ALDH Expression Characterizes G1-Phase Proliferating Beta Cells during Pregnancy

<div><p>High levels of aldehyde dehydrogenase (ALDH) activity have been detected in various progenitor and stem cells. Thus, Aldefluor fluorescence, which represents precisely the ALDH activity, has been widely used for the identification, evaluation, and isolation of stem and progenitor cells. Recently, ALDH activity was detected in embryonic and adult mouse pancreas, specifically in adult centroacinar and terminal duct cells supposed to harbor endocrine and exocrine progenitor cells in the adult pancreas. Nevertheless, ALDH activity and aldeflour fluorescence have not been examined in beta cells. Here, we report a dynamic increase in the number of aldeflour+ beta cells during pregnancy. Interestingly, nearly all these aldeflour+ beta cells are positive for Ki-67, suggesting that they are in an active cell cycle (G1, S and M phases). To determine precisely at which phase beta cells activate ALDH activity and thus become aldeflour+, we co-stained insulin with additional proliferation markers, phosphohistone3 (PHH3, a marker for M-phase proliferating cells) and Bromodeoxyuridine (BrdU, a marker for S-phase proliferating cells). Our data show little aldeflour+ beta cells that were positive for either PHH3, or BrdU, suggesting that beta cells activate ALDH and become Aldefluor+ when they enter G1-phase of active cell cycle, but may downregulate ALDH when they leave G1-phase and enter S phase. Our data thus reveal a potential change in ALDH activity of proliferating beta cells during pregnancy, which provides a novel method for isolation and analysis of proliferating beta cells. Moreover, our data also suggest that caution needs to be taken on interpretation of Aldefluor lineage-tracing data in pancreas.</p></div

ALDH+ beta cells are not in M or S phase.

<p>(A–B) To determine precisely at which phase of a cell cycle, beta cells activate ALDH activity and thus become ALDH+, we co-stained ALDH (in green), insulin (in red) with PHH3 (in blue) and BrdU (in blue). BrdU was given to the mice two hours prior to sacrifice. (A) Representative images show essentially no PHH+ALDH+ beta cells, suggesting that beta cells lose ALDH activity when they enter M phase of a cell cycle. (B) Representative images show essentially no BrdU+ALDH+ beta cells, suggesting that beta cells lose ALDH activity when they enter S phase of a cell cycle. Scale bar is 50 µm.</p

ALDH+ cells in the islets are predominantly beta cells in active cell cycle.

<p>(A–B) Representative triple staining for ALDH (in green), insulin (in red) and Ki-67 (in blue), together with nuclei staining with DAPI are shown in G9 (A) and G0 (B) pancreas. Each channel was shown independently. A merged image for ALDH, insulin and Ki-67 was also shown. The result suggests that aldefluor+ cells in the islets are predominantly proliferating beta cells. Arrows point to ALDH+ terminal cells. Scale bar is 50 µm.</p

Beta cells increase ALDH activity in G1 phase during proliferation.

<p>Our finding was summarized and illustrated. Beta cells activate ALDH and become aldefluor+ when they enter G1-phase of an active cell cycle, but may downregulate ALDH and become Aldefluor- when they leave G1-phase and enter S phase.</p

Aldefluor+ cells are detected in the islets of pregnant mice.

<p>(A) Representative flow cytometry analysis of aldefluor fluorescence in the bone marrow cells (+/− treatment with a specific ALDH inhibitor DEAB) and isolated islets from pregnant mice 9 days after pregnancy (G9), compared with non-pregnant mice (G0). While no aldefluor+ cells were detected in G0 islets, aldefluor+ cells (circled) were readily detected in the islets from G9 islets. (B) Isolated G9 aldeflouor+ cells were immunostained positive for ALDH. (C) Quantification of aldefluor+ cells in the islet fraction from pregnant mice at 3, 6, 9, 12, 15 and 18 days after pregnancy (G3, G6, G9, G12, G15 and G18, respectively). These data show that in the mouse pancreas, islet cells upregulate ALDH activity during pregnancy. SSC: side-light scatter. *: p<0.05.</p