Cardiosphere-Derived Cells

The formation of cardiospheres from human and murine heart tissue was first described in 2004 by Messina and coworkers [16]. It was demonstrated that, when placed in adherent plates, heart explants generated a layer of fibroblast-like cells over which small, phase-bright cells migrated. These phase-bright cells were collected and transferred to nonadherent plates where they originated three-dimensional structures named cardiospheres. Cardiospheres were clonogenic and when co-cultured with rat neonatal cardiomyocytes expressed troponin I and connexin 43. Additionally, there was visual evidence that cardiospheres showed synchronous contractions with cardiomyocytes. When transplanted into infarcted hearts, these cells started to express myosin heavy chain as well as -smooth muscle actin and platelet endothelial cell adhesion molecule, which resulted in functional improvement. Curiously, when the expression of surface molecules was analyzed by flow cytometry, cardiospheres showed a 25% expression of c-kit. Thus, it is possible that c-kit+ cells contribute to the characteristics observed in cardiospheres, explaining the similar findings between these two cardiac progenitor/stem cell types.

However, it was only in 2007 that Marbns group described cardiosphere-derived cells (CDCs) [19]. They slightly changed Messinas protocol by placing cardiospheres in adherent plates where cells were grown in monolayers instead of three-dimensional structures. The advantage of this step was that cell expansion in monolayers was easier and faster, which would facilitate future clinical use. Flow cytometry showed that c-kit expression was still present in similar levels to those described by Messina and coworkers. Additionally, high expression levels of CD105 and CD90 were found, indicating a mesenchymal phenotype. When co-cultured with rat neonatal cardiomyocytes, CDCs presented spontaneous intracellular calcium transients and action potentials, as well as INa, IK1 and ICa,L currents. In vivo, injection of CDCs in acute myocardial infarctions (MI) prevented further ejection fraction deterioration 3 weeks after MI when compared to placebo and fibroblast injected mice. In 2009, Marbns group also reported functional benefit and reduction of infarct size in a porcine animal model after CDC injection [37], a preclinical model that prompted a phase I clinical trial (CADUCEUS, ClinicalTrials.gov, Identifier NCT00893360).

Nonetheless, the usage of cardiospheres as a source of cardiac stem cells has been refuted. Andersen and coworkers showed that even though cardiospheres can be produced from heart specimens, they do not hold cardiomyogenic potential and simply represent aggregated fibroblasts [38]. This group also found cells that expressed cardiac contractile proteins, such as myosin heavy chain and troponin T, in cardiospheres. However, the findings were attributed to the presence of contaminating heart tissue fragments in the explant-derived cell suspension. By adding a filtration step in which explant-derived cells were passed through cell strainers prior to cardiosphere formation, the presence of cells expressing cardiac contractile proteins was eliminated. In addition, this group showed that phase-bright cells were of hematopoietic origin and did not organize into spheric structures, a characteristic attributed to the fibroblast-like cells.

In response to Andersens findings, Marbns group published a revalidation of the CDC isolation method [39]. Using a strategy identical to the one described by Hsieh and colleagues [8], cardiomyocytes were irreversibly labeled with GFP after a tamoxifen pulse (see Fig. 8.1). Isolation of CDCs from these transgenic mouse hearts did not reveal the presence of GFP+ cells, refuting the possibility that cardiac differentiation of CDCs was due to the presence of contaminating myocardial tissue fragments. Additionally, they reported that cardiospheres were consistently negative for CD45, indicating that CDCs do not contain cells of hematopoietic origin. The authors also emphasized that Andersen and coworkers used different isolation protocols, which could justify the discrepancies found in results.

Even though they demonstrated that CDCs expressed myosin heavy chain after transplantation into myocardial infarctions in mice [17], indicating that cardiomyogenic differentiation was possible in vivo, an additional mechanism was proposed to explain the improvement in cardiac function. Chimenti and colleagues studied the relative roles of direct regeneration versus paracrine effects promoted by human CDCs in a mouse infarction model [40]. The paracrine hypothesis has been used frequently to explain the beneficial effects observed with several types of adult stem cells or bone marrowderived cells used in cell therapy experiments. According to this hypothesis, stem cells could act secreting signaling molecules, which may influence cardiomyocyte survival and angiogenesis and could also recruit endogenous cardiac stem cells. Chimenti and coworkers demonstrated that CDCs secrete high levels of insulin growth factor-1 (IGF-1), hepatocyte growth factor (HGF), and vascular endothelial growth factor (VEGF). Moreover, using in vivo bioluminescence assays, the authors showed that no cells could be found in the heart 1 week after injection, even though functional improvement persisted until 3 weeks post-MI. Therefore, it seems that cell persistence is not important for functional improvement, strengthening the paracrine hypothesis. To address this issue, the authors quantified capillary density and viable myocardium analyzing the contributions of human (injected) and mouse (endogenous) cells to each of these variables 1-week post-MI [40]. They found that, for both variables, the contribution of endogenous cells was more prominent than that of injected cells. Hence, the release of factors seems to be more important than direct regeneration in the improvement of cardiac function after cell therapy with CDCs.

Recently, results of a phase I clinical trial using CDCs were published [41]. The safety of autologous intracoronary delivery of CDCs to patients 1.5 to 3 months after MI was evaluated. Cells were obtained from endomyocardial biopsies and cultured according to the protocols previously established by Eduardo Marbns group. Patients with a recent MI (less than 4 weeks) and left ventricular ejection fraction ranging from 25% to 45% were eligible for inclusion. Twelve months after cell therapy, patients treated with CDCs had a 12.3% decrease in scar size, whereas the control group had a 2.2% reduction, as measured by late enhancement after gadolinium MRI. However, no differences were detected in ejection fraction between cell-treated and control groups. It is important to note that this was a safety study; therefore, phase II double-blinded placebo-controlled clinical trials still need to be performed to access efficacy of therapy with CDCs in humans. Additionally, a more thorough cell biologic characterization of CDCs is required to understand provenience, molecular identity, and mechanism of action of these cells as potential cardioprotective agents.

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Cardiac Stem Cell - an overview | ScienceDirect Topics