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

At 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.CPCs 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.This study demonstrates that direct culture and expansion of CPCs from myocardial tissue is simple, straightforward, and reproducible when appropriate techniques are used

Cardiosphere-Derived Cells Improve Function in the Infarcted Rat Heart for at Least 16 Weeks – an MRI Study

Aims Endogenous cardiac progenitor cells, expanded from explants via cardiosphere formation, present a promising cell source to prevent heart failure following myocardial infarction. Here we used cine-magnetic resonance imaging (MRI) to track administered cardiosphere-derived cells (CDCs) and to measure changes in cardiac function over four months in the infarcted rat heart. Methods and Results CDCs, cultured from neonatal rat heart, comprised a heterogeneous population including cells expressing the mesenchymal markers CD90 and CD105, the stem cell marker c-kit and the pluripotency markers Sox2, Oct3/4 and Klf-4. CDCs (2×106) expressing green fluorescent protein (GFP+) were labelled with fluorescent micron-sized particles of iron oxide (MPIO). Labelled cells were administered to the infarcted rat hearts (n = 7) by intramyocardial injection immediately following reperfusion, then by systemic infusion (4×106) 2 days later. A control group (n = 7) was administered cell medium. MR hypointensities caused by the MPIOs were detected at all times and GFP+ cells containing MPIO particles were identified in tissue slices at 16 weeks. At two days after infarction, cardiac function was similar between groups. By 6 weeks, ejection fractions in control hearts had significantly decreased (47±2%), but this was not evident in CDC-treated hearts (56±3%). The significantly higher ejection fractions in the CDC-treated group were maintained for a further 10 weeks. In addition, CDC-treated rat hearts had significantly increased capillary density in the peri-infarct region and lower infarct sizes. MPIO-labelled cells also expressed cardiac troponin I, von Willebrand factor and smooth muscle actin, suggesting their differentiation along the cardiomyocyte lineage and the formation of new blood vessels. Conclusions CDCs were retained in the infarcted rat heart for 16 weeks and improved cardiac function

Precardiac deletion of numb and numblike reveals renewal of cardiac progenitors

Cardiac progenitor cells (CPCs) must control their number and fate to sustain the rapid heart growth during development, yet the intrinsic factors and environment governing these processes remain unclear. Here, we show that deletion of the conserved cell-fate regulator Numb and its homologue Numblike (Numbl) depletes CPCs in second pharyngeal arches (PA2s) and is associated with an atrophic heart. With histological, flow cytometric and functional analyses, we find that CPCs remain undifferentiated and expansive in the PA2, but differentiate into cardiac cells as they exit the arch. Tracing of Numb-and Numbl-deficient CPCs by lineage-specific mosaicism reveals that the CPCs normally populate in the PA2, but lose their expansion potential in the PA2. These findings demonstrate that Numb and Numbl are intrinsic factors crucial for the renewal of CPCs in the PA2 and that the PA2 serves as a microenvironment for their expansion