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.

induction-endothelial-differentiation-cardiac-progenitor-cells-under

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 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 ...

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 ...

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 ...

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 ...

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 induced differentiation of CD34+ hematopoietic stem and progenitor cells to the four myeloid lineage cells. CD34+ cells are isolated from human umbilical cord blood and co-cultured with MS-5 stromal cells in the presence of cytokines. Immunophenotypic characterization of the stem and progenitor cells, and the differentiated myeloid lineage cells are described. Using this protocol, CD34+ cells may be incubated with small molecules or transduced with lentiviruses to express myeloid disease mutations to investigate their impact on myeloid differentiation.

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

Here, we present a protocol for immunophenotypic characterization and cytokine induced differentiation of cord blood derived CD34+ hematopoietic stem and progenitor cells to the four myeloid lineages. The applications of this protocol include investigations on the effect of myeloid disease mutations or small molecules on myeloid differentiation of the CD34+ 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 presented a differentiation protocol for the derivation of autonomic neurons with sympathetic character that has been applied to patients with autonomic neuropathy. However, the protocol was built on Knock Out Serum Replacement (KSR) and feeder-based culture conditions, and to ensure high differentiation efficiency, cell sorting was necessary. These factors cause high variability, high cost, and low reproducibility. Moreover, mature sympathetic properties, including electrical activity, have not been verified. Here, we present an optimized protocol where PSC culture and differentiation are performed in feeder-free and chemically defined culture conditions. Genetic markers identifying trunk neural crest are identified. Further differentiation into postganglionic sympathetic neurons is achieved after 20 days without the need for cell sorting. Electrophysiological recording further shows the functional neuron identity. Firing detected from our differentiated neurons can be enhanced by nicotine and suppressed by the adrenergic receptor antagonist propranolol. Intermediate sympathetic neural progenitors in this protocol can be maintained as neural spheroids for up to 2 weeks, which allows expansion of the cultures. In sum, our updated sympathetic neuron differentiation protocol shows high differentiation efficiency, better reproducibility, more flexibility, and better neural maturation compared to the previous version. This protocol will provide researchers with the cells necessary to study human disorders that affect the autonomic nervous system.

In this protocol, we describe a stable, highly efficient differentiation strategy for the generation of postganglionic sympathetic neurons from human pluripotent stem cells. This model will make neurons available for the use of studies of multiple autonomic disorders.

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