Description
Cardiac progenitor cells (CPCs) present in the adult heart are used for cell-based treatment of myocardial damage but factors determining stemness, self-renewal and lineage commitment are poorly understood. Immortal DNA strands are inherited through non-random or asymmetric chromatid segregation (ACS) of non-sister chromatids containing 'old' and newly synthesized DNA strands. ACS correlates with self-renewal of adult stem cells, but whether CPCs asymmetrically segregate chromatids to retain immortal strands is unknown. Cardio protective kinase Pim-1 increases asymmetric cell division in vivo, proliferation and commitment of CPCs after adoptive transfer in pathological injury model but its role in ASC is unknown. The hypothesis of the thesis is CPCs asymmetrically segregate immortal strand inheriting chromatids and Pim-1 kinase increases asymmetric segregation. Asymmetric segregation was tracked by incorporation of bromodeoxyuridine (BrdU), a thymidine analogue labeling newly synthesized DNA strands. In label retention assay, CPCs were labeled for many generations and blocked in second cytokinesis during chase in BrdU free media to determine distribution of immortal versus newly synthesized strands. Two daughter nuclei within a CPC cell-body had asymmetric BrdU intensity. Density measurements showed 4.57% of CPCs have 70:30 or higher BrdU intensity ratio between daughter nuclei indicative of asymmetric segregation of BrdU labeled chromatids. Non-random segregation was further demonstrated by asymmetric segregation of BrdU labeled chromatids in anaphase cells from second mitotic shake off during chase. Genetic engineering of CPCs to over express Pim-1 kinase enhanced asymmetric segregation by nearly two fold (9.19% vs. 4.79% eGFP expressing cells). Future studies are warranted to understand the direct role of Pim-1 in asymmetric chromatid segregation and stem cell self-renewal and fate determination.
