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. 2013 Jun 15;304(12):G1103-16.
doi: 10.1152/ajpgi.00299.2012. Epub 2013 Apr 18.

Inflammation Increases Cells Expressing ZSCAN4 and Progenitor Cell Markers in the Adult Pancreas

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Free PMC article

Inflammation Increases Cells Expressing ZSCAN4 and Progenitor Cell Markers in the Adult Pancreas

Shigeru B H Ko et al. Am J Physiol Gastrointest Liver Physiol. .
Free PMC article

Abstract

We have recently identified the zinc finger and SCAN domain containing 4 (Zscan4), which is transiently expressed and regulates telomere elongation and genome stability in mouse embryonic stem (ES) cells. The aim of this study was to examine the expression of ZSCAN4 in the adult pancreas and elucidate the role of ZSCAN4 in tissue inflammation and subsequent regeneration. The expression of ZSCAN4 and other progenitor or differentiated cell markers in the human pancreas was immunohistochemically examined. Pancreas sections of alcoholic or autoimmune pancreatitis patients before and under maintenance corticosteroid treatment were used in this study. In the adult human pancreas a small number of ZSCAN4-positive (ZSCAN4⁺) cells are present among cells located in the islets of Langerhans, acini, ducts, and oval-shaped cells. These cells not only express differentiated cell markers for each compartment of the pancreas but also express other tissue stem/progenitor cell markers. Furthermore, the number of ZSCAN4⁺ cells dramatically increased in patients with chronic pancreatitis, especially in the pancreatic tissues of autoimmune pancreatitis actively regenerating under corticosteroid treatment. Interestingly, a number of ZSCAN4⁺ cells in the pancreas of autoimmune pancreatitis returned to the basal level after 1 yr of maintenance corticosteroid treatment. In conclusion, coexpression of progenitor cell markers and differentiated cell markers with ZSCAN4 in each compartment of the pancreas may indicate the presence of facultative progenitors for both exocrine and endocrine cells in the adult pancreas.

Keywords: autoimmune pancreatitis; facultative progenitor cells; regeneration.

Figures

Fig. 1.
Fig. 1.
Dynamic control of zinc finger and SCAN domain containing 4 (ZSCAN4) expression in adult human (h) pancreas. A: ZSCAN4 expression at RNA levels in various human organs. Low level of ZSCAN4 expression was detected in all of the human tissues examined. B: immunolocalization of ZSCAN4 protein in adult human pancreas. A very small number of cells showed strong nuclear and cytoplasmic staining for ZSCAN4 in all of the major compartments: the islet of Langerhans, acini, ducts, and oval-shaped cells (referred to as oval cells: putative quiescent pancreatic stellate cells). Immunostaining without a primary antibody against ZSCAN4 is shown as a negative control. C: immunolocalization of mouse Zscan4 in the pancreas. Most of the cells are negative for Zscan4, but a subset of cells in acini, ducts, the islet of Langerhans are positive for mouse Zscan4. Oval-shaped cells (see text) are also positive for Zscan4. Note that oval-shaped cells between acini appear as a single cell or sometimes a cluster of a few cells. D: an anti-mouse (m) Zscan4 antibody and an anti-human ZSCAN4 antibody stained identical cells in the adult human pancreas. E: an anti-mouse Zscan4 antibody and an anti-human ZSCAN4 antibody stained identical cells in a subset of cells in the islet of Langerhans of mouse pancreas. DIC, differential interference contrast microscopy.
Fig. 2.
Fig. 2.
Immunolocalization of differentiated cell markers in adult human pancreas. A: aquaporin 1 (AQP1) water channel is expressed in the plasma membrane of duct cells from centroacinar cells to the medium-sized ducts. Note that AQP1 is also expressed in pancreatic stellate cells (PSC) with cytoplasmic protrusions and round oval cells between adjacent acini (see text). B: acinar cells are positive for the digestive enzyme amylase. Note that amylase is also positive in a subset of cells in ducts (arrow), oval-shaped cells between acini. C: insulin. D: glucagon. All of these hormone-expressing cells are not only confined inside the islet of Langerhans but also in a subset of acinar cells and oval-shaped cells in the human pancreas. Note that some of the cells at the peripheral region of human pancreatic ducts are also positive for endocrine hormones. E: immunolocalization of desmin in the pancreas. Desmin is expressed not only in a subset of oval cells but in a subset of cells in ducts and the islet of Langerhans. F: immunolocalization of glial fibrillary acidic protein (GFAP) in the pancreas. GFAP is expressed in rare cells in the human pancreas, probably in a subset of oval-shaped cells. G: immunolocalization of CD163. CD163 is localized in oval cells. H: immunolocalization of α-smooth muscle actin (αSMA) in the pancreas. αSMA is localized at the peripheral region of acini. Some of the pancreatic stellate cells with cytoplasmic protrusions (black arrows) are positive for αSMA. Note that none of the oval cells (most likely quiescent stellate cells) was positive for αSMA. Bars, 50 μm.
Fig. 3.
Fig. 3.
Coexpression of ZSCAN4 and differentiated cell markers in the adult human pancreas. A: a ZSCAN4+ cell is located among pancreatic duct cells, which are positive for the duct cell marker AQP1. Note that there is a ZSCAN4+ cell without immunoreactivity for AQP1 (*). B: a subpopulation of amylase-positive cells expresses ZSCAN4. These cells are located between adjacent acini. C: an oval-shaped single cell located between acini expresses both insulin and ZSCAN4. D: glucagon and ZSCAN4 expressions were completely overlapped in a few cells scattered in the adult human pancreas. Note that these cells are easily identifiable in DIC images. E: oval-shaped cells located between adjacent acini coexpress ZSCAN4. F: a subset of oval-shaped cells (putative quiescent pancreatic stellate cells) is positive for both ZSCAN4 and GFAP. G: ZSCAN4 is not colocalized with αSMA-positive fibrotic cells in the human pancreas. Bars, 25 μm.
Fig. 4.
Fig. 4.
Immunolocalization of stem/progenitor cell markers in the adult human pancreas. A: B lymphoma Mo-MLV insertion region 1 homolog (BMI1) is expressed in a subset of cells in every compartment of the pancreas, i.e., acinar cells (arrowhead), the islet of Langerhans, ducts, and oval cells (arrowhead). B: carbonic anhydrase II (CAII) is expressed in ducts from centroacinar cells to small-sized ducts. Note that CAII is also positive in oval cells. C: aldehyde dehydrogenase 1 (ALDH1) is localized in a subset of acinar cells and oval cells (white arrow). D: neurogenin 3 (NEUROG3) is not only localized in a subset of acinar cells but also in the islet of Langerhans and centroacinar cells. E: stage-specific embryonic antigen 3 (SSEA3) is exclusively expressed in a subset of oval cells. F: leucine-rich repeat-containing G protein-coupled receptor 5 (LGR5) is expressed in a subset of cells in every compartment of the pancreas as well as BMI1: acinar cells, the islet of Langerhans, duct and centroacinar cells, and oval cells. G: rare Doublecortin and CaM kinase-like-1 (DCAMKL-1)-positive cells are localized in a subset of pancreatic acinar cells, islets of Langerhans, and duct cells. Bars, 50 μm.
Fig. 5.
Fig. 5.
Coexpression of ZSCAN4 with stem/progenitor cell markers in the adult human pancreas. A: coexpression of ZSCAN4 and BMI1 is shown in an oval cell between acini. Note that these oval cells exist as a single cell or sometimes a cluster of a few cells. White dotted lines indicate the margin of acini. B: CAII is expressed in centroacinar cells and oval cells. ZSCAN4 is colocalized with CAII in an oval cell. C: ZSCAN4 and ALDH1 are colocalized in an oval cell between acini. D: NEUROG3 is colocalized with ZSCAN4 in a subpopulation of acinar cells. White dotted lines indicate the margin of pancreatic acini. E: both ZSCAN4 and SSEA3 are expressed in the same oval cells. F: coexpression of ZSCAN4 with LGR5 in an oval cell with some cytoplasmic protrusions. White dotted lines indicate the margin of pancreatic acini. G: DCAMKL-1 is colocalized with ZSCAN4 in the pancreas (white arrows). H: SSEA3-positive oval cells are also positive for CD163. Bars, 25 μm.
Fig. 6.
Fig. 6.
ZSCAN4 expression in pancreatic cells in adult human pancreas. A: colocalization of LGR5 and ZSCAN4 in the islet of Langerhans. LGR5+ cells are located at the peripheral region of the islet. Only a small portion of LGR5+ cells are positive for ZSCAN4 in the islet of Langerhans. White dashed line indicates the margin of the islet. B: LGR5 and BMI1 expression in the islet of Langerhans. Some of the LGR5+ cells are also positive for BMI1. C: ZSCAN4 is expressed in a mature AQP1+ cell apart from pancreatic ducts. Note that ZSCAN4+ cells are clearly identifiable in a DIC image. D: AQP1, amylase, and insulin (INS) triple-positive intermediate mixed exocrine and endocrine cells located in the interstitium of the human pancreas. Note that these cells are clearly identifiable in a DIC image. E: an insulin-positive cell in pancreatic acini. F: amylase and insulin double-positive oval-shaped cell is also positive for the undifferentiated endocrine cell marker NEUROG3. G: a BMI1 and AQP1 double-positive cell at the interstitium of the human pancreas. H: AQP1 and insulin double-positive oval cells also express SSEA3. Bars, 50 μm.
Fig. 7.
Fig. 7.
Dynamic control of ZSCAN4 expression in chronic pancreatitis under inflammation and regeneration. ZSCAN4 expression patterns in tissues taken from patients with chronic alcoholic pancreatitis and autoimmune pancreatitis. A: a representative ZSCAN4 immunostaining of the pancreas from an unaffected individual. Arrow, a ZSCAN4+ cell. B: a representative ZSCAN4 immunostaining of the pancreas from a patient with chronic alcoholic pancreatitis. ZSCAN4+ cells are scattered in the pancreas. A number of ZSCAN4+ cells in the islet of Langerhans are dramatically increased in sections of alcoholic pancreatitis. C: a representative ZSCAN4 immunostaining of the duct region of the pancreas from a patient with untreated autoimmune pancreatitis. ZSCAN4+ cells increase in tissues on chronic inflammation. Arrows, a small-sized pancreatic duct containing a few ZSCAN4+ cells. D: a representative ZSCAN4 immunostaining of the acinar region of the pancreas from a patient with untreated autoimmune pancreatitis. ZSCAN4+ cells are scattered throughout the pancreas with autoimmune pancreatitis. E: a representative ZSCAN4 immunostaining of the pancreas from a patient with autoimmune pancreatitis under the maintenance corticosteroid therapy for 3 mo. A large number of ZSCAN4+ cells are seen in pancreatic ducts, the islet of Langerhans, and regenerating pancreatic acinar cells. F: a representative ZSCAN4 immunostaining of the pancreas from a patient with autoimmune pancreatitis under the maintenance corticosteroid therapy for 12 mo. Note that immunoreactivity for ZSCAN4 almost disappeared and returned to a basal level at fully regenerated tissues. G: the no. of ZSCAN4+ cells per high-power field (HPF) in each condition is summarized (n = 3–5, mean ± SD). Normal subjects, 1.8 ± 1.3; alcoholic pancreatitis (AL-CP), 118.8 ± 97.0; autoimmune pancreatitis pretreatment (AIP pre), 193.0 ± 56.0; autoimmune pancreatitis 3 mo after the initiation of corticosteroid treatment (AIP 3m Tx), 208.7 ± 23.4; autoimmune pancreatitis 1 yr after the initiation of corticosteroid treatment (AIP 1y Tx), 0.6 ± 0.9. *P < 0.01 and **P < 0.05.
Fig. 8.
Fig. 8.
Increased expression of Zscan4 and other progenitor cell markers in mouse acute pancreatitis. All of the progenitor cell markers examined are upregulated in caerulein-induced mouse acute pancreatitis at day 1 (D1) but returned to the basal levels at as early as day 4 (D4) (see text). D0, control day. A: Zscan4; B: Bmi1; C: SSEA3; D: Neurog3; E: Lgr5. The no. of marker positive cells per HPF is summarized (mean ± SD, n = 3) in the panels on the right.
Fig. 9.
Fig. 9.
Summary diagram. A: summary diagram showing the expression of various markers in each compartment of adult human pancreas at the ZSCAN4 state and the ZSCAN4+ state. B: diagram showing the demonstrated oscillation between the Zscan4 state and the Zscan4+ state in mouse ES cells (redrawn from the Ref. 28).

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