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Role of the ATP-Binding Cassette Transporter Abcg2 in Cardiac Regeneration

Final Report Summary - CARDIAC ABC'S (Role of the ATP-Binding Cassette Transporter Abcg2 in Cardiac Regeneration)

Our studies of heart-derived Sca-1+ cardiac progenitor cells with a “side population” (SP) phenotype have helped us to develop a better understanding of their multi-lineage stem cell potential and likely involvement in the natural regenerative capacity of the heart. In addition to these two cellular markers, our group more recently has been able to identify platelet-derived growth factor receptor alpha (PDGFR-α) as an important cell surface feature that correlated more precisely with the clonogenic capacity and cardiogenic transcription factor profile of the larger heterogeneous Sca-1+ population.

One fundamental goal of this research project is to understand the role of telomeres maintenance and dynamics and its relations with heart regeneration and tissue homeostasis. Our studies on the heart derived Sca-1 +cardiac progenitor cells (CPC) with an “side population” (SP) phenotype have helped us to development a better understanding on their potential stem cell potential and involvement in the natural regenerative capacity of the heart. More recently, our group have been able to identify the platelet derived growth factor alpha (PDGFR-α) as an important cellular marker that correlated with the clonogenic capacity of Sca-1 + cells. Systematic evaluation of the capacity of Sca-1 + SP cells to be cloned and indefinitely kept in cell culture in our laboratory has helped define demonstrate that the clonogenicity of these cells increases when isolated and cultured a lower that conventional oxygen concentration. Using quantitative telomere specific fluorescence in situ hybridization (FISH) we have observed that, in the absence of differences of proliferation capacity or these cells, Sca-1+ cell clones that are kept in lower oxygen concentration have significantly longer telomeres. Additional characterisation of this observations in currently been conducted to elucidate the mechanism involved in this reduced telomere attrition.
To further our understanding on the role of telomere maintenance of this cardiac progenitor cell (CPC) populations and to incorporate our new findings regarding the potential role of PDGFR- α as a maker of clonogenicity, we have isolated and produced mouse heart derived Sca-1+ PDGFR-α+ SP and non-SP cellular clones from telomerase reverse transcriptase (TERT) knock-out, heterozygous, and wild-type animals.
We have successfully isolated Sca1+ PDGFR-alpha +, side population (SP) and non side population (NSP) cardiac stem cells from telomerase deficient mice (TERT-/-) and its wild type counterparts (TERT+/+) from three back breed generations (G1-G3). As expected, clonogenicity was significantly increased in the cell populations showing a SP phenotype however, we have observed that that the absence of TERT locus in G1-G3) mouse lines do not modify the capacity of this cells to growth right after isolation, measured as the operational definition of clonogenicity. More detailed analysed of the G3 isolated CPCs suggested that the kinetic of colony growth in the third generation (G3) of TERT knockout cells maybe slightly impaired.
Characterization of the telomere length of the TERT G1 CPCs has shown that TERT G1 knockout cells exhibit an increased proportion of chromosome ends with critically short telomeres with not detectable deference in the mean telomere length. Not been able to observe a significant difference in telomere length in the analysed cells is not necessary surprising, as it has already reported that telomerase deficient mice show little or undetectable levels of telomere attrition in their first back breed generation, as well as indistinguishable ageing phenotype compare to wild-type mice. It is important to note that the consecutive TERT knockout mice generations used this project have been established in a pure C57BL/6 background as it has been previous reported that average offspring size, testes size, telomere shortening and, chromosomal signal free ends; all characteristic phenotypes of premature ageing are already observed from the TERT knockout generations 2 to 4 (G2-G4), and infertility is fully evident at generation 4 (G4). On the other hand, when a similar mating strategy has been applied in a C57BL/6/129 mixed background, these phenotypes have been observed only from generations 5 to 8 (G5-G8) [Erdmann N, Liu Y, Harrington L. Proc Natl Acad Sci U S A. 2004 Apr 20;101(16):6080-5]. Our research team is currently evaluating the effect of telomerase gene abrogation on the telomere length of CSC of second and third generations knockout mice, where difference could be expected. We are also currently characterising the potential DNA damage response associated with critically short telomeres and its relationship with a provocable phenotype of susceptibility to programmed cell death in telomerase deficient cardiac progenitor cells.
Our most significant results comprise the successful generation of telomerase knockout (TERT-/-) cardiac progenitor cells. The characterization of isolated cells will be instrumental to understand the role of telomerase in stemness potential of cell capable to contribute to heart regeneration after injury and during the decrease in regenerative capacity observed during ageing.