**Marcus-André Deutsch 1, Stefan Brunner 2, Ulrich Grabmaier 2, Robert David 3, Ilka Ott <sup>4</sup> and Bruno C. Huber 2,\***


Received: 14 January 2020; Accepted: 27 February 2020; Published: 2 March 2020

**Abstract:** Objective: The potential therapeutic role of endothelial progenitor cells (EPCs) in ischemic heart disease for myocardial repair and regeneration is subject to intense investigation. The aim of the study was to investigate the proregenerative potential of human endothelial colony-forming cells (huECFCs), a very homogenous and highly proliferative endothelial progenitor cell subpopulation, in a myocardial infarction (MI) model of severe combined immunodeficiency (SCID) mice. Methods: CD34<sup>+</sup> peripheral blood mononuclear cells were isolated from patient blood samples using immunomagnetic beads. For generating ECFCs, CD34<sup>+</sup> cells were plated on fibronectin-coated dishes and were expanded by culture in endothelial-specific cell medium. Either huECFCs (5 <sup>×</sup> 105) or control medium were injected into the peri-infarct region after surgical MI induction in SCID/beige mice. Hemodynamic function was assessed invasively by conductance micromanometry 30 days post-MI. Hearts of sacrificed animals were analyzed by immunohistochemistry to assess cell fate, infarct size, and neovascularization (huECFCs *n* = 15 vs. control *n* = 10). Flow-cytometric analysis of enzymatically digested whole heart tissue was used to analyze different subsets of migrated CD34+/CD45<sup>+</sup> peripheral mononuclear cells as well as CD34−/CD45− cardiac-resident stem cells two days post-MI (huECFCs *n* = 10 vs. control *n* = 6). Results: Transplantation of human ECFCs after MI improved left ventricular (LV) function at day 30 post-MI (LVEF: 30.43 ± 1.20% vs. 22.61 ± 1.73%, *p* < 0.001; ΔP/ΔTmax 5202.28 ± 316.68 mmHg/s vs. 3896.24 ± 534.95 mmHg/s, *p* < 0.05) when compared to controls. In addition, a significantly reduced infarct size (50.3 ± 4.5% vs. 66.1 ± 4.3%, *p* < 0.05) was seen in huECFC treated animals compared to controls. Immunohistochemistry failed to show integration and survival of transplanted cells. However, anti-CD31 immunohistochemistry demonstrated an increased vascular density within the infarct border zone (8.6 <sup>±</sup> 0.4 CD31<sup>+</sup> capillaries per HPF vs. 6.2 <sup>±</sup> 0.5 CD31<sup>+</sup> capillaries per HPF, *<sup>p</sup>* <sup>&</sup>lt; 0.001). Flow cytometry at day two post-MI showed a trend towards increased myocardial homing of CD45+/CD34<sup>+</sup> mononuclear cells (1.1 <sup>±</sup> 0.3% vs. 0.7 <sup>±</sup> 0.1%, *p* = 0.2). Interestingly, we detected a significant increase in the population of CD34−/CD45−/Sca1<sup>+</sup> cardiac resident stem cells (11.7 ± 1.7% vs. 4.7 ± 1.7%, *p* < 0.01). In a subgroup analysis no significant differences were seen in the cardioprotective effects of huECFCs derived from diabetic or nondiabetic patients. Conclusions: In a murine model of myocardial infarction in SCID mice, transplantation of huECFCs ameliorated myocardial function by attenuation of adverse post-MI remodeling, presumably through paracrine effects. Cardiac repair is enhanced by increasing myocardial neovascularization

and the pool of Sca1<sup>+</sup> cardiac resident stem cells. The use of huECFCs for treating ischemic heart disease warrants further investigation.

**Keywords:** cardiovascular diseases; adult stem cells; cardiac regeneration; myocardial infraction; angiogenesis

#### **1. Background**

Ischemic heart disease following acute myocardial infarction (AMI) is the leading cause of morbidity and mortality in the Western world [1]. Most of the clinically approved therapeutics focus on modulating hemodynamics to reduce early mortality but do not facilitate cardiac repair, which would be needed to reduce the incidence of heart failure [2]. Therefore, the concept of cell-based therapies may have the potential to transform the treatment and prognosis of heart failure through regeneration or repair of injured cardiac tissue [3,4]. Most clinical trials have used bone marrow derived mononuclear cells (BMCd), which have demonstrated inconsistent and, overall, modest efficacy [5].

In recent years, there has been an intense investigative effort to uncover the mechanism by which transplanted stem cells preserve the function of infarcted hearts. Based on these findings, the attenuation of ischemic cardiomyopathy after cell transplantation is not attributable to cardiomyocyte repopulation or transdifferentiation. Rather, functional benefits after stem cell transplantation might be attributable to an augmentation of the natural process of myocardial healing by paracrine signaling and promoting neovascularization [6].

Endothelial progenitor cells (EPCs) are a minor population of mononuclear cells migrating from the bone marrow into the bloodstream, which are able to home in on sites of injury and promote neovascularization, which finally is associated with increased blood flow and tissue repair [7]. EPCs can be isolated from peripheral or umbilical cord blood and culturing in vitro can produce two different EPC types. These two distinct EPC subtypes have been named as early EPCs (eEPCs) and late outgrowth EPCs (LOEPCs) or endothelial colony-forming cells (ECFCs). ECFCs comply with the definition of bona fide EPCs and represent a well-characterized and homogeneous cell population of endothelial origin with high proliferative capacity and inherent vasculogenic activity [8–10]. In vivo, their potential has been investigated in different mouse models where ECFCs increased neovascularization and tissue regeneration [11]. In a rat stroke model, ECFCs treatment was associated with reduced glial scarring and increased functional recovery, which could be explained by stimulation of angiogenesis and a marked reduction in apoptosis [12]. However, there are only a few studies investigating the potential of human ECFCs to regenerate ischemic myocardium. To create a clinically relevant scenario, which aims to address the effect of cell therapy after acute MI, we transplanted ECFCs from patients with coronary artery disease (CAD) into immunodeficient SCID mice and employed functional studies, immunohistology, as well as flow cytometry to assess their potential in facilitating cardiac regeneration.

#### **2. Methods**

#### *2.1. Isolation and Culture of ECFCs*

Human adult ECFCs were collected by leukapheresis after G-CSF-induced mobilization of CD34<sup>+</sup> cells from diabetic (*n* = 9) and nondiabetic (*n* = 8) patients with coronary artery disease. The diagnosis of diabetes was made in accordance with current guidelines (mean HbA1c 7.5% ± 0.3%).

For ECFC collection, mononuclear cells from leukapheresis were isolated by density gradient centrifugation for 20 min at 1000<sup>×</sup> *g* (Ficoll-Hypaque, Seromed, Berlin, Germany). CD34<sup>+</sup> cells were isolated using immunomagnetic beads (Miltenyi Biotec, Bergisch Gladbach, Germany) [13]. The purity of the isolated CD34<sup>+</sup> cells ranged between 86% and 99% as assessed by flow cytometry (EPICS XL, Couter, Hialeah, FL, USA). This study was approved by the Medical Ethics Committee of the Technical University of Munich.

CD34<sup>+</sup> cord blood (CB) and peripheral blood (PB) cells were cultured using a modified protocol as described in [14]. Briefly, CD34<sup>+</sup> cells from mobilized PB was cultured on 1% gelatin (Sigma, Hamburg, Germany) or fibronectin (10 μg/cm2, Cellsystems, St. Katharinen, Germany) in Iscove's Modified Dulbecco's Medium (IMDM, Gibco, Paisley, UK), with 10% horse serum and 10% fetal calf serum (PAN-Biotech, Aidenbach, Germany) supplemented with penicillin/streptomycin (Gibco), 50 ng/mL recombinant human stem cell factor (SCF, R&D Systems, Abingdon, UK), 50 ng/mL vascular endothelial growth factor (VEGF, R&D Systems), 20 ng/mL basic fibroblast growth factor (FGF-2, R&D Systems), and 20 ng/mL stem cell growth factor (SCGFβ, Peprotech, London, UK). This medium (ECM) was replaced 3 times a week. After 3 weeks, cells were adapted from ECM to the low-serum EGM-2 medium (Cellsystems). To analyze EC colony-forming units (CFU-EC), CD34<sup>+</sup> cells were plated in a limiting dilution series of cell concentrations in 24-well plates and treated as above. These multiwell tissue culture plates were scored for the presence ("positive") or absence ("negative") of EC colonies between 21 and 35 days. Adherent cells were cultured to confluence in 1% gelatin-coated chamber slides (Nalge Nunc, Naperville, IL, USA). Cells were washed twice in phosphate-buffered saline (PBS), fixed, and permeabilized using Fix and Perm (Dianova, Hamburg, Germany). Samples were then incubated for 2 h with primary antibodies: antihuman specific CD31 (Sertotec, Raleigh, NC, USA), anti-CD105, anti-CD144 (VE-cadherin, Coulter-Immunotech, Krefeld, Germany), anti-CD45 and anti-vWF (Dako, Hamburg, Germany), anti VEGF-R2 (KDR-1 and KDR-2, Sigma), anti-Flt1, anti-Flt4, anti-Tie-2 (Santa Cruz Biotechnologies Inc., Heidelberg, Germany), and CD146 (Chemicon, Limburg, Germany). To visualize antibody binding (mouse and rabbit antibodies), the peroxidase-labeled avidin-biotin method (Universal Dako LSAB®-Kit, Dako, Santa Clara, CA, USA) was used according to the manufacturer's recommendations. For goat primary antibodies, donkey antigoat antibodies directly conjugated to peroxidase were used (Jackson Laboratories, Dianova, Hamburg, Germany). Isotype-matched control antibodies (Coulter-Immunotech, Jackson Laboratories, Brea, CA, USA) served as negative controls. In selected experiments, nuclear staining was performed with hematoxylin staining solution (Merck, Darmstadt, Germany).
