The authors thank the support of Institutional Program E015; and Fondo Sectorial FOSSIS-CONACYT, SALUD-2014-1-233947.

This protocol meets the institutional guidelines from the human research Ethics Committee.
1. Coronary Angiography, Ultrasound, and Blood Sampling
<ol>
	<li>Request baseline clinical and demographic information before coronary intervention. Collect the individual&#39;s data: age, sex, current smoking status, body mass index (BMI), high blood pressure, dyslipidemia, diabetes mellitus, medications, and the indication for current coronary angiography.</li>
	<li>Perform coronary angiography through heart catheterization using a radial approach. This procedure should be performed under a fluoroscopy guide in the hemodynamics room by expert cardiologists. 
	NOTE: Identify evaluable vessels. For the present study, evaluable vessels were defined as arteries with sections larger than 1.5 mm and lumen stenosis of more than 50%.</li>
	<li>Advance the intravascular ultrasound catheter to the region of interest and record images. Use the appropriate software to locate and measure the smallest luminal area.</li>
	<li>Use a coronary catheter to collect 10 mL of blood from the closest location to the plaque.</li>
	<li>After patient discharge, schedule periodical medical evaluations to follow up study endpoints. If telephone contact is not possible or a physician visit is delayed for longer than 2 months, request an authorized person (previously designed) to verify the study endpoints. 
	NOTE: Consider any of the following a MACE: 1) cardiovascular death, 2) new myocardial infarction, 3) unstable angina prompting an unscheduled medical visit within 24 h, 4) stent restenosis as demonstrated by coronary angiography, 5) episodes of decompensated heart failure requiring clinical attention.</li>
</ol>
2. Determination of Circulating MPCs (Figure 2)
<ol>
	<li>Process the blood within 1 h from collection. Transfer 6 mL of the collected blood to a 15 mL conical tube and dilute 1:1 (v/v) with 1x phosphate buffered saline (PBS), pH = 7.4.</li>
	<li>Add 2 mL of density gradient medium to three test tubes. Carefully transfer three equal volume aliquots of diluted blood into each test tube containing the density gradient medium. 
	NOTE: The total volume of density gradient medium and diluted blood should not exceed three-fourths of the test tube maximal capacity.</li>
	<li>Centrifuge at 1,800 x g, 4 &#176;C for 30 min. Transfer the band at the interface between the layers into a new tube. Add 2 mL of PBS and centrifuge at 1,800 x g, 4 &#176;C for 6 min. The pellet will contain the MPCs.</li>
	<li>Wash the pellet several times. Aspirate off the previous solution and gently resuspend the cell pellet in fresh PBS. For subsequent washes, centrifuge at 1,800 x g, 4 &#176;C for 2 min. Repeat the process 6x.</li>
	<li>Resuspend the cell pellet in 1 mL of PBS. Mix 20 &#181;L of the cell suspension with 0.4% trypan blue, diluted 1:1 (v/v). Apply a drop to a hemocytometer and count the unstained cells under a light microscope.</li>
	<li>Proceed to MPCs determination. Label 5 mL flow cytometry tubes and aliquot out 1 x 106 cells per tube. Prepare the corresponding isotype-matched control antibodies. Centrifuge at 1,800 x g, 4 &#176;C for 6 min and discard the supernatant.</li>
	<li>Add the primary antibody diluted in 100 &#181;L of an antibody incubation solution consisting of 1x PBS (pH = 7.4), 2 mM ethylenediaminetetraacetic acid (EDTA), and 0.05% bovine serum albumin (BSA). Resuspend for 10 s and incubate for 20 min at 4 &#176;C, light-protected. The protocol may be paused at this step by fixing the lymphocytes in 4% paraformaldehyde in PBS and storing samples up to 24 h at 4 &#176;C. 
	NOTE: The final concentrations of the primary antibodies used in the present protocol were CD45 1:50, CD34 1:20, KDR 1:50, CD184 1:20, CD133 1:50.</li>
	<li>Centrifuge at 1,800 x g, 4 &#176;C for 2 min and discard the supernatant. Resuspend in 500 &#181;L of 1x PBS (pH = 7.4), 2 mM EDTA.</li>
	<li>Perform flow cytometry analysis. Use isotype-matched control antibodies to set up the background staining. Then, select lymphocytes spread at the FSC/SSC plot, trying to exclude residual granulocytes, cellular debris, and other particles, which are usually located in the lower, left-distributed in the plot. Such distribution is considered as 100%.</li>
	<li>Use a gate containing a high number of cells with common immunophenotype CD45+ and CD34+. For double positive immunophenotypes, use a gate previously identifying CD45+, CD34+, with the addition of either KDR (VEGFR-2)+, CD133+, or CD184+. Identify the MPC subpopulations by their specific cell surface markers. Report as the percentage of gated events.</li>
	<li>Identify the main subpopulations of MPCs. In the present study the main immunophenotypes were CD45+CD34+CD133+, CD45+CD34+CD184+, CD45+CD34+CD133+CD184+, CD45+CD34+KDR+, CD45+CD34+KDR+CD133+, and CD45+CD34+KDR+CD184. 
	NOTE: The cell surface markers used were CD45 (lymphocytes), CD34 (endothelial and/or vascular cells), KDR (VEGFR-2, membrane marker of endothelial cells), CD133 (endothelial progenitor cells), and CD184 (hematopoietic stem cells and endothelial cells).</li>
</ol>
3. Determination of Plasma Soluble Biomarkers
<ol>
	<li>Use an enzyme-linked immunosorbent assay (ELISA) to determine the concentration of SICAM-1 and MMP-9 (Figure 3, upper row).
	<ol>
		<li>Centrifuge the blood samples at 3,000 x g, room temperature for 5 min and collect the plasma.</li>
		<li>Label the standards, sample tubes, and control tubes. Equilibrate the pre-coated wells in the assay plate by washing 2x with the washing buffer provided in the ELISA kit.</li>
		<li>Transfer the standards, samples, and controls to the wells. Seal and incubate at 37 &#176;C for 90 min. 
		NOTE: Do not let the wells dry completely.</li>
		<li>Discard the contents and add the biotin-detection antibody. Seal and incubate at 37 &#176;C for 60 min.</li>
		<li>Discard the contents and wash 3x. Seal the plaque and incubate sequentially with streptavidine working solution followed by tetramethylbenzidine substrate at 37 &#176;C for 30 min, light-protected. Wash 3x between incubations. When the color develops, add the stop solution, and read the optical density absorbance in a microplate ELISA reader.</li>
	</ol>
	</li>
	<li>Use an immuno-magnetic multiplexing assay to determine the concentration of tumor necrosis factor alpha (TNF&#945;) and interleukin 1 beta (IL-1&#946;) (Figure 3, lower row).
	<ol>
		<li>Label the standards, sample tubes, and control tubes.</li>
		<li>Vortex the magnetic beads vials for 30 s. Transfer the bead suspension to appropriately sized tubes, and then to the wells in the multiplexing assay plate. Periodic vortexing avoids precipitation of the beads.</li>
		<li>Securely insert the hand-held magnetic plate washer. Wait 2 min for the beads to accumulate on the bottom of each well and quickly invert both the hand-held magnetic plate washer and plate assembly, over a sink or waste container. Remember to use the hand-held magnetic plate washer to maintain the beads inside the wells.</li>
		<li>Add 150 &#181;L of wash buffer into each well and wait 30 s to allow the beads to accumulate on the bottom. Discard the contents as in step 3.2.3. Then, add 25 &#181;L of universal assay buffer (provided in the kit) followed by 25 &#181;L of prepared standards, samples, and controls.</li>
		<li>Seal the plate and incubate for least 60 min at room temperature, light-protected, with constant shaking at 500 rpm. Alternatively, incubate overnight at 4 &#176;C, light-protected, with constant shaking at 500 rpm if possible.</li>
		<li>Wash 2x by adding 150 &#181;L of wash buffer and wait 30 s. Discard the contents by inserting the hand-held magnetic plate washer. Wait 2 min and invert over a sink or waste container.</li>
		<li>Incubate sequentially with 25 &#181;L of detection antibody mixture followed by 25 &#181;L of streptavidin-PE solution at room temperature for 30 min, sealed and light-protected, with constant shaking at 500 rpm. Wash 2x between incubations, as described in step 3.2.6.</li>
		<li>Obtain the readings. Add 120 &#181;L of reading buffer. Seal the plate and incubate 5 min at room temperature, light-protected, with constant shaking at 500 rpm. Run the reading on a multiplexing assay reader. Adjust the reading parameters according to each analyte.</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Cassar, A., Holmes, D. R., Rihal, C. S., Gersh, B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chronic+coronary+artery+disease:+diagnosis+and+management.">Chronic coronary artery disease: diagnosis and management.</a> Mayo Clinic Proceedings. 84 (12), 1130-1146 (2009).</li><li>Regueiro, A., 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=Mobilization+of+endothelial+progenitor+cells+in+acute+cardiovascular+events+in+the+PROCELL+study:+time-course+after+acute+myocardial+infarction+and+stroke.">Mobilization of endothelial progenitor cells in acute cardiovascular events in the PROCELL study: time-course after acute myocardial infarction and stroke.</a> Journal of Molecular and Cellular Cardiology. 80, 146-155 (2015).</li><li>Sen, S., McDonald, S. P., Coates, P. T., Bonder, C. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endothelial+progenitor+cells:+novel+biomarker+and+promising+cell+therapy+for+cardiovascular+disease.">Endothelial progenitor cells: novel biomarker and promising cell therapy for cardiovascular disease.</a> Clinical Science (Lond). 120 (7), 263-283 (2011).</li><li>Samman Tahhan, A., 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=Progenitor+Cells+and+Clinical+Outcomes+in+Patients+With+Acute+Coronary+Syndromes.">Progenitor Cells and Clinical Outcomes in Patients With Acute Coronary Syndromes.</a> Circulation Research. 122 (11), 1565-1575 (2018).</li><li>Tomuli&#263;, V., Gobi&#263;, D., Luli&#263;, D., &#381;idan, D., Zaputovi&#263;, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Soluble+adhesion+molecules+in+patients+with+acute+coronary+syndrome+after+percutaneous+coronary+intervention+with+drug-coated+balloon,+drug-eluting+stent+or+bare+metal+stent.">Soluble adhesion molecules in patients with acute coronary syndrome after percutaneous coronary intervention with drug-coated balloon, drug-eluting stent or bare metal stent.</a> Medical Hypotheses. 95, 20-23 (2016).</li><li>Jaumdally, R., Varma, C., Macfadyen, R. J., Lip, G. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coronary+sinus+blood+sampling:+an+insight+into+local+cardiac+pathophysiology+and+treatment?.">Coronary sinus blood sampling: an insight into local cardiac pathophysiology and treatment?.</a> European Heart Journal. 28 (8), 929-940 (2007).</li><li>Kremastinos, D. T., 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=Intracoronary+cyclic-GMP+and+cyclic-AMP+during+percutaneous+transluminal+coronary+angioplasty.">Intracoronary cyclic-GMP and cyclic-AMP during percutaneous transluminal coronary angioplasty.</a> International Journal of Cardiology. 53 (3), 227-232 (1996).</li><li>Karube, N., 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=Measurement+of+cytokine+levels+by+coronary+sinus+blood+sampling+during+cardiac+surgery+with+cardiopulmonary+bypass.">Measurement of cytokine levels by coronary sinus blood sampling during cardiac surgery with cardiopulmonary bypass.</a> American Society for Artificial Internal Organs Journal. 42 (5), M787-M791 (1996).</li><li>Truong, Q. A., 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=Coronary+sinus+biomarker+sampling+compared+to+peripheral+venous+blood+for+predicting+outcomes+in+patients+with+severe+heart+failure+undergoing+cardiac+resynchronization+therapy:+the+BIOCRT+study.">Coronary sinus biomarker sampling compared to peripheral venous blood for predicting outcomes in patients with severe heart failure undergoing cardiac resynchronization therapy: the BIOCRT study.</a> Heart Rhythm. 11 (12), 2167-2175 (2014).</li><li>Su&#225;rez-Cuenca, J. A., 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=Coronary+circulating+mononuclear+progenitor+cells+and+soluble+biomarkers+in+the+cardiovascular+prognosis+after+coronary+angioplasty.">Coronary circulating mononuclear progenitor cells and soluble biomarkers in the cardiovascular prognosis after coronary angioplasty.</a> Journal of Cellular and Molecular Medicine. 23 (7), 4844-4849 (2019).</li><li>Su&#225;rez-Cuenca, J. 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The authors have nothing to disclose.

Blood collection from the affected coronary artery may be difficult. Sometimes, the coronary artery is barely accessible. In this case, sampling from the venous sinus may be an alternative. We performed validation tests comparing circulating biomarkers in coronary artery vs. venous sinus, with no significant differences. However, the performance of circulating biomarkers was validated only for coronary sampling. Therefore, the performance of biomarkers obtained from the venous sinus remains to be explored.
<p class="...

Coronary angioplasty and stenting represent a salvage procedure for patients with coronary artery disease (CAD). However, major adverse cardiovascular events (MACEs), including cardiovascular death, myocardial infarction, coronary restenosis, and episodes of angina or decompensate heart failure, may occur months after coronary intervention, prompting unscheduled visits to the hospital. MACEs are common worldwide and their morbi-mortality is high1.
Coronary ischemic injury induces early vascular response and reparative mechanisms involving mobilization of MPCs due to their differentiation ability and/or angio-reparative potential, as well as the production of soluble molecules like intercellular adhesion molecules (ICAMs), matrix metalloproteinases (MMPs), and reactive oxygen species, reflecting cell adhesion, tissue remodeling, and oxidative stress. Although intrinsic vascular features like plaque characteristics and coronary artery complexity have been used to predict MACEs, some studies have suggested that biomarkers related to the mechanisms of injury and repair occurring in the coronary endothelium could be very useful for the early identification and prognosis of cardiovascular events in patients with CAD submitted to coronary angioplasty2,3,4,5.
Continuous interest in understanding the mechanisms underlying CAD injury and repair has motivated investigators to study intracoronary circulating biomarkers, because coronary sampling more closely reflects vascular damage and repair6. However, characterization of coronary biomarkers in human studies has been scarce7,8,9. Therefore, the purpose of the present study was to describe a method to determine the amount of coronary circulating MPCs and soluble molecules, reflecting both vascular injury and repair, and to show whether these biomarkers are associated with MACEs and the clinical prognosis of CAD patients that underwent coronary angioplasty. This method is based on the use of vascular-related, circulating MPCs and soluble molecules obtained by sampling locations closest to the vessel damage. It may also be useful for clinical studies for lower limb ischemia, stroke, vasculitis, venous thrombosis, and other injuries involving vascular injury and repair.

<table><tbody><tr><td>BSA</td><td>Roche</td><td>10735086001</td><td>Bovine Serum Albumin (BSA) as a buffering agent, stabilizer, standard and for blending.</td></tr><tr><td>Calibration Beads</td><td>Miltenyi Biotec / MACS</td><td>#130-093-607</td><td>MACQuant calibration beads are supplied in aqueous solution containing 0.05% sodium azide. 3.5 ml for up to 100 tests</td></tr><tr><td>CD133/1 (AC133)-PE</td><td>Milteny Biotec / MACS</td><td>#130-080-801</td><td>Antibody conjugated to R-Phycoerythrin in PBS/EDTA buffer</td></tr><tr><td>CD184 (CXCR4)-PE-VIO770</td><td>Miltenyi Biotec / MACS</td><td>#130-103-798</td><td>Monoclonal, Isotype recombinant human IgG1, conjugated</td></tr><tr><td>CD309 (VEGFR-2/KDR)-APC</td><td>Miltenyi Biotec / MACS</td><td>#130-093-601</td><td>Antibody conjugated to R-Phycoerythrin in PBS/EDTA buffer</td></tr><tr><td>CD34-FITC</td><td>Miltenyi Biotec / MACS</td><td>#130-081-001</td><td>The monoclonal antibody clone AC136 detecs a class III epitope of the CD34</td></tr><tr><td>CD45- VioBlue</td><td>Miltenyi Biotec / MACS</td><td>#130-092-880</td><td>Monoclonal CD45 Antibody, human conjugated</td></tr><tr><td>Conical Tubes</td><td>Thermo SCIENTIFIC</td><td>#339651</td><td>15ml conical centrifuge tubes</td></tr><tr><td>Cytometry Tubes</td><td>FALCON Corning Brand</td><td>#352052</td><td>5 mL Polystyrene Round-Bottom Tube. 12x75 style. Sterile.</td></tr><tr><td>EDTA</td><td>BIO-RAD</td><td>#161-0729</td><td>Heavy metals, (as Pb) &amp;lt;10ppm, Fe &amp;lt;0.01%, As &amp;lt;1ppm, Insolubles &amp;lt;0.005%</td></tr><tr><td>Improved Neubauer</td><td>Without brand</td><td>Without catalog number</td><td>Hemocytometer for cell counting. (range 0.1000mm, 0.0025mm&lt;sup&gt;2&lt;/sup&gt;)</td></tr><tr><td>K2 EDTA Blood Collection Tubes</td><td>BD Vacutainer</td><td>#367863</td><td>Lilac plastic vacutainer tube (K2E) 10.8mg, 6 mL.</td></tr><tr><td>Lymphoprep</td><td>Stemcell Technologies</td><td>01-63-12-002-A</td><td>Sterile and checked on the presence of endotoxins. Density: 1.077&amp;plusmn;0.001g/mL</td></tr><tr><td>Paraformaldehyde</td><td>SIGMA-ALDRICH</td><td>#SZBF0920V</td><td>Fixation of biological samples, (powder, 95%)</td></tr><tr><td>Pipette Transfer 1,3mL</td><td>CRM Globe</td><td>PF1016, PF1015</td><td>The transfer pipette is a tool that facilitates liquid transfer with greater accuracy.</td></tr><tr><td>Test Tubes</td><td>KIMBLE CHASE</td><td>45060 13100</td><td>Heat-resistant test tubes. SIZE/CAP 13 x 100 mm</td></tr></tbody></table>

coronary progenitor cells and soluble biomarkers in cardiovascular prognosis after coronary angioplasty

Major adverse cardiovascular events (MACEs) negatively impact the cardiovascular prognosis of patients undergoing coronary angioplasty due to coronary ischemic injury. The extent of coronary damage and the mechanisms of vascular repair are factors influencing the future development of MACEs. Intrinsic vascular features like the plaque characteristics and coronary artery complexity have demonstrated prognostic information for MACEs. However, the use of intracoronary circulating biomarkers has been postulated as a convenient method for the early identification and prognosis of MACEs, as they more closely reflect dynamic mechanisms involving coronary damage and repair. Determination of coronary circulating biomarkers during angioplasty, such as the number of subpopulations of mononuclear progenitor cells (MPCs) as well as the concentration of soluble molecules reflecting inflammation, cell adhesion, and repair, allows for assessment of future developments and the prognosis of MACEs 6 months post coronary angioplasty. This method is highlighted by its translational nature and better performance than peripheral blood circulating biomarkers regarding prediction of MACEs and its effect on the cardiovascular prognosis, which may be applied for risk stratification of patients with coronary artery disease undergoing angioplasty.

Coronary, venous sinus, and peripheral blood were collected from 52 patients that underwent coronary angiography (Figure 1) and showed a high prevalence of hypertension and dyslipidemia. At the clinical follow-up, 11 (21.1%) MACEs occurred 6 months after coronary angiography: death (n = 1), angina requiring hospital attendance (n = 6), myocardial infarction (n = 2), and/or evidence of heart failure (n = 4).
<p class="jove_content" fo:keep-together.within-...

Watch this Scientific Journal Video about Coronary Progenitor Cells and Soluble Biomarkers in Cardiovascular Prognosis after Coronary Angioplasty at JoVE.com

Coronary Progenitor Cells and Soluble Biomarkers in Cardiovascular Prognosis after Coronary Angioplasty

Development of major adverse cardiovascular events, which impact cardiovascular prognosis after coronary angioplasty, are influenced by the extent of coronary damage and vascular repair. The use of novel coronary cellular and soluble biomarkers, reactive to vascular damage and repair, are useful to predict the development of MACEs and prognosis.

Major adverse cardiovascular events (MACEs) negatively impact the cardiovascular prognosis of patients undergoing coronary angioplasty due to coronary ...

coronary-progenitor-cells-soluble-biomarkers-cardiovascular-prognosis

Research

JoVE Journal

Medicine

5.2K Views.  Experimental Metabolism and Clinical Research Laboratory & Regenerative Medicine and Tissue Engineering Laboratory. Hoewel angioplastie patiënten met coronaire hartziekte helpt, kunnen cardiovasculaire voorvallen nog steeds optreden na de procedure. Als MpCs en oplosbare moleculen worden verkregen uit coronaire circulatie, kunnen ze worden gecorreleerd aan de mate van vasculaire schade en reparatie. Coronaire MPC- en oplosbare bemiddelaarniveaus kunnen daarom worden gebruikt om cardiovasculaire voorvallen en prognose te schatten bij patiënten met coronaire hartziekten die coronaire angioplasties ondergaa...

Video: Coronary Progenitor Cells and Soluble Biomarkers in Cardiovascular Prognosis after Coronary Angioplasty

Isolation of Perivascular Multipotent Precursor Cell Populations from Human Cardiac Tissue

Multipotent mesenchymal stem/stromal cells (MSC) were conventionally isolated, through their plastic adherence, from primary tissue digests whilst their anatomical tissue location remained unclear. The recent discovery of defined perivascular and MSC cell marker expression by perivascular cells in multiple tissues by our group and other researchers has provided an opportunity to prospectively isolate and purify specific homogenous subpopulations of multipotent perivascular precursor cells. We have previously demonstrated the use of fluorescent activated cell sorting (FACS) to purify microvascular CD146+CD34- pericytes and vascular CD34+CD146- adventitial cells from human skeletal muscle. Herein we describe a method to simultaneously isolate these two perivascular cell subsets from human myocardium by FACS, based on the expression of a defined set of cell surface markers for positive and negative selections. This method thus makes available two specific subpopulations of multipotent cardiac MSC-like precursor cells for use in basic research and/or therapeutic investigations.

Human cardiac tissue harbours multipotent perivascular precursor cell populations that may be suitable for myocardial regeneration. The technique described here allows for the simultaneous isolation and purification of two multipotent stromal cell populations associated with native blood vessels, i.e. CD146+CD34- pericytes and CD34+CD146- adventitial cells, from the human myocardium.

Multipotent mesenchymal stem/stromal cells (MSC) were conventionally isolated, through their plastic adherence, from primary tissue digests whilst their ...

Developmental Biology

Assessing Stem Cell DNA Integrity for Cardiac Cell Therapy

Stem and stem-cell-derived cells have immense potential as a regenerative therapy for various degenerative diseases. DNA is the storehouse of genetic data in all cells, including stem cells, and its integrity is fundamental to its regenerative ability. Stem cells undergo rapid propagation in labs to achieve the necessary numbers for transplantation. Accelerated cell growth leads to the loss of DNA integrity by accumulated metabolites, such as reactive oxygen, carbonyl, and alkylating agents. Transplanting these cells would result in poor engraftment and regeneration of the deteriorating organ. Moreover, transplanting DNA-damaged cells leads to mutations, DNA instability, cellular senescence, and possibly, life-threatening diseases such as cancer. Therefore, there is an immediate need for a quality control method to evaluate the cell's suitability for transplantation. Here, we provide step-by-step protocols for the assessment of the DNA integrity of stem cells prior to cell transplantation.

Here, we provide a detailed description of an experimental setup for an analysis of the assessment of DNA integrity in stem cells prior to cell transplantation.

Stem and stem-cell-derived cells have immense potential as a regenerative therapy for various degenerative diseases. DNA is the storehouse of genetic data ...

Genetics

Pluripotent Stem Cell Derived Cardiac Cells for Myocardial Repair

Human induced pluripotent stem cells (hiPSCs) must be fully differentiated into specific cell types before administration, but conventional protocols for differentiating hiPSCs into cardiomyocytes (hiPSC-CMs), endothelial cells (hiPSC-ECs), and smooth muscle cells (SMCs) are often limited by low yield, purity, and/or poor phenotypic stability. Here, we present novel protocols for generating hiPSC-CMs, -ECs, and -SMCs that are substantially more efficient than conventional methods, as well as a method for combining cell injection with a cytokine-containing patch created over the site of administration. The patch improves both the retention of the injected cells, by sealing the needle track to prevent the cells from being squeezed out of the myocardium, and cell survival, by releasing insulin-like growth factor (IGF) over an extended period. In a swine model of myocardial ischemia-reperfusion injury, the rate of engraftment was more than two-fold greater when the cells were administered with the cytokine-containing patch comparing to the cells without patch, and treatment with both the cells and the patch, but not with the cells alone, was associated with significant improvements in cardiac function and infarct size.

We present three novel and more efficient protocols for differentiating human induced pluripotent stem cells into cardiomyocytes, endothelial cells, and smooth muscle cells and a delivery method that improves the engraftment of transplanted cells by combining cell injection with patch-mediated cytokine delivery.

Human induced pluripotent stem cells (hiPSCs) must be fully differentiated into specific cell types before administration, but conventional protocols for ...

Isolation of Endothelial Progenitor Cells from Human Umbilical Cord Blood

The existence of endothelial progenitor cells (EPCs) in peripheral blood and its involvement in vasculogenesis was first reported by Ashara and colleagues1. Later, others documented the existence of similar types of EPCs originating from bone marrow2,3. More recently, Yoder and Ingram showed that EPCs derived from umbilical cord blood had a higher proliferative potential compared to ones isolated from adult peripheral blood4,5,6. Apart from being involved in postnatal vasculogenesis, EPCs have also shown promise as a cell source for creating tissue-engineered vascular and heart valve constructs7,8. Various isolation protocols exist, some of which involve the cell sorting of mononuclear cells (MNCs) derived from the sources mentioned earlier with the help of endothelial and hematopoietic markers, or culturing these MNCs with specialized endothelial growth medium, or a combination of these techniques9. Here, we present a protocol for the isolation and culture of EPCs using specialized endothelial medium supplemented with growth factors, without the use of immunosorting, followed by the characterization of the isolated cells using Western blotting and immunostaining.

The goal of this protocol is to isolate endothelial progenitor cells from umbilical cord blood. Some of the applications include using these cells as a biomarker for identifying patients with cardiovascular risk, treating ischemic diseases, and creating tissue-engineered vascular and heart valve constructs.

The existence of endothelial progenitor cells (EPCs) in peripheral blood and its involvement in vasculogenesis was first reported by Ashara and ...

Suppression of Pro-fibrotic Signaling Potentiates Factor-mediated Reprogramming of Mouse Embryonic Fibroblasts into Induced Cardiomyocytes

Trans-differentiation of one somatic cell type into another has enormous potential to model and treat human diseases. Previous studies have shown that mouse embryonic, dermal, and cardiac fibroblasts can be reprogrammed into functional induced-cardiomyocyte-like cells (iCMs) through overexpression of cardiogenic transcription factors including GATA4, Hand2, Mef2c, and Tbx5 both in vitro and in vivo. However, these previous studies have shown relatively low efficiency. In order to restore heart function following injury, mechanisms governing cardiac reprogramming must be elucidated to increase efficiency and maturation of iCMs.
We previously demonstrated that inhibition of pro-fibrotic signaling dramatically increases reprogramming efficiency. Here, we detail methods to achieve a reprogramming efficiency of up to 60%. Furthermore, we describe several methods including flow cytometry, immunofluorescent imaging, and calcium imaging to quantify reprogramming efficiency and maturation of reprogrammed fibroblasts. Using the protocol detailed here, mechanistic studies can be undertaken to determine positive and negative regulators of cardiac reprogramming. These studies may identify signaling pathways that can be targeted to promote reprogramming efficiency and maturation, which could lead to novel cell therapies to treat human heart disease.

Here we present a robust method to reprogram primary embryonic fibroblasts into functional cardiomyocytes through overexpression of GATA4, Hand2, Mef2c, Tbx5, miR-1, and miR-133 (GHMT2m) alongside inhibition of TGF-&#946; signaling. Our protocol generates beating cardiomyocytes as early as 7 days post-transduction with up to 60% efficiency.

Trans-differentiation of one somatic cell type into another has enormous potential to model and treat human diseases. Previous studies have shown that ...

Induction of Endothelial Differentiation in Cardiac Progenitor Cells Under Low Serum Conditions

Cardiac progenitor cells (CPCs) may have therapeutic potential for cardiac regeneration after injury. In the adult mammalian heart, intrinsic CPCs are extremely scarce, but expanded CPCs could be useful for cell therapy. A prerequisite for their use is their ability to differentiate in a controlled manner into the various cardiac lineages using defined and efficient protocols. In addition, upon in vitro expansion, CPCs isolated from patients or preclinical disease models may offer fruitful research tools for the investigation of disease mechanisms.
Current studies use different markers to identify CPCs. However, not all of them are expressed in humans, which limits the translational impact of some preclinical studies. Differentiation protocols that are applicable irrespective of the isolation technique and marker expression will allow for the standardized expansion and priming of CPCs for cell therapy purpose. Here we describe that the priming of CPCs under a low fetal bovine serum (FBS) concentration and low cell density conditions facilitates the endothelial differentiation of CPCs. Using two different subpopulations of mouse and rat CPCs, we show that laminin is a more suitable substrate than fibronectin for this purpose under the following protocol: after culturing for 2 - 3 days in medium including supplements that maintain multipotency and with 3.5% FBS, CPCs are seeded on laminin at &#60;60% confluence and cultured in supplement-free medium with low concentrations of FBS (0.1%) for 20 - 24 hours before differentiation in endothelial differentiation medium. Because CPCs are a heterogeneous population, serum concentrations and incubation times may need to be adjusted depending on the properties of the respective CPC subpopulation. Considering this, the technique can be applied to other types of CPCs as well and provides a useful method to investigate the potential and mechanisms of differentiation and how they are affected by disease when using CPCs isolated from respective disease models.

This protocol describes an endothelial differentiation technique for cardiac progenitor cells. It particularly focuses on how serum concentration and cell-seeding density affect the endothelial differentiation potential.

Cardiac progenitor cells (CPCs) may have therapeutic potential for cardiac regeneration after injury. In the adult mammalian heart, intrinsic CPCs are ...

Protocol for MicroRNA Transfer into Adult Bone Marrow-derived Hematopoietic Stem Cells to Enable Cell Engineering Combined with Magnetic Targeting

While CD133+ hematopoietic stem cells (SCs) have been proven to provide high potential in the field of regenerative medicine, their low retention rates after injection into injured tissues as well as the observed massive cell death rates lead to very restricted therapeutic effects. To overcome these limitations, we sought to establish a non-viral based protocol for suitable cell engineering prior to their administration. The modification of human CD133+ expressing SCs using microRNA (miR) loaded magnetic polyplexes was addressed with respect to uptake efficiency and safety as well as the targeting potential of the cells. Relying on our protocol, we can achieve high miR uptake rates of 80&#8211;90% while the CD133+ stem cell properties remain unaffected. Moreover, these modified cells offer the option of magnetic targeting. We describe here a safe and highly efficient procedure for the modification of CD133+ SCs. We expect this approach to provide a standard technology for optimization of therapeutic stem cell effects and for monitoring of the administered cell product via magnetic resonance imaging (MRI).

This protocol illustrates a safe and efficient procedure to modify CD133+ hematopoietic stem cells. The presented non-viral, magnetic polyplex-based approach may provide a basis for the optimization of therapeutic stem cell effects as well as for monitoring the administered cell product via magnetic resonance imaging.

While CD133+ hematopoietic stem cells (SCs) have been proven to provide high potential in the field of regenerative medicine, their low retention rates ...

Bioengineering

Isolation of Endothelial Progenitor Cells from Healthy Volunteers and Their Migratory Potential Influenced by Serum Samples After Cardiac Surgery

Endothelial progenitor cells (EPCs) are recruited from the bone marrow under pathological conditions like hypoxia and are crucially involved in the neovascularization of ischemic tissues. The origin, classification and characterization of EPCs are complex; notwithstanding, two prominent sub-types of EPCs have been established: so-called &#34;early&#34; EPCs (subsequently referred to as early-EPCs) and late-outgrowth EPCs (late-EPCs). They can be classified by biological properties as well as by their appearance during in vitro culture. While &#34;early&#34; EPCs appear in less than a week after culture of peripheral blood-derived mononuclear cells in EC-specific media, late-outgrowth EPCs can be found after 2-3 weeks. Late-outgrowth EPCs have been recognized to be directly involved in neovascularization, mainly through their ability to differentiate into mature endothelial cells, whereas &#34;early&#34; EPCs express various angiogenic factors as endogenous cargo to promote angiogenesis in a paracrine manner. During myocardial ischemia/reperfusion (I/R), various factors control the homing of EPCs to regions of blood vessel formation.
Macrophage migration inhibitory factor (MIF) is a chemokine-like pro-inflammatory and ubiquitously expressed cytokine and was recently described to function as key regulator of EPCs migration at physiological concentrations1. Interestingly, MIF is stored in intracellular pools and can rapidly be released into the blood stream after several stimuli (e.g. myocardial infarction). 
This protocol describes a method for the reliable isolation and culture of early-EPCs from adult human peripheral blood based on CD34-positive selection with subsequent culture in medium containing endothelial growth factors on fibronectin-coated plates for use in in vitro migration assays against serum samples of cardiac surgical patients. Furthermore, the migratory influence of MIF on chemotaxis of EPCs compared to other well-known angiogenesis-stimulating cytokines is demonstrated.

Endothelial progenitor cells (EPCs) are crucially involved in the neovascularization of ischemic tissues. This method describes the isolation of human EPCs from peripheral blood, as well as the identification of their migratory potential against serum samples of cardiac surgical patients.

Endothelial progenitor cells (EPCs) are recruited from the bone marrow under pathological conditions like hypoxia and are crucially involved in the ...

In Vitro Model of Coronary Angiogenesis

Here, we describe an in vitro culture assay to study coronary angiogenesis. Coronary vessels feed the heart muscle and are of clinical importance. Defects in these vessels represent severe health risks such as in atherosclerosis, which can lead to myocardial infarctions and heart failures in patients. Consequently, coronary artery disease is one of the leading causes of death worldwide. Despite its clinical importance, relatively little progress has been made on how to regenerate damaged coronary arteries. Nevertheless, recent progress has been made in understanding the cellular origin and differentiation pathways of coronary vessel development. The advent of tools and technologies that allow researchers to fluorescently label progenitor cells, follow their fate, and visualize progenies in vivo have been instrumental in understanding coronary vessel development. In vivo studies are valuable, but have limitations in terms of speed, accessibility, and flexibility in experimental design. Alternatively, accurate in vitro models of coronary angiogenesis can circumvent these limitations and allow researchers to interrogate important biological questions with speed and flexibility. The lack of appropriate in vitro model systems may have hindered the progress in understanding the cellular and molecular mechanisms of coronary vessel growth. Here, we describe an in vitro culture system to grow coronary vessels from the sinus venosus (SV) and endocardium (Endo), the two progenitor tissues from which many of the coronary vessels arise. We also confirmed that the cultures accurately recapitulate some of the known in vivo mechanisms. For instance, we show that the angiogenic sprouts in culture from SV downregulate COUP-TFII expression similar to what is observed in vivo. In addition, we show that VEGF-A, a well-known angiogenic factor in vivo, robustly stimulates angiogenesis from both the SV and Endo cultures. Collectively, we have devised an accurate in vitro culture model to study coronary angiogenesis.

In vitro models of coronary angiogenesis can be utilized for the discovery of the cellular and molecular mechanisms of coronary angiogenesis. In vitro explant cultures of sinus venosus and endocardium tissues show robust growth in response to VEGF-A and display a similar pattern of COUP-TFII expression as in vivo.

Here, we describe an in vitro culture assay to study coronary angiogenesis. Coronary vessels feed the heart muscle and are of clinical importance. Defects ...

Predicting Amputation using Local Circulating Mononuclear Progenitor Cells in Angioplasty-treated Patients with Critical Limb Ischemia

Critical limb ischemia (CLI) represents an advanced stage of the peripheral arterial disease. Angioplasty improves the blood flow to the lower limb; however, some patients irreversibly progress to limb amputation. The extent of vascular damage and the mechanisms of vascular repair are factors affecting post-angioplasty outcome. Mononuclear Progenitor Cells (MPCs) are reactive to vascular damage and repair, with the ability to reflect vascular diseases. The present protocol describes quantification of MPCs obtained from blood circulation from vessel close to the angioplasty site, as well as its relationship with endothelial dysfunction and its predictive ability for limb amputation in the next 30 days after angioplasty in patients with CLI.

Lower limb amputation may occur even after angioplasty of obstructed vessels in Critical Limb Ischemia (CLI). Mononuclear Progenitor Cells (MPCs) reflect vascular repair. The present protocol describes the quantification of MPCs from circulation close to angioplasty, and its relationship with endothelial dysfunction and prediction of lower limb amputation.

Critical limb ischemia (CLI) represents an advanced stage of the peripheral arterial disease. Angioplasty improves the blood flow to the lower limb; ...

Coronary Progenitor Cells and Soluble Biomarkers in Cardiovascular Prognosis after Coronary Angioplasty

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