Asymmetric chromatid segregation in cardiac progenitor cells is enhanced by Pim-1 kinase

Abstract

Rationale: Cardiac progenitor cells (CPCs) 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 inherited through asymmetric chromatid segregation correlate with self-renewal of adult stem cells, but the capacity of CPCs for asymmetric segregation to retain immortal strands is unknown. Cardioprotective kinase Pim-1 increases asymmetric cell division in vivo, but the ability of Pim-1 to enhance asymmetric chromatid segregation is unknown.

Objective: We aimed to demonstrate immortal strand segregation in CPCs and the enhancement of asymmetric chromatid distribution by Pim-1 kinase.

Methods and results: Asymmetric segregation is tracked by incorporation of bromodeoxyuridine. The CPC DNA was labeled for several generations and then blocked in second cytokinesis during chase to determine distribution of immortal versus newly synthesized strands. Intensity ratios of binucleated CPCs with bromodeoxyuridine of ≥70:30 between daughter nuclei indicative of asymmetric chromatid segregation occur with a frequency of 4.57, and asymmetric chromatid segregation is demonstrated at late mitotic phases. Asymmetric chromatid segregation is significantly enhanced by Pim-1 overexpression in CPCs (9.19 versus 4.79 in eGFP-expressing cells; P=0.006).

Conclusions: Asymmetric segregation of chromatids in CPCs is increased nearly two-fold with Pim-1 kinase overexpression, indicating that Pim-1 promotes self-renewal of stem cells.

Figure 1. CPCs retain unlabeled immortal strands in label release assay

A, Binucleated cell with one daughter nuclei exhibiting low BrdU immuno-staining (red arrowhead) and another showing intense BrdU signal. B, Binucleated cell with daughter nuclei exhibiting symmetric BrdU immunostaining. Binucleated CPCs are confirmed by reflection scanning (Cell). Scale bar = 10 μm.

Figure 2. CPCs asymmetrically segregate BrdU labeled chromatids

A and B, BrdU intensity distribution among daughter nuclei in binucleated cells at first (A) and second (B) mitosis during chase in label retention assay. 70:30 distribution threshold for BrdU intensity ratio is shown as dotted box in panel B and events above this threshold are considered asymmetrically segregating cells. C and D, Representative binucleated cells with symmetric (C) and asymmetric (D) BrdU immuno-stain.

Figure 3. Asymmetric BrdU segregation at late mitotic stages

A, A telophase cell with symmetric BrdU staining. B, An anaphase cell with asymmetric BrdU segregation. C, Z stacks of BrdU immuno-staining of B. Mitotic phases are confirmed by differential interference contrast (DIC) images, labeled as Cell.

Figure 4. Pim-1 kinase increases asymmetric DNA segregation

A, Representative binucleated cell at second mitosis for CPCe (upper) and CPCeP (lower) with asymmetric BrdU staining indicated by red arrow head. B and C, BrdU distribution at second mitosis between daughter nuclei of CPCe and CPCeP, respectively. Dotted box represents 70:30 threshold of BrdU intensity ratio. D, Percentage of asymmetrically segregating cells from five independent experiments. Asterisk indicates p=0.006 compared to CPCe.