Human Cardiac Stem Cells Isolated from Atrial Appendages Stably Express c-kit

Jia-Qiang He,Duc Minh Vu,Greg Hunt,Aruni Bhatnagar,Atul Chugh,Roberto Bolli,Marcello Rota

doi:10.1371/journal.pone.0027719

Abstract

The in vivo studies of myocardial infarct using c-kit+/Lin− cardiac stem cells (CSCs) are still in the early stage with margin or no beneficial effects for cardiac function. One of the potential reasons may be related to the absence of fully understanding the properties of these cells both in vitro and in vivo. In the present study, we aimed to systematically examine how CSCs adapted to in vitro cell processes and whether there is any cell contamination after long-term culture. Human CSCs were enzymatically isolated from the atrial appendages of patients. The fixed tissue sections, freshly isolated or cultured CSCs were then used for identification of c-kit+/Lin− cells, detection of cell contamination, or differentiation of cardiac lineages. By specific antibody staining, we demonstrated that tissue sections from atrial appendages contained less than 0.036% c-kit+/Lin− cells. For the first time, we noted that without magnetic activated cell sorting (MACS), the percentages of c-kit+/Lin− cells gradually increased up to ∼40% during continuously culture between passage 2 to 8, but could not exceed >80% unless c-kit MACS was carried out. The resulting c-kit+/Lin− cells were negative for CD34, CD45, CD133, and Lin markers, but positive for KDR and CD31 in few patients after c-kit MACS. Lin depletion seemed unnecessary for enrichment of c-kit+/Lin− cell population. Following induced differentiation, c-kit+/Lin− CSCs demonstrated strong differentiation towards cardiomyocytes but less towards smooth and endothelial cells. We concluded that by using an enzymatic dissociation method, a large number, or higher percentage, of relative pure human CSCs with stable expression of c-kit+ could be obtained from atrial appendage specimens within ∼4 weeks following c-kit MACS without Lin depletion. This simple but cost-effective approach can be used to obtain enough numbers of stably-expressed c-kit+/Lin− cells for clinical trials in repairing myocardial infarction.

Highlights

It is a long-held belief that mammalian cardiomyocytes withdraw from the cell cycle during the perinatal period and that the mammalian heart is a terminal post-mitotic organ incapable of selfregeneration after myocardial injury
We examined cardiac stem cells (CSCs) markers adapted to post-isolation, expansion, purification, Lin depletion (Lin-dep); their vitro differentiation properties, and potential contamination by other cell types, such as cardiac fibroblasts and mast cells
We found that a small proportion of un-fractional cells isolated from atrial appendages were fibroblast positive cells (22.760.9%, n = 3 patients); these fibroblasts were entirely removed by c-kit magnetic activated cell sorting (MACS) procedure (Figure 6)

Summary

Introduction

It is a long-held belief that mammalian cardiomyocytes withdraw from the cell cycle during the perinatal period and that the mammalian heart is a terminal post-mitotic organ incapable of selfregeneration after myocardial injury. This paradigm has been challenged by the work of Beltrami and colleagues [1] who for the first time, discovered specialized cells within the heart tissue expressing stem cell markers (c-kit, Sca-1, and MDR1). The origin and the function of these cells remain unclear, different putative adult CSCs most likely represent different developmental and/or physiological stages of a unique CSC population in the adult mammalian heart [20]

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Conclusion