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Summary

Abstract

Introduction

Protocol

Representative Results

Discussion

Acknowledgements

Materials

References

Developmental Biology

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

Published: January 7th, 2019

DOI:

10.3791/58370

1Department of Biomedicine, University Hospital and University of Basel, 2Division of Cardiology, University Hospital Basel
* These authors contributed equally

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 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% 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.

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 he....

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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.......

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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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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.......

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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).

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Name Company Catalog Number Comments
Culture medium
Iscove's Modified Dulbecco's Medium (IMDM) ThermoFisher #12440
Dulbecco's Modified Eagle's Medium (DMEM)/Nutrient Mixture F12 Ham Merck #D8437
Penicillin-Streptomycin (P/S) ThermoFisher #15140122
Fetal Bovine Serum (FBS) Hyclone #SH30071 3.5% (0.1% for lineage induction)
L-Glutamine  ThermoFisher #25030 Final concentration 2 mM
Glutathione Merck #G6013
Recombinant Human Epidermal Growth Factor (EGF) Peprotech #AF-100-15
Recombinant Basic Fibroblast Growth Factor (FGF) Peprotech #AF-100-18B
B27 Supplement ThermoFisher #17504044
Cardiotrophin 1  Peprotech #250-25
Thrombin Diagontech AG, Switzerland #100-125
Hanks' Balanced Salt Solution (HBSS) CaCl2(-), MgCl2(-) ThermoFisher #14170
0.05 % Trypsin-EDTA ThermoFisher #25300
T75 Flask Sarstedt #83.3911
Endothelial differentiation  
Endothelial Cell Growth Medium (EGM)-2 BulletKit Lonza #CC-3162
Ham's F-12K (Kaighn's) Medium ThermoFisher #21127
Laminin  Merck #L2020
Fibronectin  Merck #F4759 Dilute in ddH2O
6 Well Plate Falcon #353046
Formaldehyde Solution Merck #F1635 Diluite 1:10 in PBS (3.7% final concentration)
Triton X-100 Merck #93420 0.1% in ddH2O
Normal Goat Serum (10%) ThermoFisher #50062Z
Anti-von Willebrand Factor antibody Abcam #ab6994 1:100 in 10% goat serum
Goat anti-Rabbit IgG, Alexa Fluor 546 ThermoFisher #A11010 1:500 in 10% goat serum
4',6-diamidino-2-phenylindole, dihydrochloride (DAPI) ThermoFisher #62247 1:500 in ddH2O 
SlowFade Antifade Kit ThermoFisher #S2828
BX63 widefield microscope Olympus
Tube formation
96 Well Plate Falcon #353072
5 ml Round Bottom Tube with Strainer Cap Falcon #352235
Matrigel Growth Factor Reduced Corning #354230
IX50 widefield microscope Olympus
Sca-1+/CD31- cardiac side population isolation34 
Reagents
Pentobarbital Natrium 50 mg/ml ad usum vet. in house hospital pharmacy #9077862 Working solution: 200 mg/kg
Phosphate Buffered Saline (PBS) CaCl2(-), MgCl2(-) ThermoFisher #20012
Hanks' Balanced Salt Solution (HBSS) CaCl2(-), MgCl2(-), phenol red (-) ThermoFisher #14175 Prepare HBSS 500 mL + 2% FBS  for quenching Collagenase B activity 
Dulbecco's Modified Eagle's Medium (DMEM) 1g/L of D-Glucose, L-Glutamine, Pyruvate ThermoFisher #331885 Prepare DMEM + 10% FBS  + 25 mM HEPES+ P/S for Hoechst stanining
Penicillin-Streptomycin  (P/S) ThermoFisher #15140122
HEPES 1 M ThermoFisher #15630080 Final concentration 25 mM
Fetal Bovine Serum (FBS) Hyclone #SH30071
RBC LysisBuffer (10X) BioLegend #420301/100mL Dilute to 1X in ddH2O and filter through a 0.2 µm filter
Collagenase B Merck #11088807001 Final concentration 1 mg/mL in HBSS, filtered through a 0.2 µm filter
bisBenzimide H33342 Trihydrochloride (Hoechst) Merck #B2261 Prepare 1 mg/mL in ddH2O
Verapamil-hydrochloride  Merck #V4629 Final concentration 83.3 µM 
APC Rat Anti-Mouse CD31 BD Biosciences #551262 0.25 µg/107cells
FITC Rat Anti-Mouse Ly-6A/E (Sca-1) BD Biosciences #557405 0.6 µg/107cells
7-Aminoactinomycin D (7-ADD) ThermoFisher #A1310 0.15 µg/106cells
APC rat IgG2a k Isotype Control BD Biosciences #553932 0.25 µg/107cells
FITC Rat IgG2a k Isotype Control BD Biosciences #554688 0.6 µg/107cells
Material
Needles 27G Terumo #NN-2719R
Needles 18G Terumo #NN-1838S
Single Use Syringes 1 mL sterile CODAN #62.1640
Transferpipette 3.5 mL Sarstedt #86.1171.001
Cell Strainer 40 µm blue BD Biosciences  #352340
Cell Strainer 100 µm yellow BD Biosciences #352360
Lumox Dish 50 Sarstedt #94.6077.305
Culture Dishes P100 Corning #353003
Culture Dishes P60 Corning #353004
Mouse
Line Age Breeding
C57BL/6NRj / male 12 weeks in house
Product Name Company Catalogue No.
Reagents
Iscove's Modified Dulbecco's Medium (IMDM) ThermoFisher #12440
Dulbecco's Modified Eagle's Medium (DMEM)/Nutrient Mixture F12 Ham Merck #D8437
Penicillin-Streptomycin  (P/S) ThermoFisher #15140122
Fetal Bovine Serum (FBS) Hyclone #SH30071
L-Glutamine  ThermoFisher #25030
Glutathione Merck #G6013
B27 Supplement ThermoFisher #17504044
Recombinant Human Epidermal Growth Factor (EGF) Peprotech #AF-100-15
Recombinant Basic Fibroblast Growth Factor (FGF) Peprotech #AF-100-18B
Cardiotrophin 1  Peprotech #250-25
Thrombin Diagontech AG, Switzerland  #100-125
Endothelial Cell Growth Medium (EGM)-2 BulletKit Lonza #CC-3162
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. 
Product Name Medium 18 Medium 2 Medium 3
Reagents Culture Lineage induction Endothelial diff.
Iscove's Modified Dulbecco's Medium (IMDM) 35% 35%
Dulbecco's Modified Eagle's Medium (DMEM)/Nutrient Mixture F12 Ham 65% 65%
Penicillin-Streptomycin  (P/S) 1% 1%
Fetal Bovine Serum (FBS) 3.5% ≤0.1%
L-Glutamine  2 mM 2 mM
Glutathione 0.2 nM 0.2 nM
B27 Supplement 1.3%
Recombinant Human Epidermal Growth Factor (EGF) 6.5 ng/mL
Recombinant Basic Fibroblast Growth Factor (FGF) 13 ng/mL
Cardiotrophin 1  0.65 ng/mL

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