Name: induction of endothelial differentiation in cardiac progenitor cells under low serum conditions
Uploaded: 2019-01-07T14:00:00
Description: Watch this Scientific Journal Video about Induction of Endothelial Differentiation in Cardiac Progenitor Cells Under Low Serum Conditions at JoVE.com

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, card...

<ol>
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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 ...

Differentiation of cardiac progenitor cells, or CPCs, upon amplification requires a phase switch from a proliferating to a committed stage, and this protocol uses efficient endothelial lineage in CPCs. A major advantage of the technique is that it is applicable to CPCs isolated based on several techniques and from different species. This protocol can be used to enhance the therapeutic potentials of CPCs for cell therapy.It can also be used to investigate the potentials and mechanisms of differentiation and how these are affected by cardiac disease. CPCs are an inhomogeneous population of cells that may differ in shape, size, and potency. Therefore, the protocol has to be adjusted according to the specific subpopulation of CPCs used.After anesthetizing the mouse, wipe down its chest with 70%ethanol. Using scissors, cut the skin and the thoracic wall to expose the thoracic cavity. Use forceps to lift the heart, and then use scissors to cut it at the base.Transfer the heart to a P60 culture dish containing five milliliters of cold PBS, and then use forceps to pump the heart and eject the blood out of the cavities. Place the heart in a P60 dish containing five milliliters of cold PBS and wash it. Use small scissors to remove the atria, cut the heart into two longitudinal pieces, and wash them in five milliliters of ice cold PBS.Using small scissors, cut the heart pieces into smaller sections. Add a drop of a Hank's balanced salt solution containing collagenase B at a concentration of one milligram per milliliter, and use a sterile razor blade to mince the small heart pieces thoroughly. Next, add 2.5 milliliters of the collagenase B solution to the dish.Place the dish at a 30 degree angle in an incubator at 37 degrees Celsius. Let the minced heart pieces incubate for a maximum of 30 minutes while repeatedly passing them through a Pasteur pipette every 10 minutes during the incubation. First, add five milliliters of cold HBSS supplemented with 2%FBS to the minced and homogenized heart pieces.Using a 100 micrometer filter, filter the heart pieces to remove any undigested tissue. Centrifuge at 470 times G and at room temperature for five minutes. Discard the supernatant and re-suspend the pellet in five milliliters of red blood cell lysis buffer.Incubate on ice for five minutes with occasional shaking. After this, add 10 milliliters of PBS and filter the sample through a 40 micrometer filter to exclude larger cells including residual cardiomyocytes. Centrifuge at 470 times G and at room temperature for five minutes without the break.Then, re-suspend the cells in DMEM supplemented with 10%FBS, aiming for a final cell concentration of one million cells per milliliter. Distribute the cells into two tubes for staining and sorting, adding 1.5 milliliters of the cell solution to one tube and 3.5 milliliters to the second tube. Add Hoechst solution and incubate at 37 degrees Celsius for 90 minutes with occasional flipping to ensure equal distribute of the dye and prevent clotting of the cells.The side population appears at the Hoechst low lower left corner aside of the main population. In the FACS plot it can be identified based on the negative control in which verapamil prompts the side population to disappear. Warm up medium one to 37 degrees Celsius and cool down the centrifuge to four degrees Celsius.Centrifuge the sorting tubes at 470 times G and at four degrees Celsius for six minutes. Then, re-suspend the cells in medium one. Transfer the cells to a gas-permeable P60 dish containing four milliliters of medium one.Change the medium every three days until the cells have reached a confluence between 70 and 80%Culture SP-CPCs for two to three days in T75 flasks using eight milliliters of medium one in each flask. Next, carefully aspirate the medium and gently rinse the flask with five milliliters of warm HBSS. Treat the cells with five milliliters of trypsin EDTA for five minutes at 37 degrees Celsius with 5%carbon dioxide.Then, add five milliliters of medium one to stop the trypsin activity, and transfer the cell suspension to a 15 milliliter tube. Centrifuge at 470 times G for five minutes at room temperature. Next, re-suspend the cells in either medium one or medium two.Plate the cells on P60 dishes, plating 250, 000 SP-CPCs per dish with three milliliters of medium containing different serum concentrations. After two days of culturing, collect the medium from each dish into a 15 milliliter tube to collect the dead cells. Trypsinize the adherent cells as previously described and collect the cell suspension into the 15 milliliter tube that contains the collected medium.Centrifuge at 840 times G for five minutes at room temperature, then aspirate the supernatant and add one milliliter of either medium one or medium two for cell counting. Stain the SP-CPCs with trypan blue and count the viable and dead cells. First, aspirate the medium from the prepared cells, and rinse them gently with five milliliters of warm HBSS.Treat the cells with five milliliters of trypsin EDTA for five minutes in the cell incubator. Then, add five milliliters of medium one to stop the trypsin activity. Transfer the cell suspension to a 15 milliliter tube and centrifuge at 470 times G for five minutes at room temperature.Aspirate the supernatant and add medium two for cell counting. Next, seed 80, 000 cells into each well of a pre-coated six-well culture plate with three milliliters of medium two. Keep the plate at 37 degrees Celsius for 20 to 24 hours, and then change the medium in each well to three milliliters of medium three.Culture the plate for 21 days, changing the medium every three days. After this, stain the cells with an endothelial marker and perform fluorescence microscopy to verify the endothelial nature of the differentiated cells. First, aspirate the medium from the prepared cells and rinse them gently with five milliliters of warm HBSS.Treat the cells with two milliliters of trypsin EDTA for five minutes in the cell incubator, then add three milliliters of medium three to stop the trypsin activity. Transfer the cell suspension to a 15 milliliter tube and centrifuge at 470 times G for five minutes at room temperature. Aspirate the supernatant and add one milliliter of medium three, pipetting gently to mix.Count the cells and seed between 2, 000 and 4, 000 cells in 100 microliters of medium three to each matrix coated well of a 96-well plate. Incubate the plate at 37 degrees Celsius for 16 hours. After this, use a bright-field microscope at two times magnification to take a picture of the cells.In this study, suitable conditions are explored for the facilitation of endothelial lineage commitment of cardiac progenitor cells. When the cell confluency is at or below 60%no significant difference is seen in the samples treated with 3.5%FBS. When cells at this confluency are treated with 0.1%FBS, they show a decrease in cell proliferation on laminin when compared to fibronectin, but show no increase in cell death.In contrast, cells at a high confluency show no significant difference in either cell proliferation or cell death between the two conditions. Changes in cell shape appear to be an indicator of suitable culture conditions in which endothelial differentiation is going to be successful. Within seven to 14 days in the endothelial differentiation medium, successful cultures are seen to contain larger cells with different morphology.Interestingly, these cells disappeared towards the end of the differentiation phase and tended to appear in lower numbers and at later time points in high-density cultures. Tube formation is consistently successful in cells plated at low density and differentiated on laminin, while it is seen to mostly fail in cells plated at high density. While tube formation is mostly unsuccessful in cells cultured on fibronectin irrespective of cell density, those differentiated at a low density sometimes form rudimentary tubes.The serum concentration and cell density to induce endothelial lineage are crucial, and they have to be adjusted to guarantee slowing of proliferation while maintaining viability. Upon lineage induction, CPCs from humans or preclinical disease models may be used for cell transplantation studies to assess their in vivo differentiation potential. This protocol could help to study or establish novel cardiac regeneration tools, and it was previously used to study molecular mechanism of early lineage commitment.

Research

JoVE Journal

Developmental Biology

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 ...

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 ...

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

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Initiating Differentiation in Immortalized Multipotent Otic Progenitor Cells

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Epigenetic Regulation of Cardiac Differentiation of Embryonic Stem Cells and Tissues

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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 ...

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 ...

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 ...

Pan-myeloid Differentiation of Human Cord Blood Derived CD34+ Hematopoietic Stem and Progenitor Cells

Pan-myeloid Differentiation of Human Cord Blood Derived CD34+ Hematopoietic Stem and Progenitor Cells

Ex vivo differentiation of human hematopoietic stem cells is a widely used model for studying hematopoiesis. The protocol described here is for cytokine ...

Efficient Differentiation of Postganglionic Sympathetic Neurons using Human Pluripotent Stem Cells under Feeder-free and Chemically Defined Culture Conditions

Human pluripotent stem cells (hPSCs) have become a powerful tool for disease modeling and the study of human embryonic development in vitro. We previously ...