The Isolation and Culture of Primary Epicardial Cells Derived from Human Adult and Fetal Heart Specimens

Podsumowanie

The epicardium plays a crucial role in the development and repair of the heart by providing cells and growth factors to the myocardial wall. Here, we describe a method to culture human primary epicardial cells that enables the study and comparison of their developmental and adult characteristics.

Streszczenie

The epicardium, an epithelial cell layer covering the myocardium, has an essential role during cardiac development, as well as in the repair response of the heart after ischemic injury. When activated, epicardial cells undergo a process known as epithelial to mesenchymal transition (EMT) to provide cells to the regenerating myocardium. Furthermore, the epicardium contributes via secretion of essential paracrine factors. To fully appreciate the regenerative potential of the epicardium, a human cell model is required. Here we outline a novel cell culture model to derive primary epicardial derived cells (EPDCs) from human adult and fetal cardiac tissue. To isolate EPDCs, the epicardium is dissected from the outside of the heart specimen and processed into a single cell suspension. Next, EPDCs are plated and cultured in EPDC medium containing the ALK 5-kinase inhibitor SB431542 to maintain their epithelial phenotype. EMT is induced by stimulation with TGFβ. This method enables, for the first time, the study of the process of human epicardial EMT in a controlled setting, and facilitates gaining more insight in the secretome of EPDCs that may aid heart regeneration. Furthermore, this uniform approach allows for direct comparison of human adult and fetal epicardial behavior.

Wprowadzenie

The epicardium, a single-cell epithelial layer that envelopes the heart, is of vital importance for cardiac development and repair (reviewed in Smits et al.¹). Developmentally, the epicardium arises from the proepicardial organ, a small structure located at the base of the developing heart. Around developmental day E9.5 in mouse, and 4 weeks post-conception in human, cells start to migrate from this cauliflower structure and cover the developing myocardium². Once a single epithelial cell layer is formed, a portion of the epicardial cells undergoes epithelial to mesenchymal transition (EMT). During EMT, cells lose their epithelial characteristics, such as cell-cell adhesions, and obtain a mesenchymal phenotype which gives them the capacity to migrate into the developing myocardium. The formed epicardial derived cells (EPDCs) can differentiate into several cardiac cell types including fibroblasts, smooth muscle cells, and potentially cardiomyocytes and endothelial cells³, although differentiation of the latter two cell populations remains subject to debate (reviewed in Smits et al.⁴). Furthermore, the epicardium provides instructive paracrine signals to the myocardium to regulate its growth and vascularization^5,6,7,8. Multiple studies have demonstrated that impaired epicardial formation leads to developmental defects in cardiac muscle^9,10, vasculature¹¹, and conduction system¹², emphasizing the essential contribution of the epicardium to the formation of the heart.

Although in the adult heart the epicardium is present as a dormant layer, it becomes reactivated upon ischemia¹³. Epicardial reactivation post-injury recapitulates several of the processes described for cardiac development, including proliferation and EMT¹⁴, albeit less efficiently. Interestingly, although the exact mechanism is not fully understood, the epicardial contribution to repair can be improved by treatment with, e.g., Thymosin β4¹⁵ or modified VEGF-A mRNA¹⁶, resulting in ameliorated cardiac function after myocardial infarction. The epicardium is therefore considered an interesting cell source to enhance endogenous repair of the injured heart.

Mechanisms of cardiac development are often recapitulated during injury, although in a less efficient manner. In search of epicardial activators, it is paramount that we can determine and compare the full capacity of the fetal and adult epicardium. Moreover, from a therapeutic point of view, it is important that, in addition to animal experiments, we extend knowledge regarding the response of the human epicardium. Here, we describe a method to isolate and culture human adult and fetal epicardial derived cells (EPDCs) in an epithelial-cell-like morphology and to induce EMT. With this model, we aim to explore and compare adult and fetal epicardial cell behavior.

The main advantage of this protocol is the use of human epicardial material, which has not been thoroughly studied. Importantly, the described isolation and cell culture protocol provides a single uniform method to derive both fetal and adult cobble EPDCs, enabling a direct comparison between these two cell sources. Additionally, since the epicardium is isolated based on its location, it is ensured that the cells are actually epicardially derived¹⁷.

While human EPDC isolation methods have been established previously, these mostly rely on outgrowth protocols where pieces of cardiac or epicardial tissue are plated onto a cell culture dish^18,19. This approach thereby selects specifically for cells that partially lose their epithelial phenotype in order to migrate, and that are more prone to undergo spontaneous EMT. In the current protocol, the epicardium is first processed into a single cell solution which allows the isolated EPDCs to maintain their epithelial state. This method therefore provides a solid in vitro model to study epicardial EMT.

Protokół

All experiments with human tissue specimens were approved by the ethics committee of the Leiden University Medical Center and conforms to the Declaration of Helsinki. All steps are performed with sterile equipment in a cell culture flow cabinet.

1. Preparations

1. Prepare EPDC medium by mixing Dulbecco's modified Eagle's medium (DMEM low- glucose) and Medium 199 (M199) in a 1:1 ratio. Add 10% heat inactivated fetal bovine serum (FBS, heat inactivated for 25 min at 56 °C) and supplement with 100 U/mL penicillin and 100 mg/mL streptomycin. Pre-warm the EPDC medium in a 37 °C water bath.
2. Pre-warm Trypsin 0.25%/EDTA (1:1) in a 37 °C water bath.
3. Coat wells with gelatin. Add 0.1% gelatin/PBS to each well and incubate the plates for at least 15 min at 37 °C. Guidelines for the required cell culture plate are summarized in Table 1. Carefully remove all fluid before plating cells.
4. Prepare a stock solution of 10 mM SB431542 (SB), diluted in DMSO (CAUTION). Make 50 µL aliquots in conical bottom polypropylene centrifuge tubes, like Eppendorf tubes, and store them at -20 °C. Note that SB aliquots can be thawed only once.

2. Retrieval and Storage of Adult and Fetal Heart Specimens

1. Store human adult auricles directly upon removal during surgery in a 50-mL tube with 15 mL high-glucose DMEM containing 10% FBS, 100 U/mL penicillin, and 100 mg/mL streptomycin at 4 °C. Samples are generally 3 - 5 cm in size and can be stored up to 48 h after dissection.
2. Store fetal hearts obtained from elective abortion material in EPDC medium at 4 °C up to 24 h after isolation. For this protocol, use samples with a gestational age between 12 - 22 weeks. Note that the whole heart can be used for isolation of the epicardium.

3. Isolation of the Epicardial Layer

1. In the laminar flow cabinet, prepare a 100-mm cell-culture dish with PBS and place a separate droplet (~200 µL) of PBS in the lid. Fill a 15-mL tube with 5 mL pre-warmed EPDC medium.
2. Place the tissue in the cell-culture dish filled with PBS. Make sure that the tissue is moistened frequently during the procedure.
3. Using a stereomicroscope, remove as much of the epicardial layer from the tissue sample as possible by peeling it off using forceps.
   Note: The epicardium can be recognized as a very thin, transparent layer tightly adhered to the outside of the heart (Figure 1A). Try to avoid contamination with epicardial adipose tissue and blood vessels since this will hamper the isolation.
4. Collect pieces of epicardial tissue in the droplet of PBS on the lid.
5. Cut the epicardial tissue into small pieces (0.5 mm³) using a scalpel (Figure 1B), or using the sharp tips of small forceps.Note that the pieces should be able to pass a P1,000 pipet tip (step 3.6).
6. Add 1 mL of trypsin to the epicardial tissue and collect the pieces and trypsin with a P1,000 pipet tip. Transfer the tissue into a 1.5-mL conical centrifuge tube (Figure 1C).
7. Incubate the tube in a 37 °C water bath for 10 min, while shaking at ~60 rpm.
8. Remove the tube from the water bath, and clean the outside of the tube with 70% ethanol.
9. Allow the tissue to sink to the bottom of the tube (Figure 1D) and carefully transfer the supernatant containing epicardial cells to the 15-mL tube containing 5 mL EPDC medium to inactivate the trypsin.
10. Replenish the remaining tissue in the conical centrifuge tube with 1 mL trypsin, and mix by gently pipetting up and down.
11. Repeat step 3.7 to 3.10 two times. At the final step, after a total of 30 min of trypsin incubation, transfer both the supernatant and the remaining epicardial tissue into the tube containing EPDC medium.
12. When all cells are collected in EPDC medium, gently pass the suspension through a 10-mL syringe with a 19-gauge needle into a new 15-mL tube to mechanically dissociate the cells (Figure 1E).
    Note: Make sure to homogenize the suspension by pipetting up and down before passing the solution through the needle to prevent the needle getting obstructed.
13. To further dissociate the cells, repeat step 3.12 with a 21-gauge needle.
14. Place a 100-µm cell strainer on top of a 50-mL tube and transfer the medium containing the epicardial cells onto the strainer using a 10-mL pipette to remove all remaining clumps (Figure 1F).
15. Wash the strainer to collect residual cells by pipetting 5 mL EPDC medium onto the strainer.
16. Pipet the cell suspension from the 50-mL tube into a 15-mL tube. Since cells sink to the bottom of the tube, shake the solution gently before pipetting.
    Note: While this step is not necessary, a smaller tube aids visualization of the pellet after centrifugation.
17. Centrifuge at 200 x g for 5 min at room temperature (Figure 1G).
18. Remove the supernatant and resuspend the cell pellet (Figure 1H) in the required volume of EPDC medium (Table 1).
    Note: For fetal EPDCs, directly use EPDC medium containing 10 µM of the ALK5 kinase inhibitor (EPDC+SB). Adult cells can be plated without SB during the first passage.
19. Plate the cell suspension on the gelatin coated culture plates (Figure 1I). The size of the well depends on the size of the epicardial tissue sample (Table 1). Note that low confluency will induce the occurrence of EMT.
20. Place the cells in the incubator for at least 48 h at 37 °C, 5% CO₂ to allow the cells to attach to the culture plate.

4. Culture of EPDCs

1. Replenish the EPDC+SB medium at least every 3 days.
2. Inspect the cells at least every 3 days using the microscope. When the cells reach confluency, i.e., when the culture plate is fully covered with cells, passage the cells in a 1:2 surface ratio. Note that reaching confluency can take ~5 - 10 days.
   1. Aspirate the medium using a pipet or an aspiration system with, e.g., a glass pipet.
   2. Wash the cells carefully by adding PBS to the cells. Gently swirl the plate and aspirate the PBS.
   3. Add trypsin to the plate. Gently rotate the plate to cover all cells with trypsin and incubate the plate for 1 min at 37 °C.
      NOTE: Use as little trypsin as possible (indication: 200 µL per well of a 6 well plate)
   4. Tap the plate to mechanically detach the cells from the bottom of the plate. Use the microscope to visually check if cells have detached. If not, incubate the cell culture plate for an extra minute.
   5. Add the required volume of EPDC+SB medium to the cells, resuspend by pipetting up and down, and transfer the cell suspension to a new gelatin coated culture plate.
      NOTE: In general, cells can be kept in a cobblestone morphology up to passage 8.

5. Induction of EMT in EPDCs

1. To induce EMT, dissociate the cells with trypsin and transfer the cells to new gelatin coated wells in a 1:2 surface ratio in EPDC+SB medium, as described in 4, and incubate for at least 24 h at 37 °C, 5% CO₂.
2. Check if confluency is 50 - 70% and if EPDCs have a cobblestone morphology.
   NOTE: Confluency affects the ability of EPDCs to undergo EMT.
3. Aspirate the medium from the cells and wash the cells carefully with PBS (see step 4.2.1 - 4.2.2).
4. Stimulate cells with 1 ng/mL TGFβ3 in EPDC medium and place them in an incubator at 37 °C, 5% CO₂ for 5 days. Note that during stimulation, the EPDC medium containing TGFβ3 does not have to be replenished.
5. Monitor the cells daily. After 5 days, cells that underwent EMT are recognized by a spindle-shaped morphology (Figure 2A).
6. Culture spindle-shaped EPDCs according to the method described in 4 without addition of SB or TGFβ to the EPDC medium. Note that in general, spindle-shaped EPDCs can be cultured up to passage 20.
7. Validate the occurrence of EMT with immunofluorescent staining using antibodies against the mesenchymal markers αSMA or Vimentin or by phalloidin to detect the formation of F-actin stress fibers (Figure 2B), or using qRT-PCR for EMT-related genes (e.g., WT1, Periostin, Col1A1, αSMA, N-Cadherin, MMP3, Snail, Slug) (Figure 2C and Table 2).

Wyniki

Here, we outline a straightforward protocol to isolate EPDCs from human adult and fetal cardiac tissue (Figure 1). This protocol takes advantage of the easily accessible location of the epicardium on the outside of the heart (Figure 1A). Staining of the heart auricle after dissection demonstrates that the WT1+ epicardium is removed while the underlying subepicardial extracellular matrix and myocardial tissue remain intact (

Dyskusje

Here we describe a detailed protocol to isolate and culture primary epicardial cells derived from human adult and fetal hearts. Extensive characterization of these cells has been previously published¹⁷. We have shown that both cell types can be maintained as epithelial cobblestone-like cells when cultured with the ALK5 kinase inhibitor SB431542. EMT is an integral part of epicardial activation in vivo during both development and the post-injury response. EMT can be studied using this meth...

Materiały

Name	Company	Catalog Number	Comments
Dulbecco’s modified Eagle’s medium + GlutaMAX	Gibco	21885-025
Medium 199	Gibco	31150-022
Fetal Bovine Serum	Gibco	10270-106
Trypsin 0.25%	Invitrogen	25200-056
Penicillin G sodium salt	Roth	HP48
Streptomycin sulphate	Roth	HP66
Trypsin 1:250 from bovine pancreas	Serva	37289
EDTA	Sigma	E4884
Gelatin from porcine skin	Sigma-Aldrich	G1890
Culture plates 6 well	Greiner bio-one	657160
Culture plates 12 well	Corning	3512
Culture plates 24 well	Greiner bio-one	662160
SB 431542	Tocris	1614
Dimethyl Sulfoxide (DMSO)	Merck	102931
100-1000µL Filtered Pipet Tips	Corning	4809
10-ml pipet	Greiner bio-one	607180
5-ml pipet	Greiner bio-one	606180
Cell culture dish 100/20 mm	Greiner bio-one	664160
PBS	Gibco	10010056	Or home-made and sterilized
Eppendorf tubes 1.5 mL	Eppendorf	0030120086
15-ml centrifuge tubes	Greiner bio-one	188271
50-ml centrifuge tubes	Greiner bio-one	227261
10 mL Syringe	Becton Dickinson	305959
Needles 19 Gauge	Becton Dickinson	301700
Needles 21 Gauge	Becton Dickinson	304432
EASYstrainer Cell Sieves, 100 µm	Greiner bio-one	542000
TGFβ3	R&D systems	243-B3
Monoclonal Anti-Actin, α-Smooth Muscle	Sigma	A2547
Anti-Mouse Alexa Fluor 555	Invitrogen	A31570
Alexa Fluor 488 Phalloidin	Invitrogen	A12379
Equipment
Name	Company	Catalog Number	Comments
Pipet P1,000	Gilson	F123602
Pipet controller	Integra	155 015
Stereomicroscope	Leica	M80
Inverted Light Microscope	Olympus	CK2
Centrifuge	Eppendorf	5702
Waterbath	GFL	1083