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

Abstract

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.

Supporting data on in vitro cardioprotective and proliferative paracrine effects by the human amniotic fluid stem cell secretome

“…Cells migrating from explants were collected after 2–3 weeks. Human fetal Sca-1+ CPC and human adult epicardium derived progenitor cells (hEPDC) were obtained as previously reported [3], [4], [5] from human heart tissue, following written informed consent and according to local Medical Ethics Committee at Leiden University Medical Center (P08.087). hCPC were cultured in Iscove Modified Dulbecco's Medium (EuroClone, Milano, Italy) with 20% FBS, 1% L-glutamine, 1% penicillin/streptomycin, (all Thermo Fisher Scientific, Waltham, Massachusetts).…”

“…Human fetal Sca-1+ CPC (fSca-1+ hCPC) were obtained from human foetal heart tissue, after elective abortion without medical indication from 10 to 22 weeks of gestation and sorted for Sca-1 cross-reactivity (MACS MicroBead Kit, Miltenyi Biotechnology, Bergisch Gladbach, Germany) as previously described [3], [4] and cultured on 0.1% gelatin-coated dishes in M199 (Gibco-Thermo Fisher Scientific, Waltham, Massachusetts)/EGM (3:1) supplemented with 10% FBS (Gibco-Thermo Fisher Scientific, Waltham, Massachusetts), 10 ng/ml basic fibroblast growth factor (bFGF), 5 ng/ml epithelial growth factor (EGF), 5 ng/ml insulin-like growth factor (IGF-1) and 5 ng/ml hepatocyte growth factor (HGF). hEPDC were obtained from human atrial samples obtained during cardiac surgery, and isolated by separating the epicardium from the underlying myocardium [5]. Briefly, the tissue was processed into small pieces and digested in a 0.25% Trypsin/EDTA solution (Serva, Heidelberg, Germany).…”

Epicardial TGFβ and BMP Signaling in Cardiac Regeneration: What Lesson Can We Learn from the Developing Heart?

“…The same approach was used to show that TGFβ induces EMT in mouse epicardial cells derived from E12.5 embryonic hearts [79]. Epicardial cells were also derived from human fetal hearts [80]. Instead of epicardial outgrowth of cardiac tissue specimens, these human fetal cells were isolated by digestion of the isolated epicardial layer, followed by culture of the resulting single cell suspension in an epicardium specific culture medium.…”

“…In this case, the deviant observations could be due to the absence of fetal bovine serum (FBS) and the relatively high concentration of TGFβ (10 ng/mL), which is different from other studies about epicardial EMT. Furthermore, culture conditions such as cell layer confluency can influence the EMT response [80]. Interesting to note is that in contrast to in vivo studies, where different TGFβ ligands have distinct and uninterchangeable functions, in vitro there does not seem to be a difference between the types of TGFβ; all three TGFβ ligands elicited a similar effect on EMT and invasion in the majority of the studies [60,79,80].…”

Generation, Characterization, and Application of Inducible Proliferative Adult Human Epicardium-Derived Cells

“…First, the relative scarcity of human epicardium and EPDC sources hampers the deeper investigation of EPDC function and their potential for cardiac repair. Furthermore, although we developed a reliable method for the isolation and expansion of EPDCs from human specimens [17], EPDCs possess limited proliferation capacity and tend to lose their epithelial phenotype within a few passages. In addition, the application of EPDCs derived from different patients and with different passage numbers, has likely contributed to the variable results obtained in in vitro experiments.…”