Abstract 18738: The Cdkn2a/2b Tumour Suppressor Locus is a Hot Spot for Genome Instability in Mouse Cardiac Progenitor Cells
Among the cells with best cardiac potential, autologous heart cells present the fewest regulatory issues, and therapeutic trials are on-going. Existing work has largely relied on purified but potentially heterogeneous populations. As ES, iPS and cardiosphere-derived cells all are known to accumulate genomic aberrations, we analyzed genome integrity in clonally derived mouse Lin-/Sca-1+/side population (SP) cardiac progenitor cells (CPCs), which stably express cardiogenic transcription factors for ≥ 35 passages. DNA content analysis showed progressive aneuploidy, with >90% of clones abnormal by passage 11. By multicolour spectral karyotyping (SKY), even those with normal DNA content had aberrations, though no recurrent anomalies were seen; genome instability was not contingent on clonal isolation per se or Hoechst 33342. High-resolution array CGH (aCGH) was performed on 15 clones, using 1 million probe arrays. Numerous aberrations were found, most 100-fold below the limits of SKY. Interestingly, copy number variation did not explain the observed differences in cardiac transcription factors. After passage 10, recurrent deletions were seen in Cdkn2a/2b, encoding the tumour suppressors p16Ink4a, p15Ink4b, and p19ARF. QPCR confirmed this in >80% of clones. Integrity of Cdkn2a/2b through passage 10 indicates its deletion is not required for clonal growth. Because genome instability has been ascribed to hyperoxic stress in ambient (21%) [O2], we derived and propagated clones in physiological (2 or 7%) [O2]. Reduced [O2] increased (>3x) and accelerated (2x) clone formation, reduced aneuplody, reduced chromosomal aberrations, but did not prevent loss of Cdkn2a/2b. Low [O2] also enabled generating clones readily from Lin-/Sca1+/SP- CPCs, for which the cloning efficiency was previously reported as < 0.1%. Thus, genomic instability of propagated CPCs was demonstrated, including recurrent deletion of Cdkn2a/2b. Reduced [O2] conferred partial protection from genomic instability. Clonal analysis unveiled heterogeneities in genome structure that could be obscured in a mixed population. These data highlight the need to develop bioprocesses that optimize genomic stability.
- © 2012 by American Heart Association, Inc.