted DCFH-DA into each well and incubate at 37 &#176;C for 20 min. Wash the cells three times with a serum-free medium and capture images under a fluorescence microscope (see Table of Materials).</li>
</ol>
5. Detection of mitochondrial membrane potential
<ol>
	<li>Detect the mitochondrial membrane potential using a JC-1 mitochondrial membrane potential assay kit25 (see Table of Materials). Discard the culture medium and wash them once with serum-free medium.</li>
	<li>Add JC-1 working solution to each tube and mix them well. Incubate at 37 &#176;C for 20 min and wash the cells twice with JC-1 staining buffer. Add complete medium to each well and capture photographs under a fluorescence microscope.</li>
</ol>
6. TUNEL staining assay
<ol>
	<li>Use a TUNEL apoptosis assay kit (see Table of Materials) to determine apoptosis rates26. Discard the supernatant and wash the pellet once with serum-free medium.</li>
	<li>Add cell fixation solution and wash them with PBS once after incubating at room temperature for 30 min.</li>
	<li>Add 0.3% triton-100 to each well and incubate at room temperature for 5 min. Wash the cells with PBS twice. Add TUNEL detection working solution and incubate at 37 &#176;C in the dark for 60 min.</li>
	<li>Add an anti-fluorescence quenching sealing tablet containing 4&#8242;,6-diamidino-2-phenylindole (DAPI; see Table of Materials) and capture photographs under a fluorescence microscope.</li>
</ol>
7. Monitoring autophagic flow by transfection of mCherry GFP-LC3B adenovirus
<ol>
	<li>Replace half of the medium in each well with fresh medium. Add Ad-mCherry GFP-LC3B adenovirus (multiplicity of infection [MOI] of 2) to the culture medium and add 5 &#181;g/mL polybrene (see Table of Materials) to improve the infection efficiency.</li>
	<li>Replace the fresh medium after 24 h and observe the expression of the fluorescent protein under a confocal microscope.</li>
	<li>Culture the cells with the same conditions as section 1 after confirming successful virus infection and capture images under the confocal microscope27. 
	&#8203;NOTE: More than 20% of cells showed green fluorescent protein (GFP) fluorescence, indicating a successful infection.</li>
</ol>
8. Western blot analysis
<ol>
	<li>Collect cells for whole protein extraction and lyse them (RIPA, protease inhibitor, and phosphatase inhibitor = 100:1:1; see Table of Materials) on ice for 30 min.</li>
	<li>Add 5x protein loading buffer to the protein sample after protein quantification and boil the samples for 10 min.</li>
	<li>Electrophorese the samples that contain equal amounts of protein with sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gels28. Then, transfer the samples onto polyvinylidene difluoride (PVDF) membranes.</li>
	<li>Block the PVDF membranes for 1 h with 5% skim milk powder and incubate with the primary antibody (PINK1, Parkin; see Table of Materials) at 4 &#176;C overnight and the secondary antibody at room temperature for 1 h.</li>
	<li>Detect the target bands using enhanced chemiluminescence (ECL) solution and the chemiluminescence detection system. Use Image J software to quantify the bands via densitometry28.</li>
</ol>
9. Detection of Parkin&#39;s mitochondrial translocation by immunofluorescence
<ol>
	<li>Observe the colocalization of Parkin with mitochondria by double immunofluorescence staining. Discard the cell supernatant from each group and wash them three times with PBS.</li>
	<li>Fix the cells for 10 min at room temperature with 4% paraformaldehyde and add 0.1% triton-100 in PBS.</li>
	<li>Block with animal-free block solution (see Table of Materials) for 1 h to prevent non-specific binding between proteins and antibodies and reduce the fluorescence background.</li>
	<li>Incubate with a primary antibody (Parkin and Tom20; see Table of Materials) at 4 &#176;C overnight.</li>
	<li>Add a fluorescent secondary antibody (see Table of Materials) and incubate in the dark for 1 h.</li>
	<li>Add the DAPI-containing anti-fluorescence quenching tablets and capture images under the confocal microscope.</li>
</ol>
10. Statistical analysis
<ol>
	<li>Perform statistical analysis using graphing and analysis software (see Table of Materials).</li>
	<li>Compare continuous variables between groups using one-way ANOVA. p &#60; 0.05 was considered statistically significant.</li>
</ol>

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M., Yanagawa, T., Iijima, M., Sesaki, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SQSTM1/p62+promotes+mitochondrial+ubiquitination+independently+of+PINK1+and+PRKN/parkin+in+mitophagy.">SQSTM1/p62 promotes mitochondrial ubiquitination independently of PINK1 and PRKN/parkin in mitophagy.</a> Autophagy. 15 (11), 2012-2018 (2019).</li><li>Klionsky, D. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Guidelines+for+the+use+and+interpretation+of+assays+for+monitoring+autophagy+(4th+edition).">Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition).</a> Autophagy. 17 (1), 1 (2021).</li></ol>

The authors have no conflicts of interest to declare.

The exploration of effective ingredients from complex compounds of natural drugs through advanced technology has been a hotspot of TCM research29, and can provide laboratory evidence for future drug development after verification. Safflower is a representative drug in the treatment of &#34;promoting blood circulation and minimizing blood stasis&#34; and is widely used in the treatment of myocardial infarction30,31. Saffron is believed to h...

Acute myocardial infarction (AMI) is a life-threatening myocardial necrosis caused by severe and persistent ischemia and hypoxia to coronary arteries1,2. Percutaneous coronary intervention (PCI) is one of the first-line therapeutic strategies for AMI, and usually protects cardiomyocytes from ischemic damage3,4. The distal myocardium will lack blood and oxygen supply if not promptly and effectively treated after AMI, which leads to ischemic necrosis and further cardiovascular complications5,6. Promoting cardiomyocyte recovery and minimizing irreversible myocardial damage after missing the PCI surgical opportunity has been a research hotspot. After AMI, cardiomyocytes are in a state of ischemia and hypoxia, resulting in the inhibition of mitochondrial oxidative phosphorylation, reduction of NAD+ to NADPH, and increased single electron reduction7. As a result, the incomplete reduction reaction of oxygen generates an excess of reactive oxygen species (ROS) and ultimately leads to oxidative stress damage to cardiomyocytes8. An excessive accumulation of ROS triggers lipid peroxidation, further disrupting the structure and function of mitochondrial membranes. The result is a continuous opening of mitochondrial permeability transition pores and a decrease in mitochondrial membrane potential, inducing apoptosis and necrosis.
Angiotensin-converting enzyme (ACE) inhibitors, angiotensin-receptor blockers (ARBs), the inhibitors of &#946;-adrenoceptors, aldosterone antagonists, and other standard drugs in AMI can help enhance heart function after myocardial infarction and prevent the occurrence of malignant events, such as arrhythmias and left ventricular remodeling9. However, postinfarction survival and prognosis are greatly affected by infarct size, and satisfactory results have not been achieved for reducing cardiomyocyte apoptosis10,11. Thus, the development of drugs to promote cardiomyocyte recovery after myocardial infarction has become an urgent issue.
Traditional medicine has been a source of inspiration for modern pharmaceutical research for many years12,13,14,15. Traditional Chinese medicine (TCM) has a long history in the treatment of AMI, and a series of randomized control trials in recent years have confirmed that TCM can indeed improve the prognosis of patients16,17. According to TCM theory, AMI is caused by blood stasis18,19, so drugs for promoting blood circulation are usually used for the treatment of AMI in the acute phase20. Among them, saffron is believed to have a powerful effect on blood activation and stasis, and is often used in the acute treatment of AMI. Crocetin, a major component of saffron, may play a key role in protecting cardiomyocytes21.
In this study, H9c2 myocardial cells were induced by H2O2 to simulate myocardial ischemia/reperfusion, which causes a cardiomyocyte injury of AMI, and crocetin was used as an intervention to investigate its protective effect against oxidative stress-induced myocardial injury. The mechanism of crocetin protecting cardiomyocytes was further explored through mitophagy. More importantly, this article provides a reference for the technical approach to the study of mitophagy and describes the entire experimental procedure in detail.

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			<td>0.25% trypsin</td>
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			<td>1% Penicillin-streptomycin</td>
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			<td>V900929</td>
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			<td>5x protein loading buffer</td>
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			<td>B1030-5</td>
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			<td>Ad-mCherry GFP-LC3B adenovirus</td>
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			<td>Alexa Fluor 488-conjugated goat anti-rabbit IgG (H+L)&#160;</td>
			<td>Zhongshan Golden Bridge Biotechnology Co., Ltd.</td>
			<td>ZF-0514</td>
			<td></td>
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			<td>Alexa Fluor 594-conjugated goat anti-mouse IgG (H+L)</td>
			<td>Zhongshan Golden Bridge Biotechnology Co., Ltd.</td>
			<td>ZF-0513</td>
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			<td>Animal-free blocking solution</td>
			<td>CST</td>
			<td>15019s</td>
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			<td>Anti-Parkin antibody</td>
			<td>Santa Cruz</td>
			<td>sc-32282</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-PINK1 antibody</td>
			<td>ABclonal</td>
			<td>A11435</td>
			<td></td>
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			<td>Anti-TOM20 antibody</td>
			<td>ABclonal</td>
			<td>A19403</td>
			<td></td>
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			<td>Anti-&#946;-actin&#160; antibody</td>
			<td>ABclonal</td>
			<td>AC026</td>
			<td></td>
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			<td>BCA protein assay kit</td>
			<td>KeyGEN Biotech</td>
			<td>KGP902</td>
			<td></td>
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			<td>Blood cell counting plate</td>
			<td>Servicebio</td>
			<td>WG607</td>
			<td></td>
		</tr>
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			<td>CAT assay kits</td>
			<td>Nanjing Jiancheng Bioengineering Institute</td>
			<td>A007-1-1</td>
			<td></td>
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		<tr>
			<td>Chemiluminescence detection system</td>
			<td>Shanghai Qinxiang Scientific Instrument Factory</td>
			<td>ChemiScope 6100</td>
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			<td>CK assay kits</td>
			<td>Nanjing Jiancheng Bioengineering Institute</td>
			<td>A032-1-1</td>
			<td></td>
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		<tr>
			<td>Coenzyme Q10 (CoQ 10)</td>
			<td>Macklin</td>
			<td>C6129</td>
			<td></td>
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			<td>Crocetin</td>
			<td>Chengdu Ruifensi Biotechnology Co., Ltd.</td>
			<td>RFS-Z01802006012</td>
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		<tr>
			<td>DAPI-containing antifluorescence quenching tablets</td>
			<td>Zhongshan Golden Bridge Biotechnology Co., Ltd.</td>
			<td>ZLI-9557</td>
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			<td>DCFH-DA</td>
			<td>Beyotime</td>
			<td>S0033S</td>
			<td></td>
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			<td>DMSO</td>
			<td>Solarbio</td>
			<td>D8371</td>
			<td></td>
		</tr>
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			<td>Dulbecco&#39;s modified eagle medium (DMEM)</td>
			<td>Gibco</td>
			<td>8122091</td>
			<td></td>
		</tr>
		<tr>
			<td>Enhanced Chemiluminescence (ECL) solution</td>
			<td>NCM Biotech</td>
			<td>P10100</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum (FBS)</td>
			<td>Corning-Cellgro</td>
			<td>35-081-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>GraphPad Prism 7.0&#160;</td>
			<td>https://www.graphpad.com/</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>GSH-Px assay kits</td>
			<td>Nanjing Jiancheng Bioengineering Institute</td>
			<td>A005-1-2</td>
			<td></td>
		</tr>
		<tr>
			<td>H9c2 myocardial cells</td>
			<td>Beijing Dingguochangsheng Biotech Co., Ltd.</td>
			<td>CS0062</td>
			<td></td>
		</tr>
		<tr>
			<td>Horseradish peroxidase (HRP)-conjugated goat anti-goat IgG (H+L)&#160;</td>
			<td>Zhongshan Golden Bridge Biotechnology Co., Ltd.</td>
			<td>ZB-2305</td>
			<td></td>
		</tr>
		<tr>
			<td>Horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (H+L)&#160;</td>
			<td>Zhongshan Golden Bridge Biotechnology Co., Ltd.</td>
			<td>ZB-2301</td>
			<td></td>
		</tr>
		<tr>
			<td>JC-1 mitochondrial membrane potential assay kit</td>
			<td>LABLEAD</td>
			<td>J22202</td>
			<td></td>
		</tr>
		<tr>
			<td>LDH assay kits</td>
			<td>Nanjing Jiancheng Bioengineering Institute</td>
			<td>A020-2-2</td>
			<td></td>
		</tr>
		<tr>
			<td>MDA assay kits</td>
			<td>Nanjing Jiancheng Bioengineering Institute</td>
			<td>A003-2-2</td>
			<td></td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>Aladdin</td>
			<td>A2114057</td>
			<td></td>
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		<tr>
			<td>MTS assay</td>
			<td>Promega</td>
			<td>G3581</td>
			<td></td>
		</tr>
		<tr>
			<td>Perhydrol</td>
			<td>G-clone</td>
			<td>CS7730</td>
			<td></td>
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		<tr>
			<td>Phosphatase inhibitor</td>
			<td>CWBIO</td>
			<td>CW2383</td>
			<td></td>
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			<td>Polybrene</td>
			<td>Beyotime</td>
			<td>C0351</td>
			<td></td>
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			<td>Polyvinylidene difluoride (PVDF) membranes</td>
			<td>Millipore</td>
			<td>ISEQ00010</td>
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			<td>Radioimmunoprecipitation assay (RIPA) lysis buffer</td>
			<td>Solarbio</td>
			<td>R0010</td>
			<td></td>
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		<tr>
			<td>SDS-PAGE gels</td>
			<td>Shanghai Epizyme Biomedical Technology</td>
			<td>PG112</td>
			<td></td>
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		<tr>
			<td>SDS-PAGE running buffer powder</td>
			<td>Servicebio</td>
			<td>G2018-1L</td>
			<td></td>
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		<tr>
			<td>SDS-PAGE transfer buffer powder</td>
			<td>Servicebio</td>
			<td>G2017-1L</td>
			<td></td>
		</tr>
		<tr>
			<td>SOD assay kits</td>
			<td>Nanjing Jiancheng Bioengineering Institute</td>
			<td>A001-2-2</td>
			<td></td>
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		<tr>
			<td>Tris-buffered saline powder</td>
			<td>Servicebio</td>
			<td>G0001-2L</td>
			<td></td>
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			<td>Triton X-100</td>
			<td>Sigma</td>
			<td>SLCC9172</td>
			<td></td>
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		<tr>
			<td>TUNEL apoptosis assay kit</td>
			<td>Beyotime</td>
			<td>C1086</td>
			<td></td>
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		<tr>
			<td>Tween-20</td>
			<td>Solarbio</td>
			<td>T8220</td>
			<td></td>
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protection of h9c2 myocardial cells from oxidative stress by crocetin via pink1/parkin pathway-mediated mitophagy

This study aimed to explore the oxidative stress-protective effect of crocetin on H2O2-mediated H9c2 myocardial cells through in vitro experiments, and further explore whether its mechanism is related to the impact of mitophagy. This study also aimed to demonstrate the therapeutic effect of safflower acid on oxidative stress in cardiomyocytes and explore whether its mechanism is related to the effect of mitophagy. Here, an H2O2-based oxidative stress model was constructed and assessed the degree of oxidative stress injury of cardiomyocytes by detecting the levels of lactate dehydrogenase (LDH), creatine kinase (CK), malondialdehyde (MDA), superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH Px). Reactive oxygen species (ROS)-detecting fluorescent dye DCFH-DA, JC-1 dye, and TUNEL dye were employed to assess mitochondrial damage and apoptosis. Autophagic flux was measured by transfecting Ad-mCherry-GFP-LC3B adenovirus. Mitophagy-related proteins were then detected via western blotting and immunofluorescence. However, crocetin (0.1-10 &#181;M) could significantly improve cell viability and reduce apoptosis and oxidative stress damage caused by H2O2. In cells with excessive autophagic activation, crocetin could also reduce autophagy flow and the expression of mitophagy-related proteins PINK1 and Parkin, and reverse the transfer of Parkin to mitochondria. Crocetin could reduce H2O2-mediated oxidative stress damage and the apoptosis of H9c2 cells, and its mechanism was closely related to mitophagy.

Effects of crocetin on cell viability 
Crocetin at 0.1 &#181;M, 0.5 &#181;M, 1 &#181;M, 5 &#181;M, 10 &#181;M, 50 &#181;M, and 100 &#181;M had a significant proliferative effect on cells, while crocetin at concentrations above 200 &#181;M significantly inhibited the proliferation of H9c2 cells (Figure 1A). After 4 h of treatment with 400 &#181;M H2O2, the cell viability was reduced considerably, and crocetin could reverse this change to a certain e...

Watch this Scientific Journal Video about Protection of H9c2 Myocardial Cells from Oxidative Stress by Crocetin via PINK1/Parkin Pathway-Mediated Mitophagy at JoVE.com

Protection of H9c2 Myocardial Cells from Oxidative Stress by Crocetin via PINK1/Parkin Pathway-Mediated Mitophagy

Based on in vitro experiments, this study revealed the mechanism of crocetin in repairing oxidative stress damage of cardiomyocytes by influencing mitophagy, in which the PINK1/Parkin signaling pathway plays an important role.

Protection of H9c2 Myocardial Cells from Oxidative Stress by Crocetin via PINK1/Parkin Pathway-Mediated Mitophagy

This study aimed to explore the oxidative stress-protective effect of crocetin on H2O2-mediated H9c2 myocardial cells through in vitro experiments, and ...

protection-h9c2-myocardial-cells-from-oxidative-stress-crocetin-via

Research

JoVE Journal

Medicine

975 Views.  Beijing University of Chinese Medicine. Based on in vitro experiments, this study revealed the mechanism of crocetin in repairing oxidative stress damage of cardiomyocytes by influencing mitophagy, in which the PINK1/Parkin signaling pathway plays an important role.

Video: Protection of H9c2 Myocardial Cells from Oxidative Stress by Crocetin via PINK1/Parkin Pathway-Mediated Mitophagy

Isolation, Characterization and Comparative Differentiation of Human Dental Pulp Stem Cells Derived from Permanent Teeth by Using Two Different Methods

Developing wisdom teeth are easy-accessible source of stem cells during the adulthood which could be obtained by routine orthodontic treatments. Human pulp-derived stem cells (hDPSCs) possess high proliferation potential with multi-lineage differentiation capacity compare to the ordinary source of adult stem cells1-8; therefore, hDPSCs could be the good candidates for autologous transplantation in tissue engineering and regenerative medicine. Along with these benefits, possessing the mesenchymal stem cells (MSC) features, such as immunolodulatory effect, make hDPSCs more valuable, even in the case of allograft transplantation6,9,10. Therefore, the primary step for using this source of stem cells is to select the best protocol for isolating hDPSCs from pulp tissue. In order to achieve this goal, it is crucial to investigate the effect of various isolation conditions on different cellular behaviors, such as their common surface markers &amp; also their differentiation capacity.
Thus, here we separate human pulp tissue from impacted third molar teeth, and then used both existing protocols based on literature, for isolating hDPSCs,11-13 i.e. enzymatic dissociation of pulp tissue (DPSC-ED) or outgrowth from tissue explants (DPSC-OG). In this regards, we tried to facilitate the isolation methods by using dental diamond disk. Then, these cells characterized in terms of stromal-associated Markers (CD73, CD90, CD105 &amp; CD44), hematopoietic/endothelial Markers (CD34, CD45 &amp; CD11b), perivascular marker, like CD146 and also STRO-1. Afterwards, these two protocols were compared based on the differentiation potency into odontoblasts by both quantitative polymerase chain reaction (QPCR) &amp; Alizarin Red Staining. QPCR were used for the assessment of the expression of the mineralization-related genes (alkaline phosphatase; ALP, matrix extracellular phosphoglycoprotein; MEPE &amp; dentin sialophosphoprotein; DSPP).14

The method described isolation and characterization of human Dental Pulp Stem Cells (hDPSCs) by using either enzymatic dissociation of pulp (DPSC-ED) or direct outgrowth of stem cells from pulp tissue explants (DPSC-OG). Then followed by in vitro comparative differentiation of both types of hDPSCs into odontoblasts.

Developing wisdom teeth are easy-accessible source of stem cells during the adulthood which could be obtained by routine orthodontic treatments. Human ...

Cardiac Stress Test Induced by Dobutamine and Monitored by Cardiac Catheterization in Mice

Dobutamine is a &beta;-adrenergic agonist with an affinity higher for receptor expressed in the heart (&beta;1) than for receptors expressed in the arteries (&beta;2). When systemically administered, it increases cardiac demand. Thus, dobutamine unmasks abnormal rhythm or ischemic areas potentially at risk of infarction. 
Monitoring of heart function during a cardiac stress test can be performed by either ecocardiography or cardiac catheterization. The latter is an invasive but more accurate and informative technique that the former.
Cardiac stress test induced by dobutamine and monitored by cardiac catheterization accomplished as described here allows, in a single experiment, the measurement of the following hemodynamic parameters: heart rate (HR), systolic pressure, diastolic pressure, end-diastolic pressure, maximal positive pressure development (dP/dtmax) and maximal negative pressure development (dP/dtmin), at baseline conditions and under increasing doses of dobutamine.
As expected, in normal mice we observed a dobutamine dose-related increase in HR, dP/dtmax and dP/dtmin. Moreover, at the highest dose tested (12 ng/g/min) the cardiac decompensation of high fat diet-induced obese mice was unmasked.

We describe the protocol to perform a cardiac stress test induced by dobutamine and monitored by cardiac catheterization in normal mice. Also we show its application to unmask subclinical cardiac disease in high fat diet-induced obese mice.

Dobutamine is a &beta;-adrenergic agonist with an affinity higher for receptor expressed in the heart (&beta;1) than for receptors expressed in the ...

Ultrasonic Assessment of Myocardial Microstructure

Echocardiography is a widely accessible imaging modality that is commonly used to noninvasively characterize and quantify changes in cardiac structure and function. Ultrasonic assessments of cardiac tissue can include analyses of backscatter signal intensity within a given region of interest. Previously established techniques have relied predominantly on the integrated or mean value of backscatter signal intensities, which may be susceptible to variability from aliased data from low frame rates and time delays for algorithms based on cyclic variation. Herein, we describe an ultrasound-based imaging algorithm that extends from previous methods, can be applied to a single image frame&#160;and accounts for the full distribution of signal intensity values derived from a given myocardial sample. When applied to representative mouse and human imaging data, the algorithm distinguishes between subjects with and without exposure to chronic afterload resistance. The algorithm offers an enhanced surrogate measure of myocardial microstructure and can be performed using open-access image analysis software.

Echocardiography is commonly used to noninvasively characterize and quantify changes in cardiac structure and function. We describe an ultrasound-based imaging algorithm that offers an enhanced surrogate measure of myocardial microstructure and can be performed using open-access image analysis software.

Echocardiography is a widely accessible imaging modality that is commonly used to noninvasively characterize and quantify changes in cardiac structure and ...

Studying Pancreatic Cancer Stem Cell Characteristics for Developing New Treatment Strategies

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor initiation, metastasis and resistance to radio- and chemotherapy. Here we describe a specific methodology for culturing primary human pancreatic CSCs as tumor spheres in anchorage-independent conditions. Cells are grown in serum-free, non-adherent conditions in order to enrich for CSCs while their more differentiated progenies do not survive and proliferate during the initial phase following seeding of single cells. This assay can be used to estimate the percentage of CSCs present in a population of tumor cells. Both size (which can range from 35 to 250 micrometers) and number of tumor spheres formed represents CSC activity harbored in either bulk populations of cultured cancer cells or freshly harvested and digested tumors 1,2. Using this assay, we recently found that metformin selectively ablates pancreatic CSCs; a finding that was subsequently further corroborated by demonstrating diminished expression of pluripotency-associated genes/surface markers and reduced in vivo tumorigenicity of metformin-treated cells. As the final step for preclinical development we treated mice bearing established tumors with metformin and found significantly prolonged survival. Clinical studies testing the use of metformin in patients with PDAC are currently underway (e.g., NCT01210911, NCT01167738, and NCT01488552). Mechanistically, we found that metformin induces a fatal energy crisis in CSCs by enhancing reactive oxygen species (ROS) production and reducing mitochondrial transmembrane potential. In contrast, non-CSCs were not eliminated by metformin treatment, but rather underwent reversible cell cycle arrest. Therefore, our study serves as a successful example for the potential of in vitro sphere formation as a screening tool to identify compounds that potentially target CSCs, but this technique will require further in vitro and in vivo validation to eliminate false discoveries.

Pancreatic cancer stem cells (CSCs) can be expanded in vitro using the anchorage-independent sphere culture technique, which represents a powerful tool to study CSC biology and can serve as the first step to develop novel CSC-targeting therapies. Here the methodology for expanding, analyzing and targeting of pancreatic CSCs is provided.

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor ...

In Vitro and In Vivo Detection of Mitophagy in Human Cells, C. Elegans, and Mice

Mitochondria are the powerhouses of cells and produce cellular energy in the form of ATP. Mitochondrial dysfunction contributes to biological aging and a wide variety of disorders including metabolic diseases, premature aging syndromes, and neurodegenerative diseases such as Alzheimer's disease (AD) and Parkinson's disease (PD). Maintenance of mitochondrial health depends on mitochondrial biogenesis and the efficient clearance of dysfunctional mitochondria through mitophagy. Experimental methods to accurately detect autophagy/mitophagy, especially in animal models, have been challenging to develop. Recent progress towards the understanding of the molecular mechanisms of mitophagy has enabled the development of novel mitophagy detection techniques. Here, we introduce several versatile techniques to monitor mitophagy in human cells, Caenorhabditis elegans (e.g., Rosella and DCT-1/ LGG-1 strains), and mice (mt-Keima). A combination of these mitophagy detection techniques, including cross-species evaluation, will improve the accuracy of mitophagy measurements and lead to a better understanding of the role of mitophagy in health and disease.

In Vitro and In Vivo Detection of Mitophagy in Human Cells, C. Elegans, and Mice

Mitophagy, the process of clearing damaged mitochondria, is necessary for mitochondrial homeostasis and health maintenance. This article presents some of the latest mitophagy detection methods in human cells, Caenorhabditis elegans, and mice.

Mitochondria are the powerhouses of cells and produce cellular energy in the form of ATP. Mitochondrial dysfunction contributes to biological aging and a ...

Improvement of a Closed Chest Porcine Myocardial Infarction Model by Standardization of Tissue and Blood Sampling Procedures

Myocardial ischemia reperfusion (I/R) injury contributes to almost half of the necrotic area after myocardial infarction. To date there is no approved drug to prevent or reduce myocardial I/R injury. The study and understanding of the pathophysiological mechanisms of myocardial I/R injury is essential to develop successful treatments. Large animal experiments are an important step in translational methods. The porcine model of acute myocardial infarction has been established and described by ourselves and others. We aimed to further improve the value of the model by focusing in detail on the sampling techniques for use in future experiments. Furthermore, we emphasize small but important steps that can affect the quality of the final results. To mimic the clinical situation of myocardial I/R injury, a percutaneous coronary intervention (PCI) catheter was inserted into the left anterior descending coronary artery (LAD) of an anesthetized pig. &#176;&#176;&#176;This model mimics acute myocardial infarction and PCI treatment in humans with the possibility of accurately determining the area at risk as well as the necrotic- and viable ischemic tissue. Here the model was used to investigate the effect of a bicyclic peptide inhibitor of FXIIa. The model can also be modified to allow longer reperfusion times to study later effects of myocardial infarction.

Here we demonstrate a protocol to standardize sampling procedures of an established porcine model of acute myocardial infarction in order to increase its translational value in the understanding of the pathophysiology of myocardial ischemia/reperfusion injury and to test novel drug candidates.

Myocardial ischemia reperfusion (I/R) injury contributes to almost half of the necrotic area after myocardial infarction. To date there is no approved ...

Visualization of Endogenous Mitophagy Complexes In Situ in Human Pancreatic Beta Cells Utilizing Proximity Ligation Assay

Mitophagy is an essential mitochondrial quality control pathway, which is crucial for pancreatic islet beta cell bioenergetics to fuel glucose-stimulated insulin release. Assessment of mitophagy is challenging and often requires genetic reporters or multiple complementary techniques not easily utilized in tissue samples, such as primary human pancreatic islets. Here we demonstrate a robust approach to visualize and quantify formation of key endogenous mitophagy complexes in primary human pancreatic islets. Utilizing the sensitive proximity ligation assay technique to detect interaction of the mitophagy regulators NRDP1 and USP8, we are able to specifically quantify formation of essential mitophagy complexes in situ. By coupling this approach to counterstaining for the transcription factor PDX1, we can quantify mitophagy complexes, and the factors that can impair mitophagy, specifically within beta cells. The methodology we describe overcomes the need for large quantities of cellular extracts required for other protein-protein interaction studies, such as immunoprecipitation (IP) or mass spectrometry, and is ideal for precious human islet&#160;samples generally not available in sufficient quantities for these approaches. Further, this methodology obviates the need for flow sorting techniques to purify beta cells from a heterogeneous islet population for downstream protein applications. Thus, we describe a valuable protocol for visualization of mitophagy highly compatible for use in heterogeneous and limited cell populations.

This protocol outlines a method for quantitative analysis of mitophagy protein complex formation specifically in beta cells from primary human islet samples. This technique thus allows analysis of mitophagy from limited biological material, which are crucial in precious human pancreatic beta cell samples.

Mitophagy is an essential mitochondrial quality control pathway, which is crucial for pancreatic islet beta cell bioenergetics to fuel glucose-stimulated ...

In vitro Assessment of Myocardial Protection following Hypothermia-Preconditioning in a Human Cardiac Myocytes Model

Ischemia/reperfusion-derived myocardial dysfunction is a common clinical scenario in patients after cardiac surgery. In particular, the sensitivity of cardiomyocytes to ischemic injury is higher than that of other cell populations. At present, hypothermia affords considerable protection against an expected ischemic insult. However, investigations into complex hypothermia-induced molecular changes remain limited. Therefore, it is essential to identify a culture condition similar to in vivo conditions that can induce damage similar to that observed in the clinical condition in a reproducible manner. To mimic ischemia-like conditions in vitro, the cells in these models were treated by oxygen/glucose deprivation (OGD). In addition, we applied a standard time-temperature protocol used during cardiac surgery. Furthermore, we propose an approach to use a simple but comprehensive method for the quantitative analysis of myocardial injury. Apoptosis and expression levels of apoptosis-associated proteins were assessed by flow cytometry and using an ELISA kit. In this model, we tested a hypothesis regarding the effects of different temperature conditions on cardiomyocyte apoptosis in vitro. The reliability of this model depends on strict temperature control, controllable experimental procedures, and stable experimental results. Additionally, this model can be used to study the molecular mechanism of hypothermic cardioprotection, which may have important implications for the development of complementary therapies for use with hypothermia.

The distinct effects of different degrees of hypothermia on myocardial protection have not been thoroughly evaluated. The goal of the present study was to quantify the levels of cell death following different hypothermia treatments in a human cardiomyocyte-based model, laying the foundation for future in-depth molecular research.

Ischemia/reperfusion-derived myocardial dysfunction is a common clinical scenario in patients after cardiac surgery. In particular, the sensitivity of ...

Induction and Analysis of Oxidative Stress in Sleeping Beauty Transposon-Transfected Human Retinal Pigment Epithelial Cells

Oxidative stress plays a critical role in several degenerative diseases, including age-related macular degeneration (AMD), a pathology that affects ~30 million patients worldwide. It leads to a decrease in retinal pigment epithelium (RPE)-synthesized neuroprotective factors, e.g., pigment epithelium-derived factor (PEDF) and granulocyte-macrophage colony-stimulating factor (GM-CSF), followed by the loss of RPE cells, and eventually photoreceptor and retinal ganglion cell (RGC) death. We hypothesize that the reconstitution of the neuroprotective and neurogenic retinal environment by the subretinal transplantation of transfected RPE cells overexpressing PEDF and GM-CSF has the potential to prevent retinal degeneration by mitigating the effects of oxidative stress, inhibiting inflammation, and supporting cell survival. Using the Sleeping Beauty transposon system (SB100X) human RPE cells have been transfected with the PEDF and GM-CSF genes and shown stable gene integration, long-term gene expression, and protein secretion using qPCR, western blot, ELISA, and immunofluorescence. To confirm the functionality and the potency of the PEDF and GM-CSF secreted by the transfected RPE cells, we have developed an in vitro assay to quantify the reduction of H2O2-induced oxidative stress on RPE cells in culture. Cell protection was evaluated by analyzing cell morphology, density, intracellular level of glutathione, UCP2 gene expression, and cell viability. Both, transfected RPE cells overexpressing PEDF and/or GM-CSF and cells non-transfected but pretreated with PEDF and/or GM-CSF (commercially available or purified from transfected cells) showed significant antioxidant cell protection compared to non-treated controls. The present H2O2-model is a simple and effective approach to evaluate the antioxidant effect of factors that may be effective to treat AMD or similar neurodegenerative diseases.

Induction and Analysis of Oxidative Stress in Sleeping Beauty Transposon-Transfected Human Retinal Pigment Epithelial Cells

We present a protocol for the development and use ofan oxidative stress-model by treating retinal pigment epithelial cells with H2O2, analyzing cell morphology, viability, density, glutathione, and UCP-2 level. It is a useful model to investigate the antioxidant effect of proteins secreted by transposon-transfected cells to treat neuroretinal degeneration.

Oxidative stress plays a critical role in several degenerative diseases, including age-related macular degeneration (AMD), a pathology that affects ~30 ...

Utilizing Percutaneous Ventricular Assist Devices in Acute Myocardial Infarction Complicated by Cardiogenic Shock

Cardiogenic shock is defined as persistent hypotension, accompanied by evidence of end organ hypo-perfusion. Percutaneous ventricular assist devices (PVADs) are used for the treatment of cardiogenic shock in an effort to improve hemodynamics. Impella is currently the most common PVAD and actively pumps blood from the left ventricle into the aorta. PVADs unload the left ventricle, increase cardiac output and improve coronary perfusion. PVADs are typically placed in the cardiac catheterization laboratory under fluoroscopic guidance via the femoral artery when feasible. In cases of severe peripheral arterial disease, PVADs can be implanted through an alternative access. In this article, we summarize the mechanism of action of PVAD and the data supporting their use in the treatment of cardiogenic shock.

Percutaneous ventricular assist devices are increasingly being utilized in patients with acute myocardial infarction and cardiogenic shock. Herein, we discuss the mechanism of action and hemodynamic effects of such devices. We also review algorithms and best practices for the implantation, management and weaning of these complex devices.

Cardiogenic shock is defined as persistent hypotension, accompanied by evidence of end organ hypo-perfusion. Percutaneous ventricular assist devices ...