Validation of the Cardiosphere Method to Culture Cardiac Progenitor Cells from Myocardial Tissue

Abstract

BackgroundAt least four laboratories have shown that endogenous cardiac progenitor cells (CPCs) can be grown directly from adult heart tissue in primary culture, as cardiospheres or their progeny (cardiosphere-derived cells, CDCs). Indeed, CDCs are already being tested in a clinical trial for cardiac regeneration. Nevertheless, the validity of the cardiosphere strategy to generate CPCs has been called into question by reports based on variant methods. In those reports, cardiospheres are argued to be cardiomyogenic only because of retained cardiomyocytes, and stem cell activity has been proposed to reflect hematological contamination. We use a variety of approaches (including genetic lineage tracing) to show that neither artifact is applicable to cardiospheres and CDCs grown using established methods, and we further document the stem cell characteristics (namely, clonogenicity and multilineage potential) of CDCs.Methodology/Principal FindingsCPCs were expanded from human endomyocardial biopsies (n = 160), adult bi-transgenic MerCreMer-Z/EG mice (n = 6), adult C57BL/6 mice (n = 18), adult GFP+ C57BL/6 transgenic mice (n = 3), Yucatan mini pigs (n = 67), adult SCID beige mice (n = 8), and adult Wistar-Kyoto rats (n = 80). Cellular yield was enhanced by collagenase digestion and process standardization; yield was reduced in altered media and in specific animal strains. Heparinization/retrograde organ perfusion did not alter the ability to generate outgrowth from myocardial sample. The initial outgrowth from myocardial samples was enriched for sub-populations of CPCs (c-Kit+), endothelial cells (CD31+, CD34+), and mesenchymal cells (CD90+). Lineage tracing using MerCreMer-Z/EG transgenic mice revealed that the presence of cardiomyocytes in the cellular outgrowth is not required for the generation of CPCs. Rat CDCs are shown to be clonogenic, and cloned CDCs exhibit spontaneous multineage potential.Conclusions/SignificanceThis study demonstrates that direct culture and expansion of CPCs from myocardial tissue is simple, straightforward, and reproducible when appropriate techniques are used.

Spheroid three-dimensional culture enhances Notch signaling in cardiac progenitor cells

“…[8,21] The 3D environment and enhanced cell-cell and cell-ECM contacts offered by CSps can stimulate several molecular signaling and cellular functions. Notch is a highly conserved pathway that plays a key role in determining cell fate decisions in the heart.…”

Migration of Resident Cardiac Stem Cells in Myocardial Infarction

“…These cells were defined as cardiosphere-derived cells (CDCs), another source of CSCs from adult heart (Messina et al, 2004). Cardiosphere-derived cells are a heterogeneous population, which are comprised of c-kit þ , CD31 þ , and CD34 þ cells (Davis et al, 2009). They are clonogenic and capable of long-term self-renewal.…”

“…Mouse and human c-kit þ , CD31 þ , CD34 þ c-kit, CXCR4 Messina et al, 2004;Davis et al, 2009;Smith et al, 2007;Tang et al, 2009 Isl Laugwitz et al, 2005;Weinberger et al, 2012 CSCs, cardiac stem cells; CSP, cardiac side population.…”

Durable Benefits of Cellular Postconditioning: Long‐Term Effects of Allogeneic Cardiosphere‐Derived Cells Infused After Reperfusion in Pigs with Acute Myocardial Infarction

“…CDCs have been argued to be cardiac progenitor cells,29, 30, 31 but that feature of CDCs is an epiphenomenon when it comes to mechanism of action. In the extreme, allo‐CDCs are cleared completely within several weeks, but their functional and structural benefits persist at least 6 months 14.…”