P78Activity status of resident adult cardiac stem cells determines their stem cell properties and growth kinetics

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Abstract

Purpose

The identification of a small population of stem cells resident in the adult mammalian heart, with robust regenerative capacity, indicates the potential to develop new strategies using these cells for cardioprotective and regenerative purposes. Endogenous cardiac stem cells (eCSCs) positive for c-kit, isolated from the adult mammalian heart, are clonogenic, self-renewing and multipotent. Here we compared the properties and growth kinetics of quiescent eCSCs with activated eCSCs in vitro.

Methods

To induce tissue damage and resultant eCSC activation, 5mg kg-1 isoproterenol (ISO) was injected (s.c.) into 2-month-old adult male Wistar rats (∼250g). Saline was injected as control (CTRL; quiescent eCSC group). c-kitpos CD45neg eCSCs were isolated by retrograde coronary enzymatic perfusion, 24 hours post-ISO or saline injection. Quiescent and activated eCSCs were analysed for number, proliferative markers, multipotency genes, growth kinetics, clonogenicity and proliferation index using flow cytometry, immunocytochemistry, qRT-PCR, real time lapse cell culture imaging, and BrdU incorporation assay, respectively.

Results

ISO-induced injury significantly (P < 0.05) increased c-kitpos eCSC number (33 ± 7% of the CD45neg small cardiac cell population, compared to 8 ± 2% in controls). Most of these cells were actively proliferating, being 56 ± 7% Ki67 positive, compared to only 10 ± 1% Ki67 positive in the quiescent CTRL eCSCs. Activated c-kitpos eCSCs showed increased clonogenicity (24 ± 4% vs. 2 ± 4%), cardiosphere formation (563 ± 58 per 100,000 cells vs. 59 ± 39) and proliferation, measured by BrdU incorporation over 48 hours in culture (32 ± 8% vs. 5 ± 2%). Furthermore, activated and quiescent c-kitpos eCSCs showed differential multipotency gene expression and real time-lapse growth kinetics.

Conclusions

Activated c-kitpos CD45neg eCSCs show enhanced stem cell properties and growth kinetics, compared to quiescent c-kitpos CD45neg eCSCs, in vitro. These data imply that pre-activation of stem cells could improve their cardioprotective and regenerative ability. Identifying factors that influence the activation of eCSCs and the maintenance of this state is of paramount importance for the design and optimisation of cardiac regenerative therapies.

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