Bmi1 lineage tracing identifies a self-renewing pancreatic acinar cell subpopulation capable of maintaining pancreatic organ homeostasis

Abstract

A central question in stem cell biology is whether organ homeostasis is maintained in adult organs through undifferentiated stem cells or self-duplication of specialized cell populations. To address this issue in the exocrine pancreas we analyzed the Bmi1-labeled cell lineage of pancreatic acinar cells. Previously, we had shown that inducible linage tracing with Bmi1-Cre-estrogen receptor (ER) in the small intestine specifically, labels "classical" undifferentiated intestinal stem cells. In this article we demonstrate that the Bmi1-Cre-ER system labels a subpopulation of differentiated acinar cells in the exocrine pancreas whose derivatives are still present, at a steady-state level, 1 year after a single TM pulse. This study suggests that Bmi1 is a marker for a subpopulation of self-renewing acinar cells, indicating that self-renewal is not an exclusive feature of adult undifferentiated stem cells. Further, the extended period that Bmi1-labeled acinar cells retain a pulse of BrdU suggests that some of this subpopulation of cells are not continuously replicating, but rather are set aside until needed. This cellular behavior is again reminiscent of behavior normally associated with more classical adult stem cells. Setting aside cells capable of self-renewal until needed retains the advantage of protecting this subpopulation of cells from DNA damage induced during replication.

Fig. 1.

Bmi1 lineage analysis in pancreas. (A) More than 70 Bmi1^Cre-ER/+;Rosa26^YFP/+ mice (or Rosa26^LacZ/+) were generated, injected at weaning with TM, and harvested at different time points from 21 h up to 1 year. (B) Whole-mount LacZ staining showing the pattern of the lineage obtained 5 days after TM treatment. (C) Age-matched control Bmi1^Cre-ER/+;Rosa26^LacZ/+ pancreas without TM showed no activation of the LacZ reporter. (D and E) Pancreatic sections showing the Bmi1⁺ lineage. (E) High magnification of an YFP⁺ differentiated acinar cell. (Scale bars: 50 μm.)

Fig. 2.

Early Bmi1⁺ lineage is present only in differentiated cells. (A–D) Bmi1 is expressed in acinar cells (amylase⁺), glucagon⁺ cells (white arrowhead), and endothelial cells (PECAM⁺) (white arrowheads). (E–I) Bmi1 is largely absent from other cells in the islets (ChromograninA⁺ and C-peptide⁺), ducts (DBA⁺), and centroacinar cells (Sox9⁺) (white arrowheads). In A, B, G, and H E-cadherin staining was used to identify the cell boundaries. (J–K) Bmi1⁺ lineage is negative for Pdx1 and Nestin (white arrowheads). (L) Representative microscopic field of our analysis, the higher magnification inset shows an endothelial cell (PECAM⁺) and some acinar cells (amylase⁺). (M) At 5, 15, and 30 days pancreas was harvested from Bmi1^Cre-ER/+;Rosa26^FP/+ mice. All differentiated double positive cells were counted for each time point. (N and O) Representative pictures of 2 of the 100 cells analyzed at 5 days after TM treatment to show the perfect colocalization between YFP⁺ cell and amylase. E-cadherin staining was used to identify the cell boundaries. (Scale bars: 50 μm.)

Fig. 3.

Bmi1 long-term lineage analysis. (A and B) Bmi1^Cre-ER/+;Rosa26^YFP/+ mice were injected at weaning with TM and followed for >1 year. (A) Representative microscopic fields of pancreas harvested at different time points showing YFP⁺ cells. Control pancreas with the same genotype without TM, showing no YFP⁺ cells. (B) Graph showing the number of YFP⁺ cells present at the different time points analyzed. (C) Graph showing the percentage of clones at 5, 15, and 30 days that underwent replication. YFP⁺ cells with 1 or more labeled adjacent cells (expanded clones) increased over time. (D–I) Representative pictures of mostly isolated cells at 5 days (D) and expanded clones at 15 and 30 days (E–I). (Insets) High-magnification images of an isolated cell (D) and 3 YFP⁺ adjacent cells (E). (Scale bars: 50 μm.)

Fig. 4.

Proliferation analysis of the Bmi1 lineage. (A) Amylase⁺ cells are Ki-67⁺ indicating proliferation potential. (B and C) Bmi1 lineage is also Ki-67⁺. (D–I) Short-term analysis of BrdU incorporation. (D) Schematic representation of the BrdU and the TM treatment. (E) Percentage of YFP⁺BrdU⁺ cells at 15, 30, and 120 days. (F) The Bmi1⁺ lineage in mice fed with BrdU was reduced, with respect to mice never treated with BrdU, at 15 and 30 days, but was comparable at 120 days. (G–I) Representative microscopic fields showing YFP⁺BrdU⁺ (white arrowhead), amylase⁺BrdU⁺, and amylase⁺YFP⁺BrdU⁺ cells (white arrowhead). (J–N) Long-term BrdU chase to show the persistence of the BrdU lineage. (J) Schematic representation of the whole experiment. (K) Percentage of YFP⁺BrdU⁺ cells at 7 months. (L) Comparison of the lineage at 1 and 7 months after 1 TM injection at weaning and at 7 months after 1 injection at 6 months (with and without BrdU). (M and N) Representative pictures of YFP⁺BrdU⁺ and amylase⁺YFP⁺BrdU⁺(white arrowhead) cells indicating that those cells were differentiated. (O–Q) BrdU treatment at 8 months after TM treatment. (O) Schematic representation of the TM and BrdU treatments. (P) Percentage of YFP⁺BrdU⁺ cells at 9 months. (Q) Representative pancreatic section showing 4 YFP⁺BrdU⁺ cells. (Scale bars: 25 μm.)

Fig. 5.

Evaluation of the Bmi1 lineage after different TM treatments. (A) Schematic representation of a single treatment, 3 consecutive injections, and 3 injections spaced every 5 or 10 days. All Bmi1^Cre-ER/+;Rosa26^YFP/+ mice were analyzed 30 days after the first injection. (B and C) Representative pictures and quantitative analysis of pancreatic sections after different treatments. (D) Different representation of counted cells. The continuous curve represents the number of cells present at different days after 1 TM injection. This curve was interpolated by using the cells counted at 5, 15, 30, and 60 days, assuming a constant increase in the number of cells. The black and pink triangles represent the mean of the YFP⁺ cells counted after 3 injections at 5-day intervals and 3 injections at 10-day intervals. (E) Proposed model showing that if each TM injection activates the same number of cells (blue, red, and green lines) after 30 days the number of expected labeled cells matches the number of obtained cells. The black and pink lines indicate at each day the expected sum of all of the cells present in the pancreas as a result of the 3 different waves. (Scale bars: 100 μm.)

Fig. 6.

Bmi1 lineage ablation. (A) Bmi1^Cre-ER/+;Rosa26^DTA/+ mice were injected 3 times every 5 days before harvesting pancreas 5 and 12 days after last injection. (B) After 5 days there were many cells in mitosis. Amylase⁺Ki-67⁺ cells were counted, compared at 5 and 12 days after injection, and compared with wild-type age-matched control pancreas. (C–H) Representative pictures at low and high magnification showing Ki-67⁺ cells actively dividing. E and F are low and high magnification, respectively, of amylase⁺Ki-67⁺ cells. It was common to observe chromosomes in metaphase (G and H)(red arrowheads). (I–L) Bmi1 lineage after mild pancreatitis. (I) Schematic diagram showing the caerulein treatment followed by TM administration in Bmi1^Cre-ER/+;Rosa26^YFP/+ mice. (J–L) The Bmi1 lineage was increased compared with the lineage without caerulein treatment. (J and K) Representative pictures at 5 days after TM without and with caerulein. (M–O) Representative pictures showing that after caerulein treatment the Bmi1 lineage is present only in differentiated cells. (Scale bars: 50 μm.)