The authors thank Vera Lorenz for her helpful support during the experiments and the staff from the Flow Cytometry Facility from the Department of Biomedicine (DBM), University and University Hospital Basel. This work was supported by the Stay-on track program from the University of Basel (to Michika Mochizuki). Gabriela M. Kuster is supported by a grant from the Swiss National Science Foundation (grant number 310030_156953).

The use of mice for cell isolation purpose was in accordance with the Guide for the Care and Use of Laboratory Animals and with the Swiss Animal Protection Law and was approved by the Swiss Cantonal Authorities.
NOTE: The isolation of Sca-1+/CD31- SP-CPCs from the mouse heart was essentially done as previously described34 with some modifications. For the materials and reagents used, see Table of Materials. For all experiments, cardiac SP-CPCs isolated from mice were amplified, passaged, and used in a cell line-like manner. Passages 7 - 20 were used for this study.
1. Tissue Preparation
NOTE: All experiments using mice must be carried out according to the guidelines and regulations. This protocol uses four mice. Culture plates that are 100 mm in diameter are described as P100 and culture plates that are 60 mm in diameter are described as P60 for the following parts of the protocol.
<ol>
	<li>Inject each mouse with 200 mg/kg of pentobarbital intraperitoneally (i.p.) and wait until it is fully anesthetized by checking its response to toe pinching.</li>
	<li>Wipe the chest with 70% ethanol. Cut the skin and thoracic wall with scissors to expose the thoracic cavity.</li>
	<li>Lift the heart with forceps and cut it at the base using scissors. Put the heart in a P100 with 25 mL (5 mL for P60) of cold phosphate-buffered saline (PBS) (three to five hearts per P100 or one to two hearts per P60).</li>
	<li>Pump the heart with forceps to eject the blood out of the cavities (Figure 1A).</li>
	<li>Put the heart in a P100 with 25 mL (5 mL for P60) of cold PBS for washing. Remove the atria using small scissors. Cut the heart into two longitudinal pieces and wash them again in ice-cold PBS (25 mL/P100, 5 mL/P60).</li>
	<li>Transfer the pieces to a new P100 with 25 mL (5 mL for P60) of cold PBS. Cut the pieces into smaller pieces using small scissors (Figure 1B). Add a drop of 1 mg/mL collagenase B diluted in Hank&#8217;s balanced salt solution (HBSS) and mince the small pieces thoroughly with a sterile razor blade (Figure 1C).</li>
</ol>
2. Digestion
<ol>
	<li>Add 10 mL (2.5 mL for P60) of the 1 mg/mL collagenase B solution to the dish from step 1.6.</li>
	<li>Put the collagenase B solution containing the minced heart pieces in a tilted (about 30&#176;) P100 (or P60) in a 37 &#176;C incubator (Figure 1D).</li>
	<li>Incubate the minced heart pieces for a maximum of 30 min; homogenize by repeatedly passing them through a Pasteur pipette every 10 min during the incubation (Figure 1E). 
	NOTE: It is important to not exceed 30 min of incubation in total for step 1.3.</li>
</ol>
3. Filtration
<ol>
	<li>Add 10 mL (5 mL for P60) of cold HBSS supplemented with 2% FBS to the minced and homogenized heart pieces. 
	NOTE: HBSS supplemented with 2% FBS quenches collagenase B activity.</li>
	<li>Filter the heart pieces through 100 &#181;m filter to remove undigested tissue and centrifuge at 470 x g for 5 min at room temperature (RT) (Figure 1F, yellow filters).</li>
	<li>Discard the supernatant and resuspend the pellet in 5 mL (3 mL for P60) of red blood cell lysis buffer, and incubate the pellet for 5 min with occasional shaking on ice.</li>
	<li>Add 10 mL (5 mL for P60) of PBS (to stop the lysis reaction) and filter the sample through a 40 &#181;m filter to exclude larger cells, including residual cardiomyocytes (Figure 1F, blue filters).</li>
	<li>Centrifuge the tube at 470 x g for 5 min at RT (without brake). Discard the supernatant and resuspend the pellet in 1 mL of DMEM including 10% FBS.</li>
	<li>Count the cells in an aliquot with a hemocytometer. Resuspend the cells with DMEM including 10% FBS, aiming at a final cell concentration of 1 x 106 cells/mL. 
	NOTE: Roughly 5 x 106 cardiomyocyte- and erythrocyte-depleted cells can be estimated per mouse.</li>
	<li>Distribute the cells into two tubes (Figure 2): in tube A, add 1.5 mL for Hoechst 33342 staining with verapamil; in tube B, add 18.5 mL for Hoechst 33342 staining and proceed to stain and sort (steps 4.1 and 4.2) cardiac SP cells by flow cytometry.</li>
</ol>
4. Sorting of Cardiac SP Cells by Flow Cytometry
NOTE: Verapamil inhibits Hoechst efflux by blocking multidrug resistance (MDR) ABC transporter activity. Hoechst 33342 is a DNA-binding dye that can be used in living cells to detect the cell cycle as it correlates with the DNA content. Hoechst-33342-extruding cells appear in the Hoechst low part of both emission channels (450 nm, Hoechst blue; 650 nm, Hoechst red), that is, aside of the Hoechst-retaining &#8220;main population&#8221;, giving them their name &#8220;side population&#8221;. SP cells are enriched in cells with progenitor properties and show a high expression of multidrug-resistant ABC transporters (such as MDR1 and ABCG2). Hoechst 33342 is written as Hoechst for the following parts of the protocol. It is important to protect light-sensitive materials for ideal results.
<ol>
	<li>Staining with Hoechst in the presence and absence of verapamil
	<ol>
		<li>Add verapamil (with a final concentration of 83.3 &#181;M) and Hoechst (with a final concentration of 5 &#181;g/106 cells) to the cell solution. For tube A, use Verapamil and Hoechst; for tube B, use Hoechst only. Incubate in a water bath (at 37 &#176;C) for 90 min and revert the tubes every 20 min (Figure 1G).</li>
		<li>Centrifuge the tubes at 470 x g for 5 min at RT. Discard the supernatant and resuspend each pellet in HBSS (1 x 106 cells/mL).</li>
		<li>Take a 1.5 mL aliquot for single stainings and negative control from tube B (Figure 2): (i) fluorescein isothiocyanate (FITC)-conjugated anti-Sca-1; (ii) allophycocyanin (APC)-conjugated anti-CD31; (iii) nonstained cells (negative control). 
		NOTE: Isotype controls are used here for setting up the isolation protocol.</li>
		<li>Centrifuge tubes A and B and the single-staining and negative control aliquots at 470 x g for 5 min at RT for washing out Hoechst and verapamil.</li>
		<li>Discard the supernatant and resuspend the pellets of tube A and (iii) the negative control in 250 &#181;L of HBSS and keep them on ice in the dark until sorting.</li>
		<li>Resuspend the pellet of tube B in 200 &#181;L of HBSS and the pellet of (i, ii) the single-staining aliquots in 100 &#181;L of HBSS. Add FITC-conjugated anti-Sca-1 (0.6 &#181;g/107 cells) and APC-conjugated anti-CD31 (0.25 &#181;g/107 cells). Incubate the pellets for 30 min on ice and shake them from time to time in the dark.</li>
		<li>Add 2 mL of HBSS to the tubes. Centrifuge the tubes at 470 x g for 5 min at RT.</li>
		<li>Discard the supernatant and resuspend all pellets with 1 mL of HBSS and centrifuge as above. Discard the supernatants. Resuspend the pellet of the single-staining aliquots in 200 &#181;L of HBSS. Resuspend the pellet of tube B in HBSS (20 x 106 cells/mL).</li>
		<li>Keep the samples on ice and protected from light until sorting.</li>
	</ol>
	</li>
	<li>Sorting with flow cytometry
	<ol>
		<li>Prepare sterile 1.5 mL sorting tubes with 500 &#181;L of HBSS including 2% FBS.</li>
		<li>Stain the cells with 7-aminoactinomycin D (7-AAD) (0.15 &#181;g/106 cells) on ice for 10 min to exclude dead cells.</li>
		<li>Sort the cardiac SP using the following settings: excite Hoechst using 350 nm (UV) excitation, collect fluorescence emission with a 450/50 nm band-pass filter (Hoechst Blue) and a 670/30 nm band-pass filter (Hoechst red), and use a nozzle size of 100 &#181;m and pressure of 15 psi.</li>
		<li>For analysis, record 5 x 105 events for the sorting samples and 1 x 105 events for the negative control, verapamil control, and single-staining samples. 
		NOTE: The cardiac SP is around 0.5% - 2% (Figure 1H) but may vary between laboratories and isolates. The Sca-1+/CD31- fraction is around 1% - 11% of the total cardiac SP (Figure 1I) but may vary between laboratories and isolates. Sca-1+/CD31- SP-CPCs are written as SP-CPCs for the following parts of the protocol.</li>
	</ol>
	</li>
</ol>
5. Primary Culture of Isolated SP-CPCs
NOTE: Three different types of media were used in this protocol. They are referred to as Medium 1 (according to Noseda et al.)8, Medium 2, and Medium 3 and are described in the Table of Materials regarding their composition.
<ol>
	<li>Warm up Medium 1 to 37 &#176;C before use and cool down the centrifuge to 4 &#176;C. Centrifuge the sorting tubes at 4 &#176;C and 470 x g for 6 min and resuspend the cells in Medium 1.</li>
	<li>Put the cells on a gas-permeable P60 dish with 4 mL of Medium 1. Change the medium every 3 d until the cells have reached 70% - 80% confluence. 
	NOTE: Step 5.2 may take around 2 - 3 weeks.</li>
</ol>
6. Expansion and Differentiation of SP-CPCs
<ol>
	<li>Cell culture
	<ol>
		<li>Culture 3 x 105 SP-CPCs in 8 mL of Medium 1 in a T75 flask.</li>
		<li>Incubate SP-CPCs at 37 &#176;C with 5% CO2 until 70% - 80% confluence, changing the medium every 2 - 3 d. 
		NOTE: The cell number should be modified according to the type of CPCs used, the doubling time, and the cell size.</li>
	</ol>
	</li>
	<li>SP-CPC growth and viability under different serum concentrations
	<ol>
		<li>Culture SP-CPCs for 2 - 3 d in T75 flasks with 8 mL of Medium 1. Carefully aspirate the medium and rinse gently with 5 mL of warm (37 &#176;C) HBSS.</li>
		<li>Treat the cells with 5 mL of Trypsin-EDTA for 5 min in the cell incubator, add 5 mL of Medium 1 to stop the Trypsin activity, and transfer the cell suspension to a 15 mL tube.</li>
		<li>Centrifuge the cells at 470 x g for 5 min at RT.</li>
		<li>Resuspend the cells in Medium 1 or Medium 2 (lineage induction medium) and plate 2.5 x 105 SP-CPCs on P60 dishes with 3 mL of medium containing different serum concentrations.</li>
		<li>Collect the medium from the dish of step 6.2.4 for the collection of dead cells into a 15 mL tube after 2 d of culturing in Medium 1 or Medium 2.</li>
		<li>Trypsinize adherent cells as in step 6.2.2 and collect the cell suspension into the 15 mL tube of step 6.2.5. Centrifuge the cells at 840 x g for 5 min at RT.</li>
		<li>Aspirate the supernatant and add 1 mL of Medium 1 or Medium 2 for cell counting. Stain SP-CPCs with trypan blue (0.4%) and count trypan blue-positive (dead) and trypan blue-negative (viable) cells. 
		NOTE: The cell viability (step 6.2.7) is given as the trypan-blue negative cell number in relation to the total cell number.</li>
	</ol>
	</li>
	<li>Induction of endothelial differentiation 
	<ol>
		<li>Precoat a (6-well) culture plate with 10 &#181;g/mL of laminin (LN) or fibronectin (FN).
		<ol>
			<li>Make a substrate solution containing 10 &#181;g/mL of LN or FN with F12 medium (or PBS). Add 2 mL of substrate solution to each well. Maintain the plate for 30 min at 37 &#176;C.</li>
			<li>Aspirate the solution from the plate and add 2 mL of PBS to each well until using it.</li>
		</ol>
		</li>
		<li>Aspirate the medium from the cells from step 6.1.2, rinse them gently with 5 mL of warm (37 &#176;C) HBSS, treat them with 5 mL of Trypsin-EDTA for 5 min in the cell incubator, add 5 mL of Medium 1 to stop the Trypsin activity, and transfer the cell suspension to a 15 mL tube.</li>
		<li>Centrifuge the cells at 470 x g for 5 min at RT. Aspirate the supernatant and add Medium 2 for cell counting.</li>
		<li>Seed 8 x 104 cells per well on the coated plate with 3 mL of Medium 2 and keep it at 37 &#176;C for 20 - 24 h. Change the medium to 3 mL of Medium 3. 
		NOTE: We recommend using medium containing a low serum concentration&#8212;and without supplements&#8212;for the first 20 - 24 h. We recommended using &#60;60% cell confluence (i.e., the cell number of step 6.3.4 has to be adjusted depending on the cell size and growth rate).</li>
		<li>Culture the cells for 21 d and change the medium every 3 d.</li>
		<li>Verify the endothelial nature of differentiated cells by staining them with an endothelial marker such as von Willebrand Factor (vWF) and performing fluorescence microscopy.
		<ol>
			<li>Wash the cells with 1 mL of PBS and fix the cells in 3.7% formaldehyde for 2 min at RT.</li>
			<li>Permeabilize the cells with 0.1% Triton X in ddH2O (or PBS) for 30 min and block it with 10% goat serum for 1 h at RT.</li>
			<li>Incubate the cells with anti-von Willebrand factor antibody (1:100) for 48 h at 4 &#176;C</li>
			<li>Wash the cells 3x with 1 mL of PBS, for 10 min each time. Incubate them with Alexa Fluor 546 goat anti-rabbit secondary antibody (1:500) for 1 h at RT in the dark.</li>
			<li>Wash again 3x, for 10 min each, with 1 mL of PBS. Stain the cell nuclei with 4&#8217;6-diamidino-2-phenylindole, dihydrochloride (DAPI; 1:500) for 5 min at RT in the dark.</li>
			<li>Wash the cells 3x, for 5 min each, with 1 mL of PBS. Mount the cells and store them at 4 &#176;C 
			NOTE: The culturing conditions (substrate, medium, supplemental reagents, and FBS concentration) of steps 6.1 and 6.3 are described in Figure 3.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Tube formation assay
	<ol>
		<li>Prepare a basement membrane matrix (e.g., Matrigel, henceforth referred to as matrix) plate.
		<ol>
			<li>Thaw the matrix at 4 &#176;C overnight.</li>
			<li>Coat a 96-well plate with 100 &#181;L of the matrix on ice. 
			NOTE: It is important to avoid any bubbles in the matrix. The plates have to be coated on ice to avoid the jellification of the matrix.</li>
			<li>Maintain the plate at 37 &#176;C for 30 min.</li>
		</ol>
		</li>
		<li>Aspirate the medium from the cells (after the completion of step 6.3.5), gently rinse the cells with 5 mL of warm (37 &#176;C) HBSS, and treat them with Trypsin-EDTA for 5 min in the cell incubator. Add Medium 3 to stop the Trypsin activity and transfer the cell suspension to a 15 mL tube.</li>
		<li>Centrifuge the cells at 470 x g for 5 min at RT. Aspirate the supernatant, add 1 mL of Medium 3, and pipet gently.</li>
		<li>Filter the cells with a 35 &#181;m cell strainer, if the cells are aggregated.</li>
		<li>Count the cells and seed 2 x 103 to 4 x 103 cells in 100 &#181;L of Medium 3 in each matrix-coated well. Keep the plate at 37 &#176;C in the cell incubator for 16 h.</li>
		<li>Take a picture with a bright-field microscope at 2X magnification. 
		NOTE: In the case of an incomplete trypsinization, prolong the exposure time to Trypsin-EDTA to a maximum of 7 - 8 min.</li>
	</ol>
	</li>
</ol>

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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Roles+of+Sca-1+in+hematopoietic+stem/progenitor+cell+function.">Roles of Sca-1 in hematopoietic stem/progenitor cell function.</a> Experimental Hematology. 33 (7), 836-843 (2005).</li><li>Goodell, M. A., Brose, K., Paradis, G., Conner, A. S., Mulligan, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+functional+properties+of+murine+hematopoietic+stem+cells+that+are+replicating+in+vivo.">Isolation and functional properties of murine hematopoietic stem cells that are replicating in vivo.</a> Journal of Experimental Medicine. 183 (4), 1797-1806 (1996).</li><li>Pfister, O., 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=Role+of+the+ATP-binding+cassette+transporter+Abcg2+in+the+phenotype+and+function+of+cardiac+side+population+cells.">Role of the ATP-binding cassette transporter Abcg2 in the phenotype and function of cardiac side population cells.</a> Circulation Research. 103 (8), 825-835 (2008).</li><li>Messina, E., 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=Isolation+and+expansion+of+adult+cardiac+stem+cells+from+human+and+murine+heart.">Isolation and expansion of adult cardiac stem cells from human and murine heart.</a> Circulation Research. 95 (9), 911-921 (2004).</li><li>Davis, D. R., 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=Validation+of+the+cardiosphere+method+to+culture+cardiac+progenitor+cells+from+myocardial+tissue.">Validation of the cardiosphere method to culture cardiac progenitor cells from myocardial tissue.</a> PLoS One. 4 (9), e7195 (2009).</li><li>Carr, C. 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=Cardiosphere-derived+cells+improve+function+in+the+infarcted+rat+heart+for+at+least+16+weeks--an+MRI+study.">Cardiosphere-derived cells improve function in the infarcted rat heart for at least 16 weeks--an MRI study.</a> PLoS One. 6 (10), e25669 (2011).</li><li>Martens, 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=Rhesus+monkey+cardiosphere-derived+cells+for+myocardial+restoration.">Rhesus monkey cardiosphere-derived cells for myocardial restoration.</a> Cytotherapy. 13 (7), 864-872 (2011).</li><li>Cheng, K., 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=Relative+roles+of+CD90+and+c-kit+to+the+regenerative+efficacy+of+cardiosphere-derived+cells+in+humans+and+in+a+mouse+model+of+myocardial+infarction.">Relative roles of CD90 and c-kit to the regenerative efficacy of cardiosphere-derived cells in humans and in a mouse model of myocardial infarction.</a> Journal of the American Heart Association. 3 (5), e001260 (2014).</li><li>Mochizuki, M., 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=Polo-Like+Kinase+2+is+Dynamically+Regulated+to+Coordinate+Proliferation+and+Early+Lineage+Specification+Downstream+of+Yes-Associated+Protein+1+in+Cardiac+Progenitor+Cells.">Polo-Like Kinase 2 is Dynamically Regulated to Coordinate Proliferation and Early Lineage Specification Downstream of Yes-Associated Protein 1 in Cardiac Progenitor Cells.</a> Journal of the American Heart Association. 6 (10), (2017).</li><li>Pfister, O., Oikonomopoulos, A., Sereti, K. I., Liao, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+resident+cardiac+progenitor+cells+by+Hoechst+33342+staining.">Isolation of resident cardiac progenitor cells by Hoechst 33342 staining.</a> Methods in Molecular Biology. 660, 53-63 (2010).</li><li>Discher, D. E., Mooney, D. J., Zandstra, P. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Growth+factors,+matrices,+and+forces+combine+and+control+stem+cells.">Growth factors, matrices, and forces combine and control stem cells.</a> Science. 324 (5935), 1673-1677 (2009).</li><li>Plaisance, I., 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=Cardiomyocyte+Lineage+Specification+in+Adult+Human+Cardiac+Precursor+Cells+Via+Modulation+of+Enhancer-Associated+Long+Noncoding+RNA+Expression.">Cardiomyocyte Lineage Specification in Adult Human Cardiac Precursor Cells Via Modulation of Enhancer-Associated Long Noncoding RNA Expression.</a> JACC: Basic to Translational Science. 1 (6), 472-493 (2016).</li></ol>

The authors have nothing to disclose.

Advantages of this Protocol:
This protocol provides an endothelial differentiation technique of CPCs. We found that a low serum concentration and low cell density could improve the efficiency of endothelial differentiation, whereby LN proved to be a more suitable substrate than FN under these conditions. We used two distinct types of CPCs: rat CPCs, which were used in a cell line-like manner, and mouse SP-CPCs, which were isolated and expanded. Notably, the protocol was applicable to both types of CPCs. Current techniques allow scientists to isolate and expand primary CPCs from genetically modified mice and from preclinical disease models, as well as from humans28,36. Using this protocol on isolated CPCs could be helpful to the investigation of not only disease mechanisms but also to the exploration of novel therapeutic targets.
CPCs are currently in clinical testing for cell therapy4,5, whereby cells are isolated from patients and transplanted back after in vitro amplification. In this regard, the protocol presented here could help identify strategies to enhance the differentiation potential of such CPCs before transplantation. However, cell therapy still suffers from limited efficacy due to low retention, survival, and engraftment of transplanted cells. Mechanistic in vitro studies based on this protocol or on adaptations thereof have the potential to contribute to a better understanding of the molecular mechanisms driving the differentiation process-in particular, mechanisms related to cell density, substrate, and growth factors. New knowledge retrieved from such studies may ultimately be used for cell reprogramming and applied to directly influence resident CPCs to differentiate after injury.
Limitations of this Protocol:
Isolated primary CPCs are heterogeneous populations that show different phenotypes with respect to cell size and cell growth rate depending on the isolation, even when using the same markers and identical isolation technique. Therefore, the following three points need to be defined: (1) the cell seeding numbers according to cell size, (2) the ideal serum concentration (&#60;0.5%), and (3) the incubation time for the first step (induction of lineage) with a very low serum concentration. Here, we show differences in differentiation efficiency depending on cell density. However, although we compared low serum (0.1% FBS) versus culture serum concentrations (3.5% FBS), we did not examine other concentrations within the &#60;0.5% FBS range. In addition, depending on the isolation markers used and the species, the efficiency of the induction of lineage commitment may vary. Therefore, the protocol should be optimized for each cell type.
Pharmacological and genetic inhibition/induction techniques could be used for examining disease mechanisms with this protocol, instead of genetically modified or disease animal models. In this case, the protocol should be redesigned: before the treatment with low-serum-containing medium, the cells will be treated with specific reagents or genetic tools. As a consequence, the cells may be more vulnerable than nontreated cells. Using low serum in the first step is the key point in this protocol. Therefore, a careful validation of the serum concentration and incubation time for the first step to allow for slowing the cell proliferation while maintaining viability under serum deprivation is critical to whether this protocol can be a success or not.
Summary:
In summary, isolated CPCs from animal models provide a valuable tool for cardiac disease and regeneration studies. Upon expansion, human CPCs may also be directly used for cell therapy. We, here, provide a protocol for the efficient endothelial lineage induction and differentiation of CPCs based on careful adaptations of cell density and serum concentrations. The advantage of this protocol is its applicability to different types of CPCs and its potential to contribute a basis for the study of and/or the establishment of other novel cardiac regeneration tools.

Recent studies support the existence of resident cardiac progenitor cells (CPCs) in the adult mammalian heart1,2,3, and CPCs could be a useful source for cell therapy after cardiac injury4,5. In addition, expanded CPCs may provide a fruitful model for drug screening and the investigation of disease mechanisms when isolated from patients with rare cardiomyopathies, or from respective disease models6,7.
CPCs isolated from the adult heart possess stem/progenitor cell characteristics1,2,3,8 as they are multipotent, clonogenic, and have the capacity for self-renewal. However, there are many different (sub)populations of CPCs exhibiting different surface marker profiles, including, for instance, c-kit, Sca-1, and others, or retrieved by different isolation techniques (Table 1). Several culture and differentiation protocols have been established1,2,8,9,10,11,12,13,14,15,16,17,18. These protocols vary mostly with respect to the growth factor and serum content, which are adjusted according to the purpose of the culturing and which can lead to differences in results and outcomes, including differentiation efficiency.
Marker-based Isolation Techniques:
CPCs can be isolated based on a specific surface marker expression1,2,8,9,10,11,12,13,14,15,16,17,18. Previous studies suggest that c-kit and Sca-1&#160;may be the best markers to isolate resident CPCs1,11,14,19,20. Because none of these markers is truly specific for CPCs, combinations of different markers are usually applied. For example, whereas CPCs express low levels of c-kit21, c-kit is also expressed by other cell types, including mast cells22, endothelial cells23, and hematopoietic stem/progenitor cells24. An additional problem is the fact that not all markers are expressed across all species. This is the case for Sca-1, which expresses in mouse but not in human25. Therefore, using protocols that are independent of isolation markers may be advantageous in view of clinical trials and studies using human samples.
Marker-independent Isolation Techniques:
There are several major techniques of CPC isolation, which are primarily independent of surface marker expression, but which can be refined by the consecutive selection of specific marker-positive subfractions as needed (see also Table 1). (1) The side population (SP) technique has originally been characterized in a primitive population of hematopoietic stem cells based on the ability to efflux the DNA dye Hoechst 3334226 by ATP-binding cassette (ABC) transporters27. Cardiac SP cells have been isolated by different groups and reported to express a variety of markers with some minor differences between reports2,8,13,14. (2) Colony-forming unit fibroblast cells (CFU-Fs) have originally been defined based on a mesenchymal stromal cell (MSC)-like phenotype. Isolated MSCs are cultured on dishes to induce colony formation. Such colony-forming MSC-like CFU-Fs can be isolated from the adult heart and are capable to differentiate into cardiac lineages15. (3) Cardiosphere-derived cells (CDC) are single cells derived from clusters of cells grown from tissue biopsies or explants28,29,30,31. It was recently shown that mostly the CD105+/CD90-/c-kit- cell fraction exhibits cardiomyogenic and regenerative potential32.
Here, using SP-CPCs isolated from mice, we provide a protocol for the efficient induction of endothelial lineage based on a previous study in rat CPCs and mouse SP-CPCs33. The protocol contains specific adaptations to the culture and expansion technique with respect to the cell density, the serum content of the medium, and the substrate. It can be applied not only to mouse SP-CPCs but to different types of CPCs for the purpose to induce a fate switch from an amplifying to an endothelial-committed CPC, be it in view of transplantation of these cells or their use for mechanistic in vitro studies.

<table><tbody><tr><td>&lt;strong&gt;Culture medium&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Iscove&#039;s Modified Dulbecco&#039;s Medium (IMDM)</td><td>ThermoFisher</td><td>#12440</td><td></td></tr><tr><td>Dulbecco&#039;s Modified Eagle&#039;s Medium (DMEM)/Nutrient Mixture F12 Ham</td><td>Merck</td><td>#D8437</td><td></td></tr><tr><td>Penicillin-Streptomycin (P/S)</td><td>ThermoFisher</td><td>#15140122</td><td></td></tr><tr><td>Fetal Bovine Serum (FBS)</td><td>Hyclone</td><td>#SH30071</td><td>3.5% (0.1% for lineage induction)</td></tr><tr><td>L-Glutamine&amp;nbsp;</td><td>ThermoFisher</td><td>#25030</td><td>Final concentration 2 mM</td></tr><tr><td>Glutathione</td><td>Merck</td><td>#G6013</td><td></td></tr><tr><td>Recombinant Human Epidermal Growth Factor (EGF)</td><td>Peprotech</td><td>#AF-100-15</td><td></td></tr><tr><td>Recombinant Basic Fibroblast Growth Factor (FGF)</td><td>Peprotech</td><td>#AF-100-18B</td><td></td></tr><tr><td>B27 Supplement</td><td>ThermoFisher</td><td>#17504044</td><td></td></tr><tr><td>Cardiotrophin 1&amp;nbsp;</td><td>Peprotech</td><td>#250-25</td><td></td></tr><tr><td>Thrombin</td><td>Diagontech AG, Switzerland</td><td>#100-125</td><td></td></tr><tr><td>Hanks&#039; Balanced Salt Solution (HBSS) CaCl2(-), MgCl2(-)</td><td>ThermoFisher</td><td>#14170</td><td></td></tr><tr><td>0.05 % Trypsin-EDTA</td><td>ThermoFisher</td><td>#25300</td><td></td></tr><tr><td>T75 Flask</td><td>Sarstedt</td><td>#83.3911</td><td></td></tr><tr><td>&lt;strong&gt;Endothelial differentiation&amp;nbsp;&amp;nbsp;&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Endothelial Cell Growth Medium (EGM)-2 BulletKit</td><td>Lonza</td><td>#CC-3162</td><td></td></tr><tr><td>Ham&#039;s F-12K (Kaighn&#039;s) Medium</td><td>ThermoFisher</td><td>#21127</td><td></td></tr><tr><td>Laminin&amp;nbsp;</td><td>Merck</td><td>#L2020</td><td></td></tr><tr><td>Fibronectin&amp;nbsp;</td><td>Merck</td><td>#F4759</td><td>Dilute in ddH2O</td></tr><tr><td>6 Well Plate</td><td>Falcon</td><td>#353046</td><td></td></tr><tr><td>Formaldehyde Solution</td><td>Merck</td><td>#F1635</td><td>Diluite 1:10 in PBS (3.7% final concentration)</td></tr><tr><td>Triton X-100</td><td>Merck</td><td>#93420</td><td>0.1% in ddH2O</td></tr><tr><td>Normal Goat Serum (10%)</td><td>ThermoFisher</td><td>#50062Z</td><td></td></tr><tr><td>Anti-von Willebrand Factor antibody</td><td>Abcam</td><td>#ab6994</td><td>1:100 in 10% goat serum</td></tr><tr><td>Goat anti-Rabbit IgG, Alexa Fluor 546</td><td>ThermoFisher</td><td>#A11010</td><td>1:500 in 10% goat serum</td></tr><tr><td>4&#039;,6-diamidino-2-phenylindole, dihydrochloride (DAPI)</td><td>ThermoFisher</td><td>#62247</td><td>1:500 in ddH2O&amp;nbsp;</td></tr><tr><td>SlowFade Antifade Kit</td><td>ThermoFisher</td><td>#S2828</td><td></td></tr><tr><td>BX63 widefield microscope</td><td>Olympus</td><td></td><td></td></tr><tr><td>&lt;strong&gt;Tube formation&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>96 Well Plate</td><td>Falcon</td><td>#353072</td><td></td></tr><tr><td>5 ml Round Bottom Tube with Strainer Cap</td><td>Falcon</td><td>#352235</td><td></td></tr><tr><td>Matrigel Growth Factor Reduced</td><td>Corning</td><td>#354230</td><td></td></tr><tr><td>IX50 widefield microscope</td><td>Olympus</td><td></td><td></td></tr><tr><td>&lt;strong&gt;Sca-1+/CD31- cardiac side population isolation&lt;sup&gt;34&lt;/sup&gt;&amp;nbsp;&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>&lt;strong&gt;Reagents&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Pentobarbital Natrium 50 mg/ml ad usum vet.</td><td>in house hospital pharmacy</td><td>#9077862</td><td>Working solution: 200 mg/kg</td></tr><tr><td>Phosphate Buffered Saline (PBS) CaCl2(-), MgCl2(-)</td><td>ThermoFisher</td><td>#20012</td><td></td></tr><tr><td>Hanks&#039; Balanced Salt Solution (HBSS) CaCl2(-), MgCl2(-), phenol red (-)</td><td>ThermoFisher</td><td>#14175</td><td>Prepare HBSS 500 mL + 2% FBS&amp;nbsp; for quenching Collagenase B activity&amp;nbsp;</td></tr><tr><td>Dulbecco&#039;s Modified Eagle&#039;s Medium (DMEM) 1g/L of D-Glucose, L-Glutamine, Pyruvate</td><td>ThermoFisher</td><td>#331885</td><td>Prepare DMEM + 10% FBS&amp;nbsp; + 25 mM HEPES+ P/S for Hoechst stanining</td></tr><tr><td>Penicillin-Streptomycin&amp;nbsp; (P/S)</td><td>ThermoFisher</td><td>#15140122</td><td></td></tr><tr><td>HEPES 1 M</td><td>ThermoFisher</td><td>#15630080</td><td>Final concentration 25 mM</td></tr><tr><td>Fetal Bovine Serum (FBS)</td><td>Hyclone</td><td>#SH30071</td><td></td></tr><tr><td>RBC LysisBuffer (10X)</td><td>BioLegend</td><td>#420301/100mL</td><td>Dilute to 1X in ddH2O and filter through a 0.2 &amp;micro;m filter</td></tr><tr><td>Collagenase B</td><td>Merck</td><td>#11088807001</td><td>Final concentration 1 mg/mL in HBSS, filtered through a 0.2 &amp;micro;m filter</td></tr><tr><td>bisBenzimide H33342 Trihydrochloride (Hoechst)</td><td>Merck</td><td>#B2261</td><td>Prepare 1 mg/mL in ddH2O</td></tr><tr><td>Verapamil-hydrochloride&amp;nbsp;</td><td>Merck</td><td>#V4629</td><td>Final concentration 83.3 &amp;micro;M&amp;nbsp;</td></tr><tr><td>APC Rat Anti-Mouse CD31</td><td>BD Biosciences</td><td>#551262</td><td>0.25 &amp;micro;g/10&lt;sup&gt;7&lt;/sup&gt;cells</td></tr><tr><td>FITC Rat Anti-Mouse Ly-6A/E (Sca-1)</td><td>BD Biosciences</td><td>#557405</td><td>0.6 &amp;micro;g/10&lt;sup&gt;7&lt;/sup&gt;cells</td></tr><tr><td>7-Aminoactinomycin D (7-ADD)</td><td>ThermoFisher</td><td>#A1310</td><td>0.15 &amp;micro;g/10&lt;sup&gt;6&lt;/sup&gt;cells</td></tr><tr><td>APC rat IgG2a k Isotype Control</td><td>BD Biosciences</td><td>#553932</td><td>0.25 &amp;micro;g/10&lt;sup&gt;7&lt;/sup&gt;cells</td></tr><tr><td>FITC Rat IgG2a k Isotype Control</td><td>BD Biosciences</td><td>#554688</td><td>0.6 &amp;micro;g/10&lt;sup&gt;7&lt;/sup&gt;cells</td></tr><tr><td>&lt;strong&gt;Material&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Needles 27G</td><td>Terumo</td><td>#NN-2719R</td><td></td></tr><tr><td>Needles 18G</td><td>Terumo</td><td>#NN-1838S</td><td></td></tr><tr><td>Single Use Syringes 1 mL sterile</td><td>CODAN</td><td>#62.1640</td><td></td></tr><tr><td>Transferpipette 3.5 mL</td><td>Sarstedt</td><td>#86.1171.001</td><td></td></tr><tr><td>Cell Strainer 40 &amp;micro;m blue</td><td>BD Biosciences&amp;nbsp;</td><td>#352340</td><td></td></tr><tr><td>Cell Strainer 100 &amp;micro;m yellow</td><td>BD Biosciences</td><td>#352360</td><td></td></tr><tr><td>Lumox Dish 50</td><td>Sarstedt</td><td>#94.6077.305</td><td></td></tr><tr><td>Culture Dishes P100</td><td>Corning</td><td>#353003</td><td></td></tr><tr><td>Culture Dishes P60</td><td>Corning</td><td>#353004</td><td></td></tr><tr><td>&lt;strong&gt;Mouse&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>&lt;strong&gt;Line&lt;/strong&gt;</td><td>&lt;strong&gt;Age&lt;/strong&gt;</td><td>&lt;strong&gt;Breeding&lt;/strong&gt;</td><td></td></tr><tr><td>C57BL/6NRj / male</td><td>12 weeks</td><td>in house</td><td></td></tr><tr><td>&lt;strong&gt;Product Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalogue No.&lt;/strong&gt;</td><td></td></tr><tr><td>&lt;strong&gt;Reagents&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Iscove&#039;s Modified Dulbecco&#039;s Medium (IMDM)</td><td>ThermoFisher</td><td>#12440</td><td></td></tr><tr><td>Dulbecco&#039;s Modified Eagle&#039;s Medium (DMEM)/Nutrient Mixture F12 Ham</td><td>Merck</td><td>#D8437</td><td></td></tr><tr><td>Penicillin-Streptomycin&amp;nbsp; (P/S)</td><td>ThermoFisher</td><td>#15140122</td><td></td></tr><tr><td>Fetal Bovine Serum (FBS)</td><td>Hyclone</td><td>#SH30071</td><td></td></tr><tr><td>L-Glutamine&amp;nbsp;</td><td>ThermoFisher</td><td>#25030</td><td></td></tr><tr><td>Glutathione</td><td>Merck</td><td>#G6013</td><td></td></tr><tr><td>B27 Supplement</td><td>ThermoFisher</td><td>#17504044</td><td></td></tr><tr><td>Recombinant Human Epidermal Growth Factor (EGF)</td><td>Peprotech</td><td>#AF-100-15</td><td></td></tr><tr><td>Recombinant Basic Fibroblast Growth Factor (FGF)</td><td>Peprotech</td><td>#AF-100-18B</td><td></td></tr><tr><td>Cardiotrophin 1&amp;nbsp;</td><td>Peprotech</td><td>#250-25</td><td></td></tr><tr><td>Thrombin</td><td>Diagontech AG, Switzerland&amp;nbsp;</td><td>#100-125</td><td></td></tr><tr><td>Endothelial Cell Growth Medium (EGM)-2 BulletKit</td><td>Lonza</td><td>#CC-3162</td><td></td></tr><tr><td>&lt;strong&gt;Overview of medium compositions. Some of this infomation is identical with the one provided above, but sorted according to the composition of Media 1-3.&amp;nbsp;&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>&lt;strong&gt;Product Name&lt;/strong&gt;</td><td>&lt;strong&gt;Medium 1&lt;sup&gt;8&lt;/sup&gt;&lt;/strong&gt;</td><td>&lt;strong&gt;Medium 2&lt;/strong&gt;</td><td>&lt;strong&gt;Medium 3&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Reagents&lt;/strong&gt;</td><td>&lt;strong&gt;Culture&lt;/strong&gt;</td><td>&lt;strong&gt;Lineage induction&lt;/strong&gt;</td><td>&lt;strong&gt;Endothelial diff.&lt;/strong&gt;</td></tr><tr><td>Iscove&#039;s Modified Dulbecco&#039;s Medium (IMDM)</td><td>35%</td><td>35%</td><td></td></tr><tr><td>Dulbecco&#039;s Modified Eagle&#039;s Medium (DMEM)/Nutrient Mixture F12 Ham</td><td>65%</td><td>65%</td><td></td></tr><tr><td>Penicillin-Streptomycin&amp;nbsp; (P/S)</td><td>1%</td><td>1%</td><td></td></tr><tr><td>Fetal Bovine Serum (FBS)</td><td>3.5%</td><td>&amp;le;0.1%</td><td></td></tr><tr><td>L-Glutamine&amp;nbsp;</td><td>2 mM</td><td>2 mM</td><td></td></tr><tr><td>Glutathione</td><td>0.2 nM</td><td>0.2 nM</td><td></td></tr><tr><td>B27 Supplement</td><td>1.3%</td><td></td><td></td></tr><tr><td>Recombinant Human Epidermal Growth Factor (EGF)</td><td>6.5 ng/mL</td><td></td><td></td></tr><tr><td>Recombinant Basic Fibroblast Growth Factor (FGF)</td><td>13 ng/mL</td><td></td><td></td></tr><tr><td>Cardiotrophin 1&amp;nbsp;</td><td>0.65 ng/mL</td><td></td><td></td></tr><tr><td>Thrombin</td><td>0.0005 U/mL</td><td></td><td>All necessary components included in the kit</td></tr><tr><td>Endothelial Cell Growth Medium (EGM)-2 BulletKit</td><td></td><td></td><td>All necessary components included in the kit</td></tr></tbody></table>

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.

Mouse SP-CPC Isolation:
In this study, we used mouse CPCs isolated according to the SP phenotype, whereas results from rat CPCs are modified and added from a previous report with permission (Figure 8)33.
Cell Proliferation Under High and Low Cell Densities and with Different Serum Concentrations:
Our previous study showed that the mRNA expression of cardiac lineage markers changed within the first 24 h culturing step. It is known that the extracellular matrix affects cell fate decisions, including endothelial differentiation35. To explore suitable conditions for the facilitation of endothelial lineage commitment of CPCs, we used FN and LN (both 10 &#181;g/mL) on a 6-well plate (growth area: 9.6 cm2/well) in this study. Because cell cycle and cell fate decisions are closely linked, we are seeking the condition that exhibits a low cell proliferation rate in the absence of cell death, as such a condition may reflect the transition from cell proliferation to differentiation. We, therefore, applied the following conditions and compared cell proliferation rates: for cell density, high (80% - 90%) confluency and low (&#60;60%) confluency, and for serum concentration, normal culture conditions (in this study&#160;3.5% FBS) and low serum conditions (&#8804;0.1% FBS). First, we tested the low cell density condition. There were no significant differences of cell viability and proliferation in the low cell density with 3.5% FBS between LN and FN, whereas a low cell density with 0.1% FBS showed a decreased cell proliferation on LN compared to FN but no increase in cell death (Figure 4). In contrast, under high cell density conditions, serum concentrations of both 3.5% and 0.1% showed no differences in cell proliferation and in cell death between the two substrates (Figure 5).
These results indicate that a low cell density on LN with 0.1% serum decreases proliferation without affecting the CPC viability.
Changes in Cell Shape in the Endothelial Differentiation Medium:
Although requiring further studies to understand the significance and underlying mechanisms, changes in the cell shape appear to be an indicator of suitable culture conditions as specific changes can be observed early on in cultures, in which endothelial differentiation is going to be successful (Figure 6). As shown in the white dashed circles in Figure 6, within 7 - 14 d in the endothelial differentiation medium, successful cultures contained cells that were larger and different in morphology to the other cells. Interestingly, these cells disappeared towards the end of the differentiation phase and also appeared in lower numbers and at later time points in high-density cultures on LN and FN. Whereas we did not further characterize these cells, this protocol suggests tracking the cell shape every 2 - 3 d until around day 14. If no such cells appear, the number of cells seeded should be decreased.
Evaluation of the Endothelial Ability with a Tube Formation Assay:
The tube formation assay is a useful technique to evaluate the efficiency of endothelial differentiation of CPCs by measuring the tube formation capacity of differentiated cells. We, therefore, performed the tube formation assay with cells differentiated according to the described conditions. Interestingly, successful tube formation was consistently shown by cells plated at low density and differentiated on LN, whereas tube formation mostly failed in cells plated on LN at a high density (Figure 7A). Similarly, tube formation was mostly unsuccessful in cells cultured on FN irrespective of cell density, although cells differentiated on FN at a low density sometimes formed rudimentary tubes, depending on the cell condition (e.g., isolate and/or passage number, Figure 7B). To confirm the endothelial nature of the cells, cells plated on coverslips were differentiated according to the described protocol and stained for vWF. Again, the vWF expression was more homogeneous and more pronounced in CPCs differentiated on LN compared to FN (Figure 7C,D). Figure 8 shows the results from the tube formation assay as performed for previous studies using rat CPCs under the same protocol as here described33. These results show that tube formation is more efficient in cells differentiated on LN as compared to FN when plated under low cell density conditions and with low serum (0.1% FBS) for 20 - 24 h before differentiation in endothelial differentiation medium. These results suggest that this protocol could be useful for various cell types and independent of species.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/58370/58370fig1.jpg" /> 
Figure 1: Illustration of specific isolation steps to obtain a cardiomyocyte-depleted cell suspension from isolated mouse hearts and representative flow cytometry readouts of the cardiac SP. (A) This panel shows the ejection of residual blood from the heart cavities through repeated slight pressure applied using small forceps. (B) This panel shows the cutting&#160;of the hearts into small pieces using small scissors. (C) This panel shows the mincing of the&#160;heart pieces using a razor blade. (D) This panel shows the incubation of the minced hearts with collagenase B in tilted plates at 37 &#176;C. (E) This panel shows the homogenization of the minced hearts using a Pasteur pipette during the incubation step. (F) This panel shows the filtering of the digested tissue through a 100 &#181;m filter (yellow) and a 40 &#181;m filter (blue) for the removal of undigested tissue residues and cardiomyocytes. (G) This panel shows the gentle reversal of a 50 mL conical tube containing the cardiomyocyte-depleted cell suspension and wrapping in tin foil for light protection after the addition of Hoechst. (H) This panel shows representative flow cytometry readouts of Hoechst-stained cells in the presence and absence of the ABC transporter inhibitor verapamil for the identification of the SP. (I) This panel shows a representative dot plot of SP cells according to CD31 and Sca-1 positivity for the identification of the Sca-1+/CD31- subfraction. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58370/58370fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/58370/58370fig2.jpg" /> 
Figure 2: Schematic overview of the sample preparation leading up to the cardiac SP sorting. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58370/58370fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/58370/58370fig3.jpg" /> 
Figure 3: Protocol for endothelial lineage induction and differentiation. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58370/58370fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/58370/58370fig4v2.jpg" /> 
Figure 4: Mouse SP-CPC proliferation when plated at a low cell density under different serum concentrations on LN and FN. (A and B) These panels show the cell numbers at day 2. (C and D) These panels show the viability at day 2. The data are shown as the mean &#177; the standard error of the mean (SEM); N = 5; different passages; * p &#60; 0.05 by Student&#39;s t-test. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58370/58370fig4v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/58370/58370fig5.jpg" /> 
Figure 5: Mouse SP-CPC proliferation when plated at a high cell density under different serum concentrations on LN and FN. (A and B) These panels show the cell numbers at day 2. (C and D) These panels show the viability at day 2. The data are shown as the mean &#177; the SEM; N = 5; different passages. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58370/58370fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/58370/58370fig6.jpg" /> 
Figure 6: Cell morphology at 14 and 17 d during the differentiation process. This figure shows bright-field (BF) images of mouse SP-CPCs seeded on LN- or FN-coated dishes with a low (Low) or a high (High) cell density and with Medium 2 for 20 h, followed by Medium 3 for 14 or 17 d. White dashed circles mark the areas containing round-shaped cells with a larger cell size. The imaging was performed with bright-field microscopy. The magnification = 2X; the scale bar = 100 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58370/58370fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/58370/58370fig7.jpg" /> 
Figure 7: Tube formation and vWF staining after endothelial differentiation of SP-CPCs on LN and FN. (A and B) These panels show the tube formation. (C and D) These panels show the vWF staining.&#160;Cells were seeded on 10 &#181;g/mL of LN- or FN-coated dishes with a low or high cell density and with Medium 2 for 20 h, followed by Medium 3 for 21 d, and then harvested with Trypsin and seeded on the basement membrane matrix. All pictures were taken after 16 h. The imaging was performed with bright-field microscopy and the magnification = 2X for panels A and B. The imaging is performed with fluorescent microscopy and the magnification = 10X for panels C and D. Panels A and C show LN-coated dishes. Panels B and D show FN-coated dishes. The scale bar = 100 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58370/58370fig7large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 8" class="xfigimg" src="/files/ftp_upload/58370/58370fig8.jpg" /> 
Figure 8: Tube formation after the endothelial differentiation of rat CPCs. Rat CPCs were seeded at a low cell density on 10 &#181;g/mL of LN- or FN-coated dishes with 0.1% FBS-containing F12 medium for 20 h, followed by another 21 d in Medium 3, and then harvested with Trypsin. On the basement membrane matrix on a 96-well plate, 4 x 104 cells were seeded in 100 &#956;L of Medium 3. The imaging was performed with bright-field microscopy. The magnification = 2X; the scale bar = 50 &#181;m. This figure is modified based on a previous study33. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58370/58370fig8large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Species</td>
			<td>Isolation, detection marker</td>
			<td>Reference</td>
		</tr>
		<tr>
			<td>Human, rat, mouse</td>
			<td>c- kit+/Lin-, c-kit+/CD45-, c-kit+/CD45-/CD31-</td>
			<td>Beltrami, Cell 2003; Bolli, Lancet 2011; Ellison, Cell 2013; Smith, Nat Protoc 2014; Vicinanza, Cell Death Differ 2017</td>
		</tr>
		<tr>
			<td>Mongrel dogs</td>
			<td>Lin-, plus: c-kit+, MDR1+ or Sca-1+</td>
			<td>Linke, PNAS 2005</td>
		</tr>
		<tr>
			<td>Mouse</td>
			<td>Sca-1+</td>
			<td>Oh, PNAS 2003</td>
		</tr>
		<tr>
			<td>Mouse</td>
			<td>Side population, plus: Abcg2+, Sca-1+/CD31-, Sca-1+/PDGFRa+</td>
			<td>Hierlihy, FEBS Lett 2002; Martin, Dev Biol 2004; Pfister, Circ Res 2005; Noseda, Nat Commun 2015</td>
		</tr>
		<tr>
			<td>Mouse</td>
			<td>Colony forming unit-fibroblast, plus: CD45-, CD31-, Sca-1+, PDGFRa+</td>
			<td>Pelekanos, Stem Cell Res 2012</td>
		</tr>
		<tr>
			<td>Mouse, rat, human</td>
			<td>Isl-1+ *, plus: CD31-, c-kit-, Sca-1-, Gata4+, Nkx2.5+</td>
			<td>Laugwitz, Nature 2005; Bu, Nature 2009</td>
		</tr>
		<tr>
			<td colspan="2">* Isl-1 is found in the embryonic or fetal myocardium, not in the adult heart.</td>
			<td></td>
		</tr>
	</tbody>
</table>
Table 1: Isolation techniques and markers of cardiac progenitor cells.

Watch this Scientific Journal Video about Induction of Endothelial Differentiation in Cardiac Progenitor Cells Under Low Serum Conditions at JoVE.com

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

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 ...

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Nanyang Technological University

Research

JoVE Journal

Developmental Biology

6.4K Views.  University Hospital and University of Basel. 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.

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

Derivation of Cardiac Progenitor Cells from Embryonic Stem Cells

Cardiac progenitor cells (CPCs) have the capacity to differentiate into cardiomyocytes, smooth muscle cells (SMC), and endothelial cells and hold great promise in cell therapy against heart disease. Among various methods to isolate CPCs, differentiation of embryonic stem cell (ESC) into CPCs attracts great attention in the field since ESCs can provide unlimited cell source. As a result, numerous strategies have been developed to derive CPCs from ESCs. In this protocol, differentiation and purification of embryonic CPCs from both mouse and human ESCs is described. Due to the difficulty of using cell surface markers to isolate embryonic CPCs, ESCs are engineered with fluorescent reporters activated by CPC-specific cre recombinase expression. Thus, CPCs can be enriched by fluorescence-activated cell sorting (FACS). This protocol illustrates procedures to form embryoid bodies (EBs) from ESCs for CPC specification and enrichment. The isolated CPCs can be subsequently cultured for cardiac lineage differentiation and other biological assays. This protocol is optimized for robust and efficient derivation of CPCs from both mouse and human ESCs.

In this protocol, derivation of cardiac progenitor cells from both mouse and human embryonic stem cells will be illustrated. A major strategy in this protocol is to enrich cardiac progenitor cells with flow cytometry using fluorescent reporters engineered into the embryonic stem cell lines.

Cardiac progenitor cells (CPCs) have the capacity to differentiate into cardiomyocytes, smooth muscle cells (SMC), and endothelial cells and hold great ...

Direct Induction of Hemogenic Endothelium and Blood by Overexpression of Transcription Factors in Human Pluripotent Stem Cells

During development, hematopoietic cells arise from a specialized subset of endothelial cells, hemogenic endothelium (HE). Modeling HE development in vitro is essential for mechanistic studies of the endothelial-hematopoietic transition and hematopoietic specification. Here, we describe a method for the efficient induction of HE from human pluripotent stem cells (hPSCs) by way of overexpression of different sets of transcription factors. The combination of ETV2 and GATA1 or GATA2 TFs is used to induce HE with pan-myeloid potential, while a combination of GATA2 and TAL1 transcription factors allows for the production of HE with erythroid and megakaryocytic potential. The addition of LMO2 to GATA2 and TAL1 combination substantially accelerates differentiation and increases erythroid and megakaryocytic cells production. This method provides an efficient and rapid means of HE induction from hPSCs and allows for the observation of the endothelial-hematopoietic transition in a culture dish. The protocol includes hPSCs transduction procedures and post-transduction analysis of HE and blood progenitors.

This protocol describes the efficient induction of hemogenic endothelium and multipotential hematopoietic progenitors from human pluripotent stem cells via the forced expression of transcription factors.

During development, hematopoietic cells arise from a specialized subset of endothelial cells, hemogenic endothelium (HE). Modeling HE development in vitro ...

In Vitro Differentiation of Human Mesenchymal Stem Cells into Functional Cardiomyocyte-like Cells

Myocardial infarction and the subsequent ischemic cascade result in the extensive loss of cardiomyocytes, leading to congestive heart failure, the leading cause of mortality worldwide. Mesenchymal stem cells (MSCs) are a promising option for cell-based therapies to replace current, invasive techniques. MSCs can differentiate into mesenchymal lineages, including cardiac cell types, but complete differentiation into functional cells has not yet been achieved. Previous methods of differentiation were based on pharmacological agents or growth factors. However, more physiologically relevant strategies can also enable MSCs to undergo cardiomyogenic transformation. Here, we present a differentiation method using MSC aggregates on cardiomyocyte feeder layers to produce cardiomyocyte-like contracting cells.
Human umbilical cord perivascular cells (HUCPVCs) have been shown to have a greater differentiation potential than commonly investigated MSC types, such as bone marrow MSCs (BMSCs). As an ontogenetically younger source, we investigated the cardiomyogenic potential of first-trimester (FTM) HUCPVCs compared to older sources. FTM HUCPVCs are a novel, rich source of MSCs that retain their in utero immunoprivileged properties when cultured in vitro. Using this differentiation protocol, FTM and term HUCPVCs achieved significantly increased cardiomyogenic differentiation compared to BMSCs, as indicated by the increased expression of cardiomyocyte markers (i.e., myocyte enhancer factor 2C, cardiac troponin T, heavy chain cardiac myosin, signal regulatory protein &#945;, and connexin 43). They also maintained significantly lower immunogenicity, as demonstrated by their lower HLA-A expression and higher HLA-G expression. Applying aggregate-based differentiation, FTM HUCPVCs showed increased aggregate formation potential and generated contracting cells clusters within 1 week of co-culture on cardiac feeder layers, becoming the first MSC type to do so. 
Our results demonstrate that this differentiation strategy can effectively harness the cardiomyogenic potential of young MSCs, such as FTM HUCPVCs, and suggests that in vitro pre-differentiation could be a potential strategy to increase their regenerative efficacy in vivo.

In Vitro Differentiation of Human Mesenchymal Stem Cells into Functional Cardiomyocyte-like Cells

Here, we present a method to efficiently harness the cardiac differentiation potential of young sources of human mesenchymal stem cells in order to generate functional, contracting, cardiomyocyte-like cells in vitro.

Myocardial infarction and the subsequent ischemic cascade result in the extensive loss of cardiomyocytes, leading to congestive heart failure, the leading ...

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 ...

Generation of First Heart Field-like Cardiac Progenitors and Ventricular-like Cardiomyocytes from Human Pluripotent Stem Cells

The generation of large amounts of functional human pluripotent stem cells-derived cardiac progenitors and cardiomyocytes of defined heart field origin is a pre-requisite for cell-based cardiac therapies and disease modeling. We have recently shown that Id genes are both necessary and sufficient to specify first heart field progenitors during vertebrate development. This differentiation protocol leverages these findings and uses Id1 overexpression in combination with Activin A as potent specifying cues to produce first heart field-like (FHF-L) progenitors. Importantly, resulting progenitors efficiently differentiate (~70&#8211;90%) into ventricular-like cardiomyocytes. Here we describe a detailed method to 1) generate Id1-overexpressing hPSCs and 2) differentiate scalable quantities of cryopreservable FHF-L progenitors and ventricular-like cardiomyocytes.

Here we describe a scalable method, using a simple combination of Activin A and lentivirus-mediated Id1-overexpression, to generate first heart field-like cardiac progenitors and ventricular-like cardiomyocytes from human pluripotent stem cells.

The generation of large amounts of functional human pluripotent stem cells-derived cardiac progenitors and cardiomyocytes of defined heart field origin is ...

Construction of Defined Human Engineered Cardiac Tissues to Study Mechanisms of Cardiac Cell Therapy

Human cardiac tissue engineering can fundamentally impact therapeutic discovery through the development of new species-specific screening systems that replicate the biofidelity of three-dimensional native human myocardium, while also enabling a controlled level of biological complexity, and allowing non-destructive longitudinal monitoring of tissue contractile function. Initially, human engineered cardiac tissues (hECT) were created using the entire cell population obtained from directed differentiation of human pluripotent stem cells, which typically yielded less than 50% cardiomyocytes. However, to create reliable predictive models of human myocardium, and to elucidate mechanisms of heterocellular interaction, it is essential to accurately control the biological composition in engineered tissues. 
To address this limitation, we utilize live cell sorting for the cardiac surface marker SIRP&#945; and the fibroblast marker CD90 to create tissues containing a 3:1 ratio of these cell types, respectively, that are then mixed together and added to a collagen-based matrix solution. Resulting hECTs are, thus, completely defined in both their cellular and extracellular matrix composition.
Here we describe the construction of defined hECTs as a model system to understand mechanisms of cell-cell interactions in cell therapies, using an example of human bone marrow-derived mesenchymal stem cells (hMSC) that are currently being used in human clinical trials. The defined tissue composition is imperative to understand how the hMSCs may be interacting with the endogenous cardiac cell types to enhance tissue function. A bioreactor system is also described that simultaneously cultures six hECTs in parallel, permitting more efficient use of the cells after sorting.

This manuscript describes the creation of defined engineered cardiac tissues using surface marker expression and cell sorting. The defined tissues can then be used in a multi-tissue bioreactor to investigate mechanisms of cardiac cell therapy in order to provide a functional, yet controlled, model system of the human heart.

Human cardiac tissue engineering can fundamentally impact therapeutic discovery through the development of new species-specific screening systems that ...

Bioengineering

In Vitro Generation of Heart Field-specific Cardiac Progenitor Cells

Pluripotent stem cells offer great potential for understanding heart development and disease and for regenerative medicine. While recent advances in developmental cardiology have led to generating cardiac cells from pluripotent stem cells, it is unclear if the two cardiac fields - the first and second heart fields (FHF and SHF) &#8212; are induced in pluripotent stem cells systems. To address this, we generated a protocol for in vitro specification and isolation of heart field-specific cardiac progenitor cells. We used embryonic stem cells lines carrying Hcn4-GFP and Tbx1-Cre; Rosa-RFP reporters of the FHF and the SHF, respectively, and live cell immunostaining of the cell membrane protein Cxcr4, a SHF marker. With this approach, we generated progenitor cells which recapitulate the functional properties and transcriptome of their in vivo counterparts. Our protocol can be utilized to study early specification and segregation of the two heart fields and to generate chamber-specific cardiac cells for heart disease modelling. Since this is an in vitro organoid system, it may not provide precise anatomical information. However, this system overcomes the poor accessibility of gastrulation-stage embryos and can be upscaled for high-throughput screens.

The purpose of this method is to generate heart field-specific cardiac progenitor cells in vitro in order to study the progenitor cell specification and functional properties, and to generate chamber specific cardiac cells for heart disease modelling.

Pluripotent stem cells offer great potential for understanding heart development and disease and for regenerative medicine. While recent advances in ...

Directed Differentiation of Hemogenic Endothelial Cells from Human Pluripotent Stem Cells

Blood vessels are ubiquitously distributed within all tissues of the body and perform diverse functions. Thus, derivation of mature vascular endothelial cells, which line blood vessel lumens, from human pluripotent stem cells is crucial for a multitude of tissue engineering and regeneration applications. In vivo, primordial endothelial cells are derived from the mesodermal lineage and are specified toward specific subtypes, including arterial, venous, capillary, hemogenic, and lymphatic. Hemogenic endothelial cells are of particular interest because, during development, they give rise to hematopoietic stem and progenitor cells, which then generate all blood lineages throughout life. Thus, creating a system to generate hemogenic endothelial cells in vitro would provide an opportunity to study endothelial-to-hematopoietic transition, and may lead to ex vivo production of human blood products and reduced reliance on human donors. While several protocols exist for the derivation of progenitor and primordial endothelial cells, generation of well-characterized hemogenic endothelial cells from human stem cells has not been described. Here, a method for the derivation of hemogenic endothelial cells from human embryonic stem cells in approximately 1 week is presented: a differentiation protocol with primitive streak cells formed in response to GSK3&#946; inhibitor (CHIR99021), then mesoderm lineage induction mediated by bFGF, followed by primordial endothelial cell development promoted by BMP4 and VEGF-A, and finally hemogenic endothelial cell specification induced by retinoic acid. This protocol yields a well-defined population of hemogenic endothelial cells that can be used to further understand their molecular regulation and endothelial-to-hematopoietic transition, which has the potential to be applied to downstream therapeutic applications.

Presented here is a simple protocol for the directed differentiation of hemogenic endothelial cells from human pluripotent stem cells in approximately 1 week.

Blood vessels are ubiquitously distributed within all tissues of the body and perform diverse functions. Thus, derivation of mature vascular endothelial ...

Efficient Derivation of Human Cardiac Precursors and Cardiomyocytes from Pluripotent Human Embryonic Stem Cells with Small Molecule Induction

To date, the lack of a suitable human cardiac cell source has been the major setback in regenerating the human myocardium, either by cell-based transplantation or by cardiac tissue engineering1-3. Cardiomyocytes become terminally-differentiated soon after birth and lose their ability to proliferate. There is no evidence that stem/progenitor cells derived from other sources, such as the bone marrow or the cord blood, are able to give rise to the contractile heart muscle cells following transplantation into the heart1-3. The need to regenerate or repair the damaged heart muscle has not been met by adult stem cell therapy, either endogenous or via cell delivery1-3. The genetically stable human embryonic stem cells (hESCs) have unlimited expansion ability and unrestricted plasticity, proffering a pluripotent reservoir for in vitro derivation of large supplies of human somatic cells that are restricted to the lineage in need of repair and regeneration4,5. Due to the prevalence of cardiovascular disease worldwide and acute shortage of donor organs, there is intense interest in developing hESC-based therapies as an alternative approach. However, how to channel the wide differentiation potential of pluripotent hESCs efficiently and predictably to a desired phenotype has been a major challenge for both developmental study and clinical translation. Conventional approaches rely on multi-lineage inclination of pluripotent cells through spontaneous germ layer differentiation, resulting in inefficient and uncontrollable lineage-commitment that is often followed by phenotypic heterogeneity and instability, hence, a high risk of tumorigenicity6-8 (see a schematic in Fig. 1A). In addition, undefined foreign/animal biological supplements and/or feeders that have typically been used for the isolation, expansion, and differentiation of hESCs may make direct use of such cell-specialized grafts in patients problematic9-11. To overcome these obstacles, we have resolved the elements of a defined culture system necessary and sufficient for sustaining the epiblast pluripotence of hESCs, serving as a platform for de novo derivation of clinically-suitable hESCs and effectively directing such hESCs uniformly towards clinically-relevant lineages by small molecules12 (see a schematic in Fig. 1B). After screening a variety of small molecules and growth factors, we found that such defined conditions rendered nicotinamide (NAM) sufficient to induce the specification of cardiomesoderm direct from pluripotent hESCs that further progressed to cardioblasts that generated human beating cardiomyocytes with high efficiency (Fig. 2). We defined conditions for induction of cardioblasts direct from pluripotent hESCs without an intervening multi-lineage embryoid body stage, enabling well-controlled efficient derivation of a large supply of human cardiac cells across the spectrum of developmental stages for cell-based therapeutics.

We have established a protocol for induction of cardioblasts direct from pluripotent human embryonic stem cells maintained under defined conditions with small molecules, which enables derivation of a large supply of human cardiac progenitors and functional cardiomyocytes for cardiovascular repair.

To date, the lack of a suitable human cardiac cell source has been the major setback in regenerating the human myocardium, either by cell-based ...

Biology

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 ...

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

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Efficient Derivation of Human Cardiac Precursors and Cardiomyocytes from Pluripotent Human Embryonic Stem Cells with Small Molecule Induction

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