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 o...

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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr >
			<td >Culture medium</td>
			<td ></td>
			<td ></td>
			<td ></td>
		</tr>
		<tr >
			<td >Iscove&#39;s Modified Dulbecco&#39;s Medium (IMDM)</td>
			<td>ThermoFisher</td>
			<td>#12440</td>
			<td></td>
		</tr>
		<tr >
			<td >Dulbecco&#39;s Modified Eagle&#39;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&#160;</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&#160;</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&#39; 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 >Endothelial differentiation&#160;&#160;</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&#39;s F-12K (Kaighn&#39;s) Medium</td>
			<td>ThermoFisher</td>
			<td>#21127</td>
			<td></td>
		</tr>
		<tr >
			<td >Laminin&#160;</td>
			<td>Merck</td>
			<td>#L2020</td>
			<td></td>
		</tr>
		<tr >
			<td >Fibronectin&#160;</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&#39;,6-diamidino-2-phenylindole, dihydrochloride (DAPI)</td>
			<td>ThermoFisher</td>
			<td>#62247</td>
			<td>1:500 in ddH2O&#160;</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 >Tube formation</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 >Sca-1+/CD31- cardiac side population isolation34&#160;</td>
			<td ></td>
			<td ></td>
			<td></td>
		</tr>
		<tr >
			<td >Reagents</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&#39; Balanced Salt Solution (HBSS) CaCl2(-), MgCl2(-), phenol red (-)</td>
			<td >ThermoFisher</td>
			<td >#14175</td>
			<td>Prepare HBSS 500 mL + 2% FBS&#160; for quenching Collagenase B activity&#160;</td>
		</tr>
		<tr >
			<td >Dulbecco&#39;s Modified Eagle&#39;s Medium (DMEM) 1g/L of D-Glucose, L-Glutamine, Pyruvate</td>
			<td >ThermoFisher</td>
			<td >#331885</td>
			<td>Prepare DMEM + 10% FBS&#160; + 25 mM HEPES+ P/S for Hoechst stanining</td>
		</tr>
		<tr >
			<td >Penicillin-Streptomycin&#160; (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 &#181;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 &#181;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&#160;</td>
			<td>Merck</td>
			<td >#V4629</td>
			<td>Final concentration 83.3 &#181;M&#160;</td>
		</tr>
		<tr >
			<td >APC Rat Anti-Mouse CD31</td>
			<td>BD Biosciences</td>
			<td>#551262</td>
			<td>0.25 &#181;g/107cells</td>
		</tr>
		<tr >
			<td >FITC Rat Anti-Mouse Ly-6A/E (Sca-1)</td>
			<td>BD Biosciences</td>
			<td>#557405</td>
			<td>0.6 &#181;g/107cells</td>
		</tr>
		<tr >
			<td >7-Aminoactinomycin D (7-ADD)</td>
			<td>ThermoFisher</td>
			<td>#A1310</td>
			<td>0.15 &#181;g/106cells</td>
		</tr>
		<tr >
			<td >APC rat IgG2a k Isotype Control</td>
			<td>BD Biosciences</td>
			<td>#553932</td>
			<td>0.25 &#181;g/107cells</td>
		</tr>
		<tr >
			<td >FITC Rat IgG2a k Isotype Control</td>
			<td>BD Biosciences</td>
			<td>#554688</td>
			<td>0.6 &#181;g/107cells</td>
		</tr>
		<tr >
			<td >Material</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 &#181;m blue</td>
			<td>BD Biosciences&#160;</td>
			<td>#352340</td>
			<td></td>
		</tr>
		<tr >
			<td >Cell Strainer 100 &#181;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 >Mouse</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >Line</td>
			<td>Age</td>
			<td>Breeding</td>
			<td></td>
		</tr>
		<tr >
			<td >C57BL/6NRj / male</td>
			<td>12 weeks</td>
			<td>in house</td>
			<td></td>
		</tr>
		<tr >
			<td >Product Name</td>
			<td>Company</td>
			<td>Catalogue No.</td>
			<td></td>
		</tr>
		<tr >
			<td >Reagents</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >Iscove&#39;s Modified Dulbecco&#39;s Medium (IMDM)</td>
			<td>ThermoFisher</td>
			<td>#12440</td>
			<td></td>
		</tr>
		<tr >
			<td >Dulbecco&#39;s Modified Eagle&#39;s Medium (DMEM)/Nutrient Mixture F12 Ham</td>
			<td>Merck</td>
			<td>#D8437</td>
			<td></td>
		</tr>
		<tr >
			<td >Penicillin-Streptomycin&#160; (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&#160;</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&#160;</td>
			<td>Peprotech</td>
			<td>#250-25</td>
			<td></td>
		</tr>
		<tr >
			<td >Thrombin</td>
			<td>Diagontech AG, Switzerland&#160;</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 colspan="4" >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.&#160;</td>
		</tr>
		<tr >
			<td >Product Name</td>
			<td>Medium 18</td>
			<td>Medium 2</td>
			<td>Medium 3</td>
		</tr>
		<tr >
			<td >Reagents</td>
			<td>Culture</td>
			<td>Lineage induction</td>
			<td>Endothelial diff.</td>
		</tr>
		<tr >
			<td >Iscove&#39;s Modified Dulbecco&#39;s Medium (IMDM)</td>
			<td>35%</td>
			<td>35%</td>
			<td></td>
		</tr>
		<tr >
			<td >Dulbecco&#39;s Modified Eagle&#39;s Medium (DMEM)/Nutrient Mixture F12 Ham</td>
			<td>65%</td>
			<td>65%</td>
			<td></td>
		</tr>
		<tr >
			<td >Penicillin-Streptomycin&#160; (P/S)</td>
			<td>1%</td>
			<td>1%</td>
			<td></td>
		</tr>
		<tr >
			<td >Fetal Bovine Serum (FBS)</td>
			<td>3.5%</td>
			<td>&#8804;0.1%</td>
			<td></td>
		</tr>
		<tr >
			<td >L-Glutamine&#160;</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&#160;</td>
			<td>0.65 ng/mL</td>
			<td></td>
			<td></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:
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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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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 Human Neural Stem Cells from Peripheral Blood Hematopoietic Progenitor Cells

Human disease specific neuronal cultures are essential for generating in vitro models for human neurological diseases. However, the lack of access to primary human adult neural cultures raises unique challenges. Recent developments in induced pluripotent stem cells (iPSC) provides an alternative approach to derive neural cultures from skin fibroblasts through patient specific iPSC, but this process is labor intensive, requires special expertise and large amounts of resources, and can take several months. This prevents the wide application of this technology to the study of neurological diseases. To overcome some of these issues, we have developed a method to derive neural stem cells directly from human adult peripheral blood, bypassing the iPSC derivation process. Hematopoietic progenitor cells enriched from human adult peripheral blood were cultured in vitro and transfected with Sendai virus vectors containing transcriptional factors Sox2, Oct3/4, Klf4, and c-Myc. The transfection results in morphological changes in the cells which are further selected by using human neural progenitor medium containing basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF). The resulting cells are characterized by the expression for neural stem cell markers, such as nestin and SOX2. These neural stem cells could be further differentiated to neurons, astroglia and oligodendrocytes in specified differentiation media. Using easily accessible human peripheral blood samples, this method could be used to derive neural stem cells for further differentiation to neural cells for in vitro modeling of neurological disorders and may advance studies related to the pathogenesis and treatment of those diseases.

A method was developed to directly derive human neural stem cells from hematopoietic progenitor cells enriched from peripheral blood cells.

Human disease specific neuronal cultures are essential for generating in vitro models for human neurological diseases. However, the lack of access to ...

Neural Differentiation of Mouse Embryonic Stem Cells in Serum-free Monolayer Culture

The ability to differentiate mouse embryonic stem cells (ESC) to neural progenitors allows the study of the mechanisms controlling neural specification as well as the generation of mature neural cell types for further study. In this protocol we describe a method for the differentiation of ESC to neural progenitors using serum-free, monolayer culture. The method is scalable, efficient and results in production of ~70% neural progenitor cells within 4 - 6 days. It can be applied to ESC from various strains grown under a variety of conditions. Neural progenitors can be allowed to differentiate further into functional neurons and glia or analyzed by microscopy, flow cytometry or molecular techniques. The differentiation process is amenable to time-lapse microscopy and can be combined with the use of reporter lines to monitor the neural specification process. We provide detailed instructions on media preparation and cell density optimization to allow the process to be applied to most ESC lines and a variety of cell culture vessels.

This protocol describes in detail the method for generating neural progenitors from embryonic stem cells using a serum-free monolayer method. These progenitors can be used to derive mature neural cell types or to study the process of neural specification and is amenable to multiwell format scaling for compound screening.

The ability to differentiate mouse embryonic stem cells (ESC) to neural progenitors allows the study of the mechanisms controlling neural specification as ...

Initiating Differentiation in Immortalized Multipotent Otic Progenitor Cells

Use of human induced pluripotent stem cells (iPSC) or embryonic stem cells (ESC) for cell replacement therapies holds great promise. Several limitations including low yields and heterogeneous populations of differentiated cells hinder the progress of stem cell therapies. A fate restricted immortalized multipotent otic progenitor (iMOP) cell line was generated to facilitate efficient differentiation of large numbers of functional hair cells and spiral ganglion neurons (SGN) for inner ear cell replacement therapies. Starting from dissociated cultures of single iMOP cells, protocols that promote cell cycle exit and differentiation by basic fibroblast growth factor (bFGF) withdrawal were described. A significant decrease in proliferating cells after bFGF withdrawal was confirmed using an EdU cell proliferation assay. Concomitant with a decrease in proliferation, successful differentiation resulted in expression of molecular markers and morphological changes. Immunostaining of Cdkn1b (p27KIP) and Cdh1 (E-cadherin) in iMOP-derived otospheres was used as an indicator for differentiation into inner ear sensory epithelia while immunostaining of Cdkn1b and Tubb3 (neuronal &#946;-tubulin) was used to identify iMOP-derived neurons. Use of iMOP cells provides an important tool for understanding cell fate decisions made by inner ear neurosensory progenitors and will help develop protocols for generating large numbers of iPSC or ESC-derived hair cells and SGNs. These methods will accelerate efforts for generating otic cells for replacement therapies.

The current protocols to maintain immortalized multipotent otic progenitor (iMOP) cells and otic differentiation are described. Culture conditions and molecular markers that indicate differentiation into sensory epithelia and spiral ganglion neurons (SGN) are highlighted.

Use of human induced pluripotent stem cells (iPSC) or embryonic stem cells (ESC) for cell replacement therapies holds great promise. Several limitations ...

Epigenetic Regulation of Cardiac Differentiation of Embryonic Stem Cells and Tissues

Specific gene transcription is a key biological process that underlies cell fate decision during embryonic development. The biological process is mediated by transcription factors which bind genomic regulatory regions including enhancers and promoters of cardiac constitutive genes. DNA is wrapped around histones that are subjected to chemical modifications. Modifications of histones further lead to repressed, activated or poised gene transcription, thus bringing another level of fine tuning regulation of gene transcription. Embryonic Stem cells (ES cells) recapitulate within embryoid bodies (i.e., cell aggregates) or in 2D culture the early steps of cardiac development. They provide in principle enough material for chromatin immunoprecipitation (ChIP), a technology broadly used to identify gene regulatory regions. Furthermore, human ES cells represent a human cell model of cardiogenesis. At later stages of development, mouse embryonic tissues allow for investigating specific epigenetic landscapes required for determination of cell identity. Herein, we describe protocols of ChIP, sequential ChIP followed by PCR or ChIP-sequencing using ES cells, embryoid bodies and cardiac specific embryonic regions. These protocols allow to investigating the epigenetic regulation of cardiac gene transcription.

A fine tuning regulation of gene transcription underlies embryonic cell fate decision. Herein, we describe chromatin immunoprecipitation assays used to investigate epigenetic regulation of both cardiac differentiation of stem cells and cardiac development of mouse embryos.

Specific gene transcription is a key biological process that underlies cell fate decision during embryonic development. The biological process is mediated ...

Aggregate Size Optimization in Microwells for Suspension-based Cardiac Differentiation of Human Pluripotent Stem Cells

Cardiac differentiation of human pluripotent stems cells (hPSCs) is typically carried out in suspension cell aggregates. Conventional aggregate formation of hPSCs involves dissociating cell colonies into smaller clumps, with size control of the clumps crudely controlled by pipetting the cell suspension until the desired clump size is achieved. One of the main challenges of conventional aggregate-based cardiac differentiation of hPSCs is that culture heterogeneity and spatial disorganization lead to variable and inefficient cardiomyocyte yield. We and others have previously reported that human embryonic stem cell (hESC) aggregate size can be modulated to optimize cardiac induction efficiency. We have addressed this challenge by employing a scalable, microwell-based approach to control physical parameters of aggregate formation, specifically aggregate size and shape. The method we describe here consists of forced aggregation of defined hPSC numbers in microwells, and the subsequent culture of these aggregates in conditions that direct cardiac induction. This protocol can be readily scaled depending on the size and number of wells used. Using this method, we can consistently achieve culture outputs with cardiomyocyte frequencies greater than 70%.

Conventional methods to initiate suspension aggregate based cardiac differentiation of human pluripotent stems cells (hPSCs) are plagued with culture heterogeneity with respect to aggregate size and shape. Here, we describe a robust method for cardiac differentiation employing microwells to generate size-controlled hPSC aggregates cultured under cardiac-promoting conditions.

Cardiac differentiation of human pluripotent stems cells (hPSCs) is typically carried out in suspension cell aggregates.  Conventional aggregate formation ...

Chondrogenic Differentiation Induction of Adipose-derived Stem Cells by Centrifugal Gravity

Impaired cartilage cannot heal naturally. Currently, the most advanced therapy for defects in cartilage is the transplantation of chondrocytes differentiated from stem cells using cytokines. Unfortunately, cytokine-induced chondrogenic differentiation is costly, time-consuming, and associated with a high risk of contamination during in vitro differentiation. However, biomechanical stimuli also serve as crucial regulatory factors for chondrogenesis. For example, mechanical stress can induce chondrogenic differentiation of stem cells, suggesting a potential therapeutic approach for the repair of impaired cartilage. In this study, we demonstrated that centrifugal gravity (CG, 2,400 &#215; g), a mechanical stress easily applied by centrifugation, induced the upregulation of sex determining region Y (SRY)-box 9 (SOX9) in adipose-derived stem cells (ASCs), causing them to express chondrogenic phenotypes. The centrifuged ASCs expressed higher levels of chondrogenic differentiation markers, such as aggrecan (ACAN), collagen type 2 alpha 1 (COL2A1), and collagen type 1 (COL1), but lower levels of collagen type 10 (COL10), a marker of hypertrophic chondrocytes. In addition, chondrogenic aggregate formation, a prerequisite for chondrogenesis, was observed in centrifuged ASCs.

Mechanical stress can induce the chondrogenic differentiation of stem cells, providing a potential therapeutic approach for the repair of impaired cartilage. We present a protocol to induce the chondrogenic differentiation of adipose-derived stem cells (ASCs) using centrifugal gravity (CG). CG-induced upregulation of SOX9 results in the development of chondrogenic phenotypes.

Impaired cartilage cannot heal naturally. Currently, the most advanced therapy for defects in cartilage is the transplantation of chondrocytes ...

Directed Differentiation of Primitive and Definitive Hematopoietic Progenitors from Human Pluripotent Stem Cells

One of the major goals for regenerative medicine is the generation and maintenance of hematopoietic stem cells (HSCs) derived from human pluripotent stem cells (hPSCs). Until recently, efforts to differentiate hPSCs into HSCs have predominantly generated hematopoietic progenitors that lack HSC potential, and instead resemble yolk sac hematopoiesis.&#160;These resulting hematopoietic progenitors may have limited utility for in vitro disease modeling of various adult hematopoietic disorders, particularly those of the lymphoid lineages. However, we have recently described methods to generate erythro-myelo-lymphoid multilineage definitive hematopoietic progenitors from hPSCs using a stage-specific directed differentiation protocol, which we outline here. Through enzymatic dissociation of hPSCs on basement membrane matrix-coated plasticware, embryoid bodies (EBs) are formed. EBs are differentiated to mesoderm by recombinant BMP4, which is subsequently specified to the definitive hematopoietic program by the GSK3&#946; inhibitor, CHIR99021. Alternatively, primitive hematopoiesis is specified by the PORCN inhibitor, IWP2. Hematopoiesis is further driven through the addition of recombinant VEGF and supportive hematopoietic cytokines. The resulting hematopoietic progenitors generated using this method have the potential to be used for disease and developmental modeling, in vitro.

Here, we present human pluripotent stem cell (hPSC) culture protocols, used to differentiate hPSCs into CD34+ hematopoietic progenitors. This method uses stage-specific manipulation of canonical WNT signaling to specify cells exclusively to either the definitive or primitive hematopoietic program.

One of the major goals for regenerative medicine is the generation and maintenance of hematopoietic stem cells (HSCs) derived from human pluripotent stem ...

Small-scale Propagation of Human iPSCs in Serum-free Conditions for Routine Immunocytochemical Characterization

There is great interest in utilizing human induced pluripotent stem cells (hiPSCs) for disease modeling and cell therapeutics due to their patient specificity and characteristic stemness. However, the pluripotency of iPSCs, which is essential to their functionality, must be confirmed before these cells can be used in such applications. While a rigorous characterization of iPSCs, through different cellular and functional assays is necessary to establish their pluripotency, routine assessment of pluripotency maintenance can be achieved more simply and effectively through immunocytochemical techniques. Here, we present a systematic protocol for culturing hiPSCs, in a scaled-down manner, to particularly facilitate the verification of their pluripotent state using immunocytochemistry. More specifically, this methodology encompasses an efficient and cost-effective means of growing iPSCs in serum-free conditions and plating them on small chamber slides or glass coverslips ideal for immunocytochemistry.

Regular characterization of induced pluripotent stem cells (iPSCs), to ascertain maintenance of their pluripotent state, is an important step before these cells are used for other applications. Here we describe a method for the small-scale propagation of human iPSCs specifically designed to enable their easy and routine characterization via immunocytochemistry.

There is great interest in utilizing human induced pluripotent stem cells (hiPSCs) for disease modeling and cell therapeutics due to their patient ...

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.