Method Article

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

DOI:

10.3791/58370

⸱

January 7th, 2019

 ,  ,  ,  , 
1Department of Biomedicine, University Hospital and University of Basel, 2Division of Cardiology, University Hospital Basel

* These authors contributed equally

In This Article

Summary

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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.

Abstract

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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.

Introduction

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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 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.

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Protocol

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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, 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.

  1. 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.
  2. Wipe the chest with 70% ethanol. Cut the skin and thoracic wall with scissors to expose the thoracic cavity.
  3. 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).
  4. Pump the heart with forceps to eject the blood out of the cavities (Figure 1A).
  5. 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).
  6. 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’s balanced salt solution (HBSS) and mince the small pieces thoroughly with a sterile razor blade (Figure 1C).

2. Digestion

  1. Add 10 mL (2.5 mL for P60) of the 1 mg/mL collagenase B solution to the dish from step 1.6.
  2. Put the collagenase B solution containing the minced heart pieces in a tilted (about 30°) P100 (or P60) in a 37 °C incubator (Figure 1D).
  3. 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.

3. Filtration

  1. 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.
  2. Filter the heart pieces through 100 µm filter to remove undigested tissue and centrifuge at 470 x g for 5 min at room temperature (RT) (Figure 1F, yellow filters).
  3. 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.
  4. Add 10 mL (5 mL for P60) of PBS (to stop the lysis reaction) and filter the sample through a 40 µm filter to exclude larger cells, including residual cardiomyocytes (Figure 1F, blue filters).
  5. 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.
  6. 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.
  7. 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.

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 “main population”, giving them their name “side population”. 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.

  1. Staining with Hoechst in the presence and absence of verapamil
    1. Add verapamil (with a final concentration of 83.3 µM) and Hoechst (with a final concentration of 5 µg/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 °C) for 90 min and revert the tubes every 20 min (Figure 1G).
    2. 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).
    3. 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.
    4. 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.
    5. Discard the supernatant and resuspend the pellets of tube A and (iii) the negative control in 250 µL of HBSS and keep them on ice in the dark until sorting.
    6. Resuspend the pellet of tube B in 200 µL of HBSS and the pellet of (i, ii) the single-staining aliquots in 100 µL of HBSS. Add FITC-conjugated anti-Sca-1 (0.6 µg/107 cells) and APC-conjugated anti-CD31 (0.25 µg/107 cells). Incubate the pellets for 30 min on ice and shake them from time to time in the dark.
    7. Add 2 mL of HBSS to the tubes. Centrifuge the tubes at 470 x g for 5 min at RT.
    8. 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 µL of HBSS. Resuspend the pellet of tube B in HBSS (20 x 106 cells/mL).
    9. Keep the samples on ice and protected from light until sorting.
  2. Sorting with flow cytometry
    1. Prepare sterile 1.5 mL sorting tubes with 500 µL of HBSS including 2% FBS.
    2. Stain the cells with 7-aminoactinomycin D (7-AAD) (0.15 µg/106 cells) on ice for 10 min to exclude dead cells.
    3. 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 µm and pressure of 15 psi.
    4. 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.

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.

  1. Warm up Medium 1 to 37 °C before use and cool down the centrifuge to 4 °C. Centrifuge the sorting tubes at 4 °C and 470 x g for 6 min and resuspend the cells in Medium 1.
  2. 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.

6. Expansion and Differentiation of SP-CPCs

  1. Cell culture
    1. Culture 3 x 105 SP-CPCs in 8 mL of Medium 1 in a T75 flask.
    2. Incubate SP-CPCs at 37 °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.
  2. SP-CPC growth and viability under different serum concentrations
    1. 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 °C) HBSS.
    2. 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.
    3. Centrifuge the cells at 470 x g for 5 min at RT.
    4. 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.
    5. 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.
    6. 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.
    7. 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.
  3. Induction of endothelial differentiation
    1. Precoat a (6-well) culture plate with 10 µg/mL of laminin (LN) or fibronectin (FN).
      1. Make a substrate solution containing 10 µg/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 °C.
      2. Aspirate the solution from the plate and add 2 mL of PBS to each well until using it.
    2. Aspirate the medium from the cells from step 6.1.2, rinse them gently with 5 mL of warm (37 °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.
    3. Centrifuge the cells at 470 x g for 5 min at RT. Aspirate the supernatant and add Medium 2 for cell counting.
    4. Seed 8 x 104 cells per well on the coated plate with 3 mL of Medium 2 and keep it at 37 °C for 20 - 24 h. Change the medium to 3 mL of Medium 3.
      NOTE: We recommend using medium containing a low serum concentration—and without supplements—for the first 20 - 24 h. We recommended using <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).
    5. Culture the cells for 21 d and change the medium every 3 d.
    6. Verify the endothelial nature of differentiated cells by staining them with an endothelial marker such as von Willebrand Factor (vWF) and performing fluorescence microscopy.
      1. Wash the cells with 1 mL of PBS and fix the cells in 3.7% formaldehyde for 2 min at RT.
      2. 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.
      3. Incubate the cells with anti-von Willebrand factor antibody (1:100) for 48 h at 4 °C
      4. 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.
      5. Wash again 3x, for 10 min each, with 1 mL of PBS. Stain the cell nuclei with 4’6-diamidino-2-phenylindole, dihydrochloride (DAPI; 1:500) for 5 min at RT in the dark.
      6. Wash the cells 3x, for 5 min each, with 1 mL of PBS. Mount the cells and store them at 4 °C
        NOTE: The culturing conditions (substrate, medium, supplemental reagents, and FBS concentration) of steps 6.1 and 6.3 are described in Figure 3.
  4. Tube formation assay
    1. Prepare a basement membrane matrix (e.g., Matrigel, henceforth referred to as matrix) plate.
      1. Thaw the matrix at 4 °C overnight.
      2. Coat a 96-well plate with 100 µL 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.
      3. Maintain the plate at 37 °C for 30 min.
    2. Aspirate the medium from the cells (after the completion of step 6.3.5), gently rinse the cells with 5 mL of warm (37 °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.
    3. Centrifuge the cells at 470 x g for 5 min at RT. Aspirate the supernatant, add 1 mL of Medium 3, and pipet gently.
    4. Filter the cells with a 35 µm cell strainer, if the cells are aggregated.
    5. Count the cells and seed 2 x 103 to 4 x 103 cells in 100 µL of Medium 3 in each matrix-coated well. Keep the plate at 37 °C in the cell incubator for 16 h.
    6. 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.

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Results

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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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Discussion

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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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Disclosures

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The authors have nothing to disclose.

Acknowledgements

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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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Materials

List of materials used in this article
NameCompanyCatalog NumberComments
Culture medium
Iscove's Modified Dulbecco's Medium (IMDM)ThermoFisher#12440
Dulbecco's Modified Eagle's Medium (DMEM)/Nutrient Mixture F12 HamMerck#D8437
Penicillin-Streptomycin (P/S)ThermoFisher#15140122
Fetal Bovine Serum (FBS)Hyclone#SH300713.5% (0.1% for lineage induction)
L-Glutamine ThermoFisher#25030Final concentration 2 mM
GlutathioneMerck#G6013
Recombinant Human Epidermal Growth Factor (EGF)Peprotech#AF-100-15
Recombinant Basic Fibroblast Growth Factor (FGF)Peprotech#AF-100-18B
B27 SupplementThermoFisher#17504044
Cardiotrophin 1 Peprotech#250-25
ThrombinDiagontech AG, Switzerland#100-125
Hanks' Balanced Salt Solution (HBSS) CaCl2(-), MgCl2(-)ThermoFisher#14170
0.05 % Trypsin-EDTAThermoFisher#25300
T75 FlaskSarstedt#83.3911
Endothelial differentiation  
Endothelial Cell Growth Medium (EGM)-2 BulletKitLonza#CC-3162
Ham's F-12K (Kaighn's) MediumThermoFisher#21127
Laminin Merck#L2020
Fibronectin Merck#F4759Dilute in ddH2O
6 Well PlateFalcon#353046
Formaldehyde SolutionMerck#F1635Diluite 1:10 in PBS (3.7% final concentration)
Triton X-100Merck#934200.1% in ddH2O
Normal Goat Serum (10%)ThermoFisher#50062Z
Anti-von Willebrand Factor antibodyAbcam#ab69941:100 in 10% goat serum
Goat anti-Rabbit IgG, Alexa Fluor 546ThermoFisher#A110101:500 in 10% goat serum
4',6-diamidino-2-phenylindole, dihydrochloride (DAPI)ThermoFisher#622471:500 in ddH2O 
SlowFade Antifade KitThermoFisher#S2828
BX63 widefield microscopeOlympus
Tube formation
96 Well PlateFalcon#353072
5 ml Round Bottom Tube with Strainer CapFalcon#352235
Matrigel Growth Factor ReducedCorning#354230
IX50 widefield microscopeOlympus
Sca-1+/CD31- cardiac side population isolation34 
Reagents
Pentobarbital Natrium 50 mg/ml ad usum vet.in house hospital pharmacy#9077862Working solution: 200 mg/kg
Phosphate Buffered Saline (PBS) CaCl2(-), MgCl2(-)ThermoFisher#20012
Hanks' Balanced Salt Solution (HBSS) CaCl2(-), MgCl2(-), phenol red (-)ThermoFisher#14175Prepare HBSS 500 mL + 2% FBS  for quenching Collagenase B activity 
Dulbecco's Modified Eagle's Medium (DMEM) 1g/L of D-Glucose, L-Glutamine, PyruvateThermoFisher#331885Prepare DMEM + 10% FBS  + 25 mM HEPES+ P/S for Hoechst stanining
Penicillin-Streptomycin  (P/S)ThermoFisher#15140122
HEPES 1 MThermoFisher#15630080Final concentration 25 mM
Fetal Bovine Serum (FBS)Hyclone#SH30071
RBC LysisBuffer (10X)BioLegend#420301/100mLDilute to 1X in ddH2O and filter through a 0.2 µm filter
Collagenase BMerck#11088807001Final concentration 1 mg/mL in HBSS, filtered through a 0.2 µm filter
bisBenzimide H33342 Trihydrochloride (Hoechst)Merck#B2261Prepare 1 mg/mL in ddH2O
Verapamil-hydrochloride Merck#V4629Final concentration 83.3 µM 
APC Rat Anti-Mouse CD31BD Biosciences#5512620.25 µg/107cells
FITC Rat Anti-Mouse Ly-6A/E (Sca-1)BD Biosciences#5574050.6 µg/107cells
7-Aminoactinomycin D (7-ADD)ThermoFisher#A13100.15 µg/106cells
APC rat IgG2a k Isotype ControlBD Biosciences#5539320.25 µg/107cells
FITC Rat IgG2a k Isotype ControlBD Biosciences#5546880.6 µg/107cells
Material
Needles 27GTerumo#NN-2719R
Needles 18GTerumo#NN-1838S
Single Use Syringes 1 mL sterileCODAN#62.1640
Transferpipette 3.5 mLSarstedt#86.1171.001
Cell Strainer 40 µm blueBD Biosciences #352340
Cell Strainer 100 µm yellowBD Biosciences#352360
Lumox Dish 50Sarstedt#94.6077.305
Culture Dishes P100Corning#353003
Culture Dishes P60Corning#353004
Mouse
LineAgeBreeding
C57BL/6NRj / male12 weeksin house
Product NameCompanyCatalogue No.
Reagents
Iscove's Modified Dulbecco's Medium (IMDM)ThermoFisher#12440
Dulbecco's Modified Eagle's Medium (DMEM)/Nutrient Mixture F12 HamMerck#D8437
Penicillin-Streptomycin  (P/S)ThermoFisher#15140122
Fetal Bovine Serum (FBS)Hyclone#SH30071
L-Glutamine ThermoFisher#25030
GlutathioneMerck#G6013
B27 SupplementThermoFisher#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
ThrombinDiagontech AG, Switzerland #100-125
Endothelial Cell Growth Medium (EGM)-2 BulletKitLonza#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 NameMedium 18Medium 2Medium 3
ReagentsCultureLineage inductionEndothelial diff.
Iscove's Modified Dulbecco's Medium (IMDM)35%35%
Dulbecco's Modified Eagle's Medium (DMEM)/Nutrient Mixture F12 Ham65%65%
Penicillin-Streptomycin  (P/S)1%1%
Fetal Bovine Serum (FBS)3.5%≤0.1%
L-Glutamine 2 mM2 mM
Glutathione0.2 nM0.2 nM
B27 Supplement1.3%
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
Thrombin0.0005 U/mLAll necessary components included in the kit
Endothelial Cell Growth Medium (EGM)-2 BulletKitAll necessary components included in the kit

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