Elsevier

International Journal of Cardiology

Volume 241, 15 August 2017, Pages 330-343
International Journal of Cardiology

Human cardiac progenitor cells with regenerative potential can be isolated and characterized from 3D-electro-anatomic guided endomyocardial biopsies

https://doi.org/10.1016/j.ijcard.2017.02.106Get rights and content

Abstract

Aims

In the present study, we aimed to develop a percutaneous approach and a reproducible methodology for the isolation and expansion of Cardiac Progenitor Cells (CPCs) from EndoMyocardial Biopsies (EMB) in vivo. Moreover, in an animal model of non-ischemic heart failure (HF), we would like to test whether CPCs obtained by this methodology may engraft the myocardium and differentiate.

Methods and results

EMB were obtained using a preformed sheath and a disposable bioptome, advanced via right femoral vein in 12 healthy mini pigs, to the right ventricle. EMB were enzymatically dissociated, cells were expanded and sorted for c-kit. We used 3D-Electro-Anatomic Mapping (3D-EAM) to obtain CPCs from 32 patients affected by non-ischemic cardiomyopathy. The in vivo regenerative potential of CPCs was tested in a rodent model of drug-induced non-ischemic cardiomyopathy. c-kit positive CPCs replicative capacity was assessed in 30 patients. Telomere length averaged 7.4 ± 0.4 kbp and telomerase activity was present in all preparations (1.7 × 105 copies). The in situ hybridization experiments showed that injected human CPCs may acquire a neonatal myocyte phenotype given the expression of the alpha-sarcomeric actin together with the presence of the Alu probe, suggesting a beneficial impact on LV performance.

Conclusions

The success in obtaining CPCs characterized by high regenerative potential, in vitro and in vivo, from EMB indicates that harvesting without thoracotomy in patients affected by either ischemic or non-ischemic cardiomyopathy is feasible. These initial results may potentially expand the future application of CPCs to all patients affected by HF not undergoing surgical procedures.

Introduction

Cardiovascular diseases are global health problems: despite advances over the past 30 years, the prognosis for patients who are admitted to hospital with advanced heart failure (HF) remains extremely poor. Moreover, as the population continues to age, the demand for costly, age-associated health care will increase rapidly, since elderly individuals are at highest risk for HF. The development of novel therapies that lead to a survival benefit and lower rate of re-hospitalization remains an unmet need in patients with HF [1]. Importantly, available therapeutic options do not address the fundamental problem of loss of cardiac tissue or of damaged myocardium replacement: ideally, cell therapy has the potential to rebuild the injured heart, by regenerating lost myocytes both morphologically and functionally and acquiring contractility and electrical coupling with the native myocardium.

The interim results of the SCIPIO and CADUCEUS [2], [3] trials are quite promising: in these phase I trials, no adverse effects attributable to the Cardiac Progenitor Cell (CPC) treatment have been noted, and CPC-treated patients showed an improvement in ejection fraction (EF) at 1 year (+ 13.7 absolute units versus baseline), followed by a 30.2% reduction in infarct size and significant improvement in the New York Heart Association (NYHA) functional class and in the quality of life, as measured by the Minnesota Living with Heart failure Questionnaire [2]. However, these are early results, conducted in a single center with a small number of patients, restricted only to individuals affected by subacute or chronic ischemic cardiomyopathy. Moreover, a very limited number of studies explored so far the possible efficacy of stem cell therapy in the setting of non-ischemic cardiomyopathy. Of note, a subpopulation of human CPCs characterized by the expression of insulin-like growth factor receptor (IGF1R) has been demonstrated to represent a pool of younger CPCs with enhanced growth reserve in vitro and in vivo, when compared with unselected c-kit positive human CPCs [4].

In the present study, we sought to determine whether functionally competent CPCs can be collected in vivo from EndoMyocardial Biopsies (EMB), utilizing a minimally-invasive, safe and reproducible technique. In order to test this hypothesis, we performed a preclinical study in a swine model and, after the assessment of the results, we applied the same approach in patients with non-ischemic cardiomyopathy, undergoing EMB for diagnostic purposes. Finally, we tested the regenerative potential of the CPCs obtained from EMB in vivo, using a rodent animal model of non-ischemic cardiomyopathy. The evaluation of the feasibility and safety of the present technique may support the possibility that EMB biopsies could be used as a source of highly proliferative autologous CPCs for the potential treatment of patients with ischemic or non-ischemic cardiomyopathy.

Section snippets

3D-electro-anatomical mapping

In the preclinical study, we performed 3D-electro-anatomical mapping (3D EAM) using the EnSite NavX System (St. Jude Medical, 123 St. Paul, MN, USA). We used the EnSite System to create a 3-D model of the right or left or both ventricles. An electrical signal is transmitted between the Ensite Navx surface electrode patches, applied on the body, and EP catheters within the ventricle sense the signal. The physician then sweeps the catheter across the heart chamber to outline the structures and

Extraction of c-kit positive CPCs from EMB

EMB were obtained from the septum or from the outflow tract of the right ventricle (Fig. 1A–B) in all minipigs. A total of 1–4 EMB per animal, yielding a total sample weight of 3.0 ± 1.7 mg were used for the isolation of CPCs (Fig. 1C–D). The un-fractionated cardiac cell population was expanded for 26 ± 2 days. c-kit positive CPCs were obtained from all animals. In all animals, a fraction varying from 20 to 38% of c-kit positive cells were positive for markers of mesenchymal/stromal cells, such as

Discussion

The current study was designed to develop a safe and reproducible protocol for the isolation of functionally competent CPCs by 3D-EAM guided EMB. In order to test this hypothesis, we conceived a two-phase study: a first phase to be performed in a preclinical animal model and, after result assessment, a second phase to be performed in patients with non-ischemic cardiomyopathy undergoing EMB for diagnostic purpose using the same approach. Importantly, the methodology used in the present study,

Conflict of interest

None declared.

Funding

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

Acknowledgements

The authors wish to thank Dr. Maria Emiliana Caristo for the pre-, peri- and post-operative management of the minipigs and St Jude Medical for the expert technical support.

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