Reduced Ki67 Staining in the Postmortem State Calls Into Question Past Conclusions About the Lack of Turnover of Adult Human β-Cells

Abstract

Some report that adult human β-cells do not replicate, but we postulate this assumption is erroneous due a postmortem decline in replication markers such as Ki67. Our earlier report showed that Ki67-marked β-cells were rarely found in human cadaveric pancreases but were in the range of 0.2-0.5% in human islets transplanted into mice. This study subjected 4-week-old mice to autopsy conditions that typically occur with humans. Mice were killed, left at room temperature for 3 h, and then placed at 4°C for 3, 9, or 21 h. There was a rapid marked fall in Ki67 staining of β-cells compared with those fixed immediately. Values at death were 6.9 ± 0.9% (n = 6) after a 24-h fast, 4.1 ± 0.9% (n = 6) at 3 h room temperature, 2.7 ± 0.7% (n = 5) at 6 h, 1.6 ± 0.6% (n = 5) at 12 h, and 2.9 ± 0.8% (n = 5) at 24 h. Similar postmortem conditions in newborn pigs resulted in very similar declines in Ki67 staining of their β-cells. These data support the hypothesis that conclusions on the lack of replication of adult human β-cells are incorrect and suggest that adult human β-cells replicate at a low but quantitatively meaningful rate.

Figure 1

Study plan for mice. Six groups of killed mice were studied at five different times —0, 3, 6, 12, and 24 h—with varying lengths of exposure to warm ischemia (RT) and cold ischemia (4°C). SAC, sacrificed.

Figure 2

Autopsy conditions show a rapid marked reduction of Ki67 staining in mouse β-cells. Confocal images show insulin (red) and Ki67 (green) staining of representative islets. Compared with immediate fixation (A), there is a marked loss of Ki67 already by 3 h of warm ischemia (B); with an additional 21 h of cold ischemia (C), even less Ki67 can be seen in the β-cells. D: With quantification of Ki67-positive insulin-positive cells, the fed and fasted controls did not differ, but reduction of Ki67 staining in β-cells was found after warm and cold ischemia (n = 5–9). Scale bar, 40 μm. *P < 0.005, **P < 0.001.

Figure 3

Pig pancreas under autopsy conditions show marked reduction of Ki67 in their β-cells. Porcine β-cells subjected to 3 h of warm ischemia at RT, followed by 21 h of cold ischemia at 4°C, showed a significant loss of Ki67 staining compared with 3 h of warm ischemia only. There was also a significant difference between the percentage of Ki67-positive β-cells in the tail and the body/head part of the pancreas after 3 h of warm ischemia. This difference is not due to overrepresentation of ventral lobe tissue in the body/head portion (n = 4). *P < 0.001, **P < 0.005.

Figure 4

Decline of Ki67 staining in β-cells after 24 h of autopsy conditions is similar in β-cells of mice (n = 5–9) and pigs (n = 4). *P < 0.005.