inﬂammation increases cells expressing zscan4 and ... · inﬂammation increases cells expressing...

Inflammation increases cells expressing ZSCAN4 and progenitor cell markersin the adult pancreas

Shigeru B. H. Ko,1 Sakiko Azuma,2,3 Yukihiro Yokoyama,4 Akiko Yamamoto,5 Kazuhiro Kyokane,2

Shumpei Niida,3 Hiroshi Ishiguro,5 and Minoru S. H. Ko1,6

1Sakaguchi Laboratory, Department of Systems Medicine, Sakaguchi Laboratory, Keio University School of Medicine, Tokyo,Japan; 2Department of Gastroenterology, National Center for Geriatrics and Gerontology, Aichi, Japan; 3Laboratory ofGenomics and Proteomics, National Center for Geriatrics and Gerontology, Aichi, Japan; 4Department of Surgery, NagoyaUniversity Graduate School of Medicine, Aichi, Japan; 5Laboratory of Human Nutrition, Nagoya University Graduate Schoolof Medicine, Aichi, Japan; and 6Developmental Genomics and Aging Section, Laboratory of Genetics, National Institute onAging, National Institutes of Health, Baltimore, Maryland

Ko SB, Azuma S, Yokoyama Y, Yamamoto A, Kyokane K,Niida S, Ishiguro H, Ko MS. Inflammation increases cells expressingZSCAN4 and progenitor cell markers in the adult pancreas. Am JPhysiol Gastrointest Liver Physiol 304: G1103–G1116, 2013. Firstpublished April 18, 2013; doi:10.1152/ajpgi.00299.2012.—We haverecently identified the zinc finger and SCAN domain containing 4(Zscan4), which is transiently expressed and regulates telomere elon-gation and genome stability in mouse embryonic stem (ES) cells. Theaim of this study was to examine the expression of ZSCAN4 in theadult pancreas and elucidate the role of ZSCAN4 in tissue inflamma-tion and subsequent regeneration. The expression of ZSCAN4 andother progenitor or differentiated cell markers in the human pancreaswas immunohistochemically examined. Pancreas sections of alcoholicor autoimmune pancreatitis patients before and under maintenancecorticosteroid treatment were used in this study. In the adult humanpancreas a small number of ZSCAN4-positive (ZSCAN4�) cells arepresent among cells located in the islets of Langerhans, acini, ducts,and oval-shaped cells. These cells not only express differentiated cellmarkers for each compartment of the pancreas but also express othertissue stem/progenitor cell markers. Furthermore, the number ofZSCAN4� cells dramatically increased in patients with chronic pan-creatitis, especially in the pancreatic tissues of autoimmune pancre-atitis actively regenerating under corticosteroid treatment. Interest-ingly, a number of ZSCAN4� cells in the pancreas of autoimmunepancreatitis returned to the basal level after 1 yr of maintenancecorticosteroid treatment. In conclusion, coexpression of progenitorcell markers and differentiated cell markers with ZSCAN4 in eachcompartment of the pancreas may indicate the presence of facultativeprogenitors for both exocrine and endocrine cells in the adult pan-creas.

autoimmune pancreatitis; facultative progenitor cells; regeneration

AUTOIMMUNE PANCREATITIS IS a form of chronic pancreatitis char-acterized by diffuse swelling of the affected gland and a highserum immunoglobulin G type 4 concentration (19). In patientswith autoimmune pancreatitis, pancreatic exocrine (and alsomost of the time endocrine) function is severely damaged byinflammation. An anti-inflammatory regimen with corticoste-roids is widely used as a treatment for autoimmune pancreatitisto ameliorate the swelling of the pancreas and obstructivejaundice (10). In a previous study we have shown that, inpatients with autoimmune pancreatitis, corticosteroid restoresseverely damaged pancreatic tissues functionally and morpho-

logically, which is accompanied by the disappearance of in-flammatory cells and massive regeneration of pancreatic tis-sues (11). We have also shown that regeneration of exocrinecells paralleled the increase of immunoreactivity for CD133(prominin-1), one of the markers for hematopoietic (27), in-testinal (29), and pancreatic (20) progenitors. These findingslead us to the hypothesis that pancreatic progenitor cells resideeven in severely damaged autoimmune pancreatitis tissues andgive rise to mature differentiated cells in the adult humanpancreas after corticosteroid treatment (11).

Recently we have identified zinc finger and SCAN domaincontaining 4 (Zscan4), a gene that is specific in mammals,expressed exclusively in late two-cell stage mouse embryos(4), and transiently expressed in mouse embryonic stem (ES)cells (28). Zscan4 can extend telomeres and plays an essentialrole in the maintenance of genome stability. TransientZscan4 expression, or oscillation between the Zscan4-neg-ative (Zscan4�) and -positive (Zscan4�) state, is indispensablefor the maintenance of a normal karyotype, telomere length,and thus the immortality of ES cells (28). In addition, mouseZscan4 gene was shown to be a potent reprogramming factorfor the generation of induced pluripotent stem (iPS) cells (8).Considering the critical function of Zscan4 in ES cells, wehypothesized that ZSCAN4 may also be expressed transientlyin stem/progenitor cells even in the adult human pancreas.

Here we have identified ZSCAN4� cells in the adult humanand mouse pancreas by immunohistochemistry. ZSCAN4�

cells are present not only in pancreatic ducts, which are thesites of the previously identified pancreatic stem/progenitorpool (5, 9, 20, 21, 24), but also in the islets of Langerhans (6),exocrine acinar cells (22), and oval-shaped cells [putativequiescent pancreatic stellate cells (14)]. ZSCAN4 was colocal-ized with known progenitor cell markers in the pancreas andother organs such as polycomb group protein BMI1 (22),carbonic anhydrate II (CAII) (6, 9), neurogenin 3 (NEUROG3)(6), leucine-rich repeat-containing, G protein-coupled receptor5 (LGR5) (2), and Doublecortin and CaM kinase-like-1(DCAMKL-1) (16) and with known differentiated cell markersfor each compartment of the pancreas such as aquaporin 1 (12)for duct cells, amylase for acinar cells, pancreatic hormones forendocrine cells, and desmin and glial fibrillary acidic protein(GFAP) for pancreatic stellate cells (1). These findings indicatethe presence of rare facultative progenitor cells in both theexocrine and endocrine compartments of the human and mousepancreas.

Address for reprint requests and other correspondence: S. B. H. Ko, Dept. ofSystems Medicine, Sakaguchi Laboratory, Keio Univ. School of Medicine, 35Shinanomachi, Shinjuku, Tokyo 160-8582, Japan (e-mail: [email protected]).

Am J Physiol Gastrointest Liver Physiol 304: G1103–G1116, 2013.First published April 18, 2013; doi:10.1152/ajpgi.00299.2012.

0193-1857/13 Copyright © 2013 the American Physiological Societyhttp://www.ajpgi.org G1103

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Glossary

ALDH1 Aldehyde dehydrogenase 1�SMA �-smooth muscle actinAQP1 Aquaporin 1BMI1 B lymphoma Mo-MLV insertion region 1

homologCAII Carbonic anhydrase II

DCAMKL-1 Doublecortin and CaM kinase-like-1ES cells Embryonic stem cells

GFAP Glial fibrillary acidic proteinLGR5 Leucine-rich repeat-containing, G protein-

coupled receptor 5NEUROG3 Neurogenin 3

SSEA3 Stage-specific embryonic-antigen 3ZSCAN4 Zinc finger and SCAN domain containing 4

MATERIALS AND METHODS

Subjects. Surgically resected pancreatic tissues and pancreatic bi-opsy samples were used for immunohistochemical analyses. Pancre-atic tissues that were resected for the treatment of biliary carcinoma(n � 3) were used as controls, and tissues resected for the treatmentof chronic alcoholic pancreatitis (n � 3) were also used. Pancreasbiopsy samples from patients with autoimmune pancreatitis werereported previously (18). All pancreatic biopsies were performed toexclude malignancy, and written informed consent was obtained fromeach patient before the procedure. Under the visual guidance ofendoscopic ultrasonography (GF-UCT240; Olympus), pancreatic tis-sues were obtained from the body of the pancreas using a 19-gaugeTrucut biopsy needle (Wilson-Cook). Patients met the following 2006revised Japanese clinical diagnostic criteria for autoimmune pancre-atitis: diffuse swelling of the pancreas, irregular narrowing of the mainpancreatic duct, and a positive test for autoantibodies or a high

Table 1. List of antibodies used in this study

Antibodies Species Manufacturer Product ID Working Dilution

Anti-ALDH Rabbit Abcam ab52492 IHC 1:100IF 1:100

Anti-AQP1 Rabbit Alpha Diagnostic AQP11-A IHC 1:500IF 1:500

Anti-�-amylase Mouse Abcam ab54765 IHC 1:300IF 1:100

Anti-�SMA Mouse Abcam ab7817 IHC 1:100IF 1:100

Anti-BMI1 Mouse Millipore 05-637 IHC 1:200IF 1:100

Anti-CAII Rabbit Abcam ab6621 IHC 1:2,000IF 1:1,000

Anti-CD163 Mouse Leica NCL-CD163 IHC 1:300IF 1:100

Anti-DCAMKL-1 Rabbit Abcam ab37994 IHC 1:100IF 1:100

Anti-desmin Rabbit Abcam ab32362 IHC 1:500IF 1:200

Anti-GFAP Rabbit Abcam ab16997 IHC 1:100IF 1:100

Anti-glucagon Rabbit Abcam ab18461 IHC 1:5,000IF 1:1,000

Mouse Abcam ab10988 IHC 1:4,000IF 1:1,000

Anti-insulin Guinea pig Abcam ab7842 IHC 1:500IF 1:2,000

Mouse Abcam ab7760 IHC 1:3,000IF 1:2,000

Anti-LGR5 Rabbit Abcam ab75732 IHC 1:300IF 1:100

Anti-NEUROG3 Rabbit Abcam ab38548 IHC 1:100IF 1:100

Anti-SSEA3 Rat Abcam ab16286 IHC 1:100IF 1:100

Anti-mouse Zscan4 Rabbit NIH IHC 1:2,000IF 1:500

Anti-human ZSCAN4 Mouse Abnova H00201516-B01P IHC 1:200IF 1:100

Alexa Fluor 488 anti-mouse Goat Invitrogen A11017 IF 1:500Alexa Fluor 488 anti-rabbit Goat Invitrogen A11070 IF 1:500Alexa Fluor 488 anti-rat Chicken Invitrogen A21470 IF 1:500Alexa Fluor 594 anti-mouse Goat Invitrogen A11020 IF 1:500Alexa Fluor 594 anti-rabbit Goat Invitrogen A11072 IF 1:500Alexa Fluor 594 anti-guinea pig Goat Invitrogen A11076 IF 1:500

ALDH, aldehyde dehydrogenase 1; AQP1, aquaporin 1; �SMA, �-smooth mucle actin; BMI1, B lymphoma Mo-MLV insertion region 1 homolog; CAII,carbonic anhydrase II; DCAMKL-1, doublecortin and CaM kinase-like-1; GFAP, glial fibrillary acidic protein; LGR5, leucine-rich repeat-containing, Gprotein-coupled receptor 5; NEUROG3, neurogenin 3; SSEA3, stage-specific embryonic-antigen 3; ZSCAN4, zinc finger and SCAN domain containing 4; IHC,immunohistochemistry; IF, immunofluorescent.

G1104 PRESENCE OF ZSCAN4� CELLS IN ADULT HUMAN PANCREAS

AJP-Gastrointest Liver Physiol • doi:10.1152/ajpgi.00299.2012 • www.ajpgi.org

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immunoglobulin G (�1,800 mg/dl)/immunoglobulin G4 concentra-tion (�135 mg/dl). Among patients with autoimmune pancreatitis,three patients were subjected to a pancreatic biopsy at the followingthree different times: at the time of diagnosis, 3 mo after the initiationof corticosteroid treatment, and after 1 yr of maintenance corticoste-

roid treatment (11). A standard protocol for oral corticosteroids was asfollows: prednisolone at 30 mg/day for 1 wk as an initial dose, 20mg/day for a second week, 10 mg/day for four additional weeks, and5 mg/day as a maintenance dose all through the observation period(11). All of the procedures for obtaining human samples were ap-

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Fig. 1. Dynamic control of zinc finger and SCAN domaincontaining 4 (ZSCAN4) expression in adult human (h) pan-creas. A: ZSCAN4 expression at RNA levels in various humanorgans. Low level of ZSCAN4 expression was detected in allof the human tissues examined. B: immunolocalization ofZSCAN4 protein in adult human pancreas. A very smallnumber of cells showed strong nuclear and cytoplasmic stain-ing for ZSCAN4 in all of the major compartments: the islet ofLangerhans, acini, ducts, and oval-shaped cells (referred to asoval cells: putative quiescent pancreatic stellate cells). Immu-nostaining without a primary antibody against ZSCAN4 isshown as a negative control. C: immunolocalization of mouseZscan4 in the pancreas. Most of the cells are negative forZscan4, but a subset of cells in acini, ducts, the islet ofLangerhans are positive for mouse Zscan4. Oval-shaped cells(see text) are also positive for Zscan4. Note that oval-shapedcells between acini appear as a single cell or sometimes acluster of a few cells. D: an anti-mouse (m) Zscan4 antibodyand an anti-human ZSCAN4 antibody stained identical cells inthe adult human pancreas. E: an anti-mouse Zscan4 antibodyand an anti-human ZSCAN4 antibody stained identical cells ina subset of cells in the islet of Langerhans of mouse pancreas.DIC, differential interference contrast microscopy.

G1105PRESENCE OF ZSCAN4� CELLS IN ADULT HUMAN PANCREAS


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proved by the Institutional Ethics Committee of the Nagoya Univer-sity Hospital and the Aichi Cancer Center Hospital.

Animals. C57BL/6 mice were purchased from Japan SLC(Hamamatsu, Japan). Male mice at 8–10 wk were used for acutepancreatitis experiments. Experimental procedures for animal sampleswere approved by the Institutional Animal Care and Use Committee ofthe Nagoya University Graduate School of Medicine.

Caerulein-induced acute pancreatitis. Pancreatitis was induced byintraperitoneal injections of 50 �g/kg caerulein (Sigma, St. Louis,MO) in 0.9% NaCl. Controls received equal volumes of 0.9% NaClinjected intraperitoneally. The pancreas was removed from mice at 1day and 4 days after the beginning of six caerulein injections at hourlyintervals.

Immunohistochemistry and immunofluorescence. Both human andmouse pancreases were fixed in 10% formalin and embedded inparaffin. Embedded tissues were thin-sliced with a Leica microtome(Leica Microsystems, Wetzlar, Germany) at 5 �m. Sections weredeparaffinized, permeabilized, and used for immunohistochemicalanalyses. The numbers of immunopositive cells in both human andmouse pancreas were counted by two independent observers whowere blinded to the conditions.

Antibodies. Antibodies used in this study are summarized in Table 1.Antibodies were diluted according to the manufacturer’s recommen-

dation. For immunohistochemistry, immunoreactions were intensifiedusing Histofine Simple Stain MAX-PO reagent (Nichirei Biosci-ences, Tokyo, Japan). Immunolabeling was visualized using 3,3=-diaminobenzidinetetrahydrochloride as substrate for horseradish per-oxidase. Sections were counterstained with Mayer’s hematoxylin. Forimmunofluorescence, Alexa Fluor 488 (green)-, Alexa Fluor 596(red)-, or Alexa Fluor 350 (blue)-labeled secondary antibodies wereused for double or triple staining as appropriate. Immunolabelingswere microphotographed with Olympus fluorescence microscopy(AX80; Olympus, Tokyo, Japan). Cell nuclei were counterstainedwith Hoechst 33258.

RT-PCR analysis of ZSCAN4 expression in human tissues. Humanmultiple tissue cDNA panel (catalog no. 636742; Takara Bio, Shiga,Japan) was subjected to the analysis of ZSCAN4 expression at theRNA level. ZSCAN4-specific primers for PCR were designed basedon the nucleotide sequence of NCBI reference sequence no.NM_152677.2.

Materials. All of the reagents in molecular biology grade wereobtained from Sigma-Aldrich unless otherwise stated.

Statistical analysis. Statistical analysis was performed with theMann-Whitney test. Differences with a P value of �0.05 were consid-ered statistically significant. All values were expressed as means � SD.

PSC PSC oval

A AQP1

oval islet

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B amylase

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C insulin

D glucagon

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F GFAP

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G CD163

Fig. 2. Immunolocalization of differentiated cell markers in adult human pancreas. A: aquaporin 1 (AQP1) water channel is expressed in the plasma membraneof duct cells from centroacinar cells to the medium-sized ducts. Note that AQP1 is also expressed in pancreatic stellate cells (PSC) with cytoplasmic protrusionsand 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 subsetof 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 isletof 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 humanpancreatic ducts are also positive for endocrine hormones. E: immunolocalization of desmin in the pancreas. Desmin is expressed not only in a subset of ovalcells 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 isexpressed 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 �-smooth muscle actin (�SMA) in the pancreas. �SMA is localized at the peripheral region of acini. Some of the pancreatic stellatecells with cytoplasmic protrusions (black arrows) are positive for �SMA. Note that none of the oval cells (most likely quiescent stellate cells) was positive for�SMA. Bars, 50 �m.



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RESULTS

Presence of ZSCAN4� cells in adult human pancreas. Al-though public database searches did not reveal any human tissueswith high ZSCAN4 expression, RT-PCR analysis detected lowlevels of expression of ZSCAN4 in all the major organs exam-

ined, including the pancreas and placenta with relatively highexpression (Fig. 1A). We reasoned that such a low level ofexpression of ZSCAN4 can be explained by its transient expres-sion in a relatively small number of cells in adult tissues. Indeed,immunohistochemical analyses of the human pancreas with anti-bodies against human ZSCAN4 revealed that, although the ma-

merge amylase ZSCAN4 DIC

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Fig. 3. Coexpression of ZSCAN4 and differen-tiated cell markers in the adult human pancreas.A: a ZSCAN4� cell is located among pancreaticduct cells, which are positive for the duct cellmarker AQP1. Note that there is a ZSCAN4�

cell without immunoreactivity for AQP1 (*).B: a subpopulation of amylase-positive cellsexpresses ZSCAN4. These cells are located be-tween adjacent acini. C: an oval-shaped singlecell located between acini expresses both insulinand ZSCAN4. D: glucagon and ZSCAN4 ex-pressions were completely overlapped in a fewcells scattered in the adult human pancreas. Notethat these cells are easily identifiable in DICimages. E: oval-shaped cells located betweenadjacent acini coexpress ZSCAN4. F: a subsetof oval-shaped cells (putative quiescent pancre-atic stellate cells) is positive for both ZSCAN4and GFAP. G: ZSCAN4 is not colocalized with�SMA-positive fibrotic cells in the human pan-creas. Bars, 25 �m.



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jority of human pancreatic tissues were negative for ZSCAN4staining, a very small number of cells showed strong nuclear andcytoplasmic staining for ZSCAN4 in all the major compartments(i.e., the islets of Langerhans, acini, and ducts) of the adult humanpancreas (Fig. 1B) and mouse pancreas (Fig. 1C). As validationfor the human ZSCAN4 antibody, we confirmed that both anti-bodies against human ZSCAN4 and mouse Zscan4 used in ourearlier studies (28) marked almost identical cells on either human(Fig. 1D) or mouse pancreas (Fig. 1E) sections.

ZSCAN4� cells express differentiated cell markers in thepancreas. These ZSCAN4� cells also expressed markers ofdifferentiated cells specific to cells in each compartment: the

expression of a duct marker, AQP1, was detected in ZSCAN4�

cells in the duct (Figs. 2A and 3A); the expression of an acinarmarker, amylase, in ZSCAN4� cells in the acinar cells (Figs.2B and 3B); the expression of endocrine markers, insulin (Figs.2C and 3C) and glucagon (Figs. 2D and 3D), in ZSCAN4�

cells in the islet; and the expression of pancreatic stellate cellmarkers, desmin (Figs. 2E and 3E), GFAP (Figs. 2F and 3F),and CD163 (Fig. 2G), but not �-smooth muscle actin (Figs. 2Hand 3G), in ZSCAN4� cells in the subset of oval-shaped cells(see below for the details). Our earlier work showed thatundifferentiated ES cells oscillate between the Zscan4� stateand the Zscan4� state (28). Therefore, these data suggest that

acini

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Fig. 4. Immunolocalization of stem/progenitor cell markers in the adult human pancreas. A: B lymphoma Mo-MLV insertion region 1 homolog (BMI1) isexpressed 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: aldehydedehydrogenase 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 subsetof 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 ofoval 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 wellas BMI1: acinar cells, the islet of Langerhans, duct and centroacinar cells, and oval cells. G: rare Doublecortin and CaM kinase-like-1 (DCAMKL-1)-positivecells are localized in a subset of pancreatic acinar cells, islets of Langerhans, and duct cells. Bars, 50 �m.



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these differentiated cells in each compartment also oscillatebetween the ZSCAN4� state and ZSCAN4� state while re-taining their differentiated cell markers. The extremely rarepresence of these ZSCAN4� cells in the adult human pancreasindicates that the differentiated cells rarely take the ZSCAN4�

state, as is also the case in mouse ES cells.

ZSCAN4� cells coexpress stem/progenitor cell markers. Tofurther characterize these rare ZSCAN4� cells in the pancreas,we examined the expression of well-established stem/progen-itor markers for each compartment of the human and mousepancreas: polycomb ring finger oncogene BMI1 for the stem/progenitor cells in acinar cells, CAII and aldehyde dehydroge-

ZSCAN4

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Fig. 5. Coexpression of ZSCAN4 with stem/progenitor cellmarkers in the adult human pancreas. A: coexpression ofZSCAN4 and BMI1 is shown in an oval cell between acini.Note that these oval cells exist as a single cell or sometimes acluster of a few cells. White dotted lines indicate the margin ofacini. B: CAII is expressed in centroacinar cells and oval cells.ZSCAN4 is colocalized with CAII in an oval cell. C: ZSCAN4and ALDH1 are colocalized in an oval cell between acini.D: NEUROG3 is colocalized with ZSCAN4 in a subpopulationof acinar cells. White dotted lines indicate the margin ofpancreatic acini. E: both ZSCAN4 and SSEA3 are expressed inthe same oval cells. F: coexpression of ZSCAN4 with LGR5 inan oval cell with some cytoplasmic protrusions. White dottedlines indicate the margin of pancreatic acini. G: DCAMKL-1is colocalized with ZSCAN4 in the pancreas (white arrows).H: SSEA3-positive oval cells are also positive for CD163.Bars, 25 �m.



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nase 1 (ALDH1) for duct progenitors, and NEUROG3 forendocrine progenitors (6, 25). We found that rare ZSCAN4�

cells and rare cells marked with the respective stem/progenitormarkers largely overlapped in each compartment of the pan-creas (Figs. 4 and 5). Interestingly, we found cells marked with

BMI1 (Figs. 4A and 5A), as well as LGR5 (Figs. 4F and 5F),a marker for tissue stem cells in the mouse small intestine,colon, and skin (2), were present not only in acinar cells butalso in a subset of all the cell types in the pancreas, i.e., isletand ducts. Moreover, we found that DCAMKL-1, a recently

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Fig. 6. ZSCAN4 expression in pancreatic cells in adult human pancreas. A: colocalization of LGR5 and ZSCAN4 in the islet of Langerhans. LGR5� cells arelocated 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 indicatesthe 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 expressedin 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 identifiablein 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 undifferentiatedendocrine cell marker NEUROG3. G: a BMI1 and AQP1 double-positive cell at the interstitium of the human pancreas. H: AQP1 and insulin double-positiveoval cells also express SSEA3. Bars, 50 �m.



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identified intestinal (15) and pancreatic (16) stem/progenitorcell marker, was also expressed in very rare cells in a subset ofboth exocrine and endocrine pancreatic cells (Fig. 4G) and wascoexpressed with ZSCAN4 (Fig. 5G). The population of rarecells marked with BMI1� and LGR5� was largely overlapped,and a fraction (�10%) of BMI1�/LGR5� cells was alsoZSCAN4� (Fig. 6, A and B). The expression patterns of markergenes in each cell type are summarized in Table 2. Takentogether, coexpression of ZSCAN4 and progenitor cell markersof each compartment (i.e., CAII in Figs. 4A and 5B, ALDH1 inFigs. 4C and 5C, and NEUROG3 in Figs. 4D and 5D) as wellas across compartments (i.e., BMI1 in Fig. 4A, LGR5 in Fig.4F, and DCAMKL-1 in Fig. 4G) suggests that Zscan4 marks aportion of pancreatic cells that have been previously ascribedas progenitor cells (7, 9, 14, 17, 21, 23, 25, 26).

A subset of oval-shaped cells express ZSCAN4 along withprogenitor cell markers and differentiated cell markers.

In addition to cells in the duct, acini, or islet, we noticed thatthe expression of ZSCAN4 was detected in oval-shaped cells(tentatively referred to as oval cells), which exist as a singlecell or sometimes a cluster of a few cells between adjacentpancreatic acini (Fig. 1, B and C). In the human pancreas, theoval cells seemed to be the only cell type that could be stainedwith a SSEA3 antibody (Fig. 4E), a marker for adult humanstem cells recently identified in skin fibroblasts and bonemarrow stromal cells (13). In addition, these oval cells werealso positive for CD163 (Fig. 2G), a marker for monocytes andmacrophages. From their location, cell morphology, and theexpression of desmin (1, 3) (Figs. 2E and 3E) and GFAP (Figs.2F and 3F), oval cells are most likely a quiescent form ofpancreatic stellate cells (3). It is worth pointing out that only asmall portion of oval cells were ZSCAN4�. Furthermore,pancreatic stellate cells of the typical morphology with protru-sions, so-called “activated pancreatic stellate cells (8),” werenot stained with either ZSCAN4 (Fig. 1B) or SSEA3 (Fig. 4E).Interestingly, these rare ZSCAN4� oval cells expressed bothdifferentiated and undifferentiated cell markers such as AQP1(Fig. 6, C, D, F, and G), amylase (Figs. 3B, 6D, and 6E),insulin (Figs. 3C, 6D, 6E, and 6G), glucagon (Fig. 3D), BMI1(Figs. 5A and 6F), SSEA3 (Figs. 5E and 6G), and LGR5 (Fig.5F). We note that a possibility of bone marrow origin of theZSCAN4� oval cells cannot be excluded at this point, sincethey expressed both SSEA3 and CD163 (Fig. 5G). This notionmay be consistent with previous reports that at least some ofthe pancreatic stellate cells originate from bone marrow (seeRefs. 14 or 26 for review).

Chronic inflammation increases the number of ZSCAN4�

cells in adult human pancreas. We further noticed thatZSCAN4 expression levels varied among tissues from differentsubjects. In sharp contrast to the normal pancreas of unaffectedindividuals, where ZSCAN4� cells were very rare and ratherdifficult to spot (Fig. 7A), a dramatic increase of ZSCAN4expression was observed in tissues with chronic alcoholicpancreatitis (Fig. 7B) and autoimmune pancreatitis (Fig. 7, Cand D). Such an increase of ZSCAN4� cells was particularlynotable in some areas (the acinar cells and the islet) that appearto be intact among the tissues damaged severely by alcoholicpancreatitis (Fig. 7B). ZSCAN4� cells were even more abun-dant in the pancreatic tissues with autoimmune pancreatitis,where pancreatic cells were actively regenerating after a 3-mocorticosteroid treatment (Fig. 7E). Interestingly, the number ofZSCAN4� cells returned to a basal level after 1 yr of mainte-nance corticosteroid treatment for autoimmune pancreatitis(Fig. 7F). To assess these changes more quantitatively, wecounted the number of ZSCAN4� cells per high-power field ineach condition (Fig. 7G). These data indicate that the inflam-mation, destruction, and restoration of pancreatic tissues areaccompanied with the increase of ZSCAN4� cells, suggestingthe involvement of the ZSCAN4� state in tissue damage(inflammatory insult) and subsequent regeneration (differenti-ation).

Acute inflammation increases the number of ZSCAN4� cellsin mouse caerulein pancreatitis. To experimentally validateour observations in human alcoholic chronic pancreatitis andautoimmune pancreatitis, we used a well-established caerulein-induced acute pancreatitis model in mouse. In control mice, thenumber of Zscan4� cells was very low, as expected (Fig. 8A).The number of Zscan4� cells dramatically increased 1 dayafter caerulein injections six times at hourly intervals, butreturned to the basal level as early as the 4th day after theinjection. Essentially, the same trends were observed forBMI1, SSEA3, NEUROG3, and LGR5 (Fig. 8, B-E). The rapidincrease and subsequent decrease of Zscan4� cells withoutnoticeable structural changes indicate that the increase ofZscan4� cells was not caused by the proliferation of Zscan4�

cells but rather caused by the rapid upregulation of Zscan4expression, which is accompanied with the rapid upregulationof other progenitor markers. These observations in mouseacute pancreatitis are quite consistent with the finding thatZSCAN4 in the human pancreas is remarkably upregulated inchronic inflammation irrespective of etiology.

Table 2. Summary of marker-gene expression patterns of cells in adult human pancreas

Stem/Progenitor Cell Markers Differentiated Cell Markers

ZSCAN4 BMI1 CAII ALDH1 NEUROG3 SSEA3 DCAMKL-1 LGR5 AQP1 Desmin GFAP CD163 Amylase Insulin Glucagon

Acinar cells Z� � � � � � � � � � � � � � �Z� � � � � � � � � � � � � � �

Duct cells Z� � � � � � � � � � � � � � �Z� � � � � � � � � � � � � � �

Endocrine cells Z� � � � � � � � � � � � � � �Z� � � � � � � � � � � � � � �

Stellate cells Z� � � � � � � � � � � � � � �Oval cells Z� � � � � � � � � � � � � � �

Z� � � � � � � � � � � � � � �



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DISCUSSION

In the current study, we have identified a rare population ofcells that express ZSCAN4 in adult human pancreas. We havealso found that the number of ZSCAN4� cells dramaticallyincreases during inflammation associated with alcoholic orautoimmune pancreatitis, which is subsequently reduced to thebasal level after the regeneration of pancreatic tissues and therestoration of their function by corticosteroid hormone treat-

ment. An increase of ZSCAN4� cells by inflammation has alsobeen confirmed by using experimental pancreatitis models inmouse. It is worth mentioning that the current study is indeedthe first report of the expression of human ZSCAN4 in anyhuman cells and tissues. Considering the demonstrated func-tion of Zscan4 in mouse ES cells (28) and during reprogram-ming (8), it is tempting to consider that ZSCAN4� cells in theadult human pancreas may undergo similar processes, such as

A

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Fig. 7. Dynamic control of ZSCAN4 expression in chronic pancreatitis under inflammation and regeneration. ZSCAN4 expression patterns in tissues taken frompatients with chronic alcoholic pancreatitis and autoimmune pancreatitis. A: a representative ZSCAN4 immunostaining of the pancreas from an unaffectedindividual. 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 increasein tissues on chronic inflammation. Arrows, a small-sized pancreatic duct containing a few ZSCAN4� cells. D: a representative ZSCAN4 immunostaining ofthe acinar region of the pancreas from a patient with untreated autoimmune pancreatitis. ZSCAN4� cells are scattered throughout the pancreas with autoimmunepancreatitis. E: a representative ZSCAN4 immunostaining of the pancreas from a patient with autoimmune pancreatitis under the maintenance corticosteroidtherapy for 3 mo. A large number of ZSCAN4� cells are seen in pancreatic ducts, the islet of Langerhans, and regenerating pancreatic acinar cells. F: arepresentative 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 perhigh-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.



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telomere elongation (28) and epigenetic reprogramming (8).However, such a possibility remains to be investigated.

Although the current study is limited primarily to the de-scriptive observational study of human pancreas in normal anddiseased states as well as mouse-induced pancreatitis, thedetailed immunohistochemical studies have revealed the pres-ence of rare and unique populations of cells that coexpressZSCAN4, stem/progenitor markers, and markers specific to thedifferentiated cells in distinct compartments of pancreas: ducts,acini, islets of Langerhans, and oval cells. Considering all thedata together, we propose that cells in each compartment (e.g.,acinar cells), which usually do not express ZSCAN4, infre-quently transition to the ZSCAN4� state while retaining theexpression of specific markers (e.g., amylase) (Fig. 9A). The

transition of differentiated pancreatic cells to the ZSCAN4�

state can be stimulated by inflammation (Fig. 9A). Importantly,this model is consistent with unique expression patterns ofmouse Zscan4 in mouse ES cells: ES cells oscillate betweenthe Zscan4� state and Zscan4� state, resulting in the mixedpopulation of �95% Zscan4� cells and �5% Zscan4� cells inundifferentiated ES cell cultures (Fig. 9B) (28). Interestingly,when the differentiated cells in each compartment infrequentlybecome ZSCAN4�, these cells also express stem/progenitorcell markers, such as BMI1 and LGR5 (Fig. 9A). The coex-pression of stem/progenitor cell markers and differentiationmarkers in the same cell suggests that there are no distinctstem/progenitor cell pools that are set aside from the differen-tiated cells in the pancreas but does suggest the existence of

D0 Pancreatitis D1 Pancreatitis D4

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Fig. 8. Increased expression of Zscan4 and other progenitor cell markers in mouse acute pancreatitis. All of the progenitor cell markers examined are upregulatedin 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.



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differentiated cells that transiently possess a stem/progenitorcell character (Fig. 9A). The infrequent presence of ZSCAN4�

cells in the unaffected pancreatic tissues is also consistent withthe notion that the pancreas is an organ that does not haveactive tissue turnover/regeneration without inflammation (4).

The model shown in Fig. 9A has been further extended bythe following additional intriguing observation: when the dif-ferentiated cells in each compartment become ZSCAN4�,these cells express not only stem/progenitor cells markers butalso differentiation markers of other cell types (Fig. 9A). Forexample, the expression of insulin was detected in ZSCAN4�

acinar cells, duct cells, and oval cells; the expression ofamylase was detected in ZSCAN4� in duct cells and oval cells.These data suggest that these ZSCAN4� cells may be able totake phenotypes that are different from the original cell typesand further suggest that these mixed endocrine-exocrine cellsare progenitors for each compartment of adult human pancreas.

These data seem to be consistent with previous reports: theexistence of intermediate endocrine-acinar pancreatic cells inthe pancreatic duct ligation condition (5); the commonality ofthe origin of exocrine and endocrine cells in the pancreas ortransdifferentiation from exocrine cells to insulin-positive en-docrine cells in a specific condition (12, 26, 41). Overall, ourfindings are consistent with the notion that facultative stem/progenitor cells (40) are present in the pancreas. Althoughfurther study will be required to obtain evidence that theseZSCAN4� cells can indeed differentiate to mature exocrineand endocrine cells in vivo and in vitro, the concept offacultative stem/progenitor cells is not only consistent with ourdata but may also help to explain some of the controversialissues regarding the stem/progenitor cells in adult pancreas: thepresence of functional stem/progenitor cells that reside in eachcompartment [i.e., duct cells (10, 12, 16, 29, 31, 32), acinarcells (14, 26, 30, 33, 39, 44), endocrine cells (34, 35), and

A human pancreas

BMI1-LGR5-SSEA3-NGN3-

Amylase+AQP1-Insulin-Desmin-

Inflammationregeneration

Acinarcells

ZSCAN4(-)

Ductcells

ZSCAN4(-)

Endocrinecells

ZSCAN4(-)

Oval cellsZSCAN4(-)

Acinarcells

ZSCAN4(+)

Ductcells

ZSCAN4(+)

Endocrinecells

ZSCAN4(+)

Ovalcells

ZSCAN4(+)


Amylase-AQP1+Insulin-Desmin-


Amylase-AQP1-Insulin+Desmin-

BMI1-LGR5-SSEA3-NGN3-GFAP-

Amylase-AQP1+Insulin-Desmin+CD163+

BMI1+LGR5+SSEA3-NGN3+

Amylase+AQP1-Insulin+Desmin-

BMI1+LGR5+SSEA3-NGN3-

Amylase-AQP1+Insulin+Desmin-

BMI1+LGR5+SSEA3-NGN3+

Amylase-AQP1-Insulin+Desmin-

BMI1+LGR5+SSEA3+NGN3+GFAP+

Amylase+AQP1+Insulin+Desmin+CD163+

B mouse ES cells

~95% ~5%

Telomereelongation

EScells

ZSCAN4(-)

EScells

ZSCAN4(+)

Fig. 9. Summary diagram. A: summary diagram show-ing the expression of various markers in each compart-ment of adult human pancreas at the ZSCAN4� stateand the ZSCAN4� state. B: diagram showing thedemonstrated oscillation between the Zscan4� stateand the Zscan4� state in mouse ES cells (redrawnfrom the Ref. 28).



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pancreatic stellate cells (23)] of the adult human and mousepancreas.

A possibility of ZSCAN4� cells being facultative stem/progenitor cells may also have important implications for thetreatment of type 1 diabetes, which is caused by a specificimmunological destruction of pancreatic -cells. Major effortsand progress have been made to produce functional -cells fortransplantation therapy (1) from human ES cells (7, 17, 21) andiPS cells (2, 37) in vitro. It has also been reported that maturedifferentiated pancreatic acinar cells were successfully repro-grammed to insulin-secreting -cells in vivo by the forcedexpression of several transcription factors with adenoviruses(45). However, the production of sufficient quantity of maturepancreatic -cells has not been reported. Considering thepresence of a rare population of ZSCAN4� cells in eachcompartment of the pancreas, use of these cells as alternativesources for producing -cells might be an attractive option forfurther study.

ACKNOWLEDGMENTS

We thank Y. Sawa and K. Kamimura (Nagoya University) for technicalassistance and N. Mizuno (Aichi Cancer Center Hospital) for providingautoimmune pancreatitis sections.

GRANTS

This work was supported in part by grants from the Japanese Ministry ofEducation, Culture, Sports, Science, and Technology; the Ministry of Health,Labor and Welfare for intractable pancreatic diseases; Nagono Medical foun-dation; and Japan Medical Association. This research was supported in part bythe Intramural Research Program of the National Institute on Aging.

DISCLOSURES

None of the authors have any conflicts of interest.

AUTHOR CONTRIBUTIONS

Author contributions: S.B.K. and M.S.K. conception and design of re-search; S.B.K., S.A., Y.Y., A.Y., K.K., S.N., and H.I. performed experiments;S.B.K. and M.S.K. analyzed data; S.B.K. and M.S.K. interpreted results ofexperiments; S.B.K. prepared figures; S.B.K. drafted manuscript; S.B.K. andM.S.K. edited and revised manuscript; S.B.K., S.A., Y.Y., A.Y., K.K., S.N.,H.I., and M.S.K. approved final version of manuscript.

REFERENCES

1. Apte MV, Haber PS, Applegate TL, Norton ID, McCaughan GW,Korsten MA, Pirola RC, Wilson JS. Periacinar stellate shaped cells in ratpancreas: identification, isolation, and culture. Gut 43: 128–133, 1998.

2. Barker N, Clevers H. Leucine-rich repeat-containing G-protein-coupledreceptors as markers of adult stem cells. Gastroenterology 138: 1681–1696, 2010.

3. Erkan M, Adler G, Apte MV, Bachem MG, Buchholz M, Detlefsen S,Esposito I, Friess H, Gress TM, Habisch HJ, Hwang RF, Jaster R,Kleeff J, Kloppel G, Kordes C, Logsdon CD, Masamune A, MichalskiCW, Oh J, Phillips PA, Pinzani M, Reiser-Erkan C, Tsukamoto H,Wilson J. StellaTUM: current consensus and discussion on pancreaticstellate cell research. Gut 61: 172–178, 2012.

4. Falco G, Lee SL, Stanghellini I, Bassey UC, Hamatani T, Ko MS.Zscan4: a novel gene expressed exclusively in late 2-cell embryos andembryonic stem cells. Dev Biol 307: 539–550, 2007.

5. Furuyama K, Kawaguchi Y, Akiyama H, Horiguchi M, Kodama S,Kuhara T, Hosokawa S, Elbahrawy A, Soeda T, Koizumi M, Masui T,Kawaguchi M, Takaori K, Doi R, Nishi E, Kakinoki R, Deng JM,Behringer RR, Nakamura T, Uemoto S. Continuous cell supply from aSox9-expressing progenitor zone in adult liver, exocrine pancreas andintestine. Nat Genet 43: 34–41, 2011.

6. Gu G, Dubauskaite J, Melton DA. Direct evidence for the pancreaticlineage: NGN3� cells are islet progenitors and are distinct from ductprogenitors. Development 129: 2447–2457, 2002.

7. Hao E, Tyrberg B, Itkin-Ansari P, Lakey JR, Geron I, Monosov EZ,Barcova M, Mercola M, Levine F. Beta-cell differentiation from non-endocrine epithelial cells of the adult human pancreas. Nat Med 12:310–316, 2006.

8. Hirata T, Amano T, Nakatake Y, Amano M, Piao Y, Hoang HG, KoMS. Zscan4 transiently reactivates early embryonic genes during thegeneration of induced pluripotent stem cells (Abstract). Sci Rep 2: 208,2012.

9. Inada A, Nienaber C, Katsuta H, Fujitani Y, Levine J, Morita R,Sharma A, Bonner-Weir S. Carbonic anhydrase II-positive pancreaticcells are progenitors for both endocrine and exocrine pancreas after birth.Proc Natl Acad Sci USA 105: 19915–19919, 2008.

10. Kamisawa T, Shimosegawa T, Okazaki K, Nishino T, Watanabe H,Kanno A, Okumura F, Nishikawa T, Kobayashi K, Ichiya T, TakatoriH, Yamakita K, Kubota K, Hamano H, Okamura K, Hirano K, Ito T,Ko SB, Omata M. Standard steroid treatment for autoimmune pancreati-tis. Gut 58: 1504–1507, 2009.

11. Ko SB, Mizuno N, Yatabe Y, Yoshikawa T, Ishiguro H, Yamamoto A,Azuma S, Naruse S, Yamao K, Muallem S, Goto H. Corticosteroidscorrect aberrant CFTR localization in the duct and regenerate acinar cellsin autoimmune pancreatitis. Gastroenterology 138: 1988–1996, 2010.

12. Ko SB, Naruse S, Kitagawa M, Ishiguro H, Furuya S, Mizuno N,Wang Y, Yoshikawa T, Suzuki A, Shimano S, Hayakawa T. Aqua-porins in rat pancreatic interlobular ducts. Am J Physiol Gastrointest LiverPhysiol 282: G324–G331, 2002.

13. Kuroda Y, Kitada M, Wakao S, Nishikawa K, Tanimura Y, Maki-noshima H, Goda M, Akashi H, Inutsuka A, Niwa A, Shigemoto T,Nabeshima Y, Nakahata T, Fujiyoshi Y, Dezawa M. Unique multipo-tent cells in adult human mesenchymal cell populations. Proc Natl AcadSci USA 107: 8639–8643, 2010.

14. Mato E, Lucas M, Petriz J, Gomis R, Novials A. Identification of apancreatic stellate cell population with properties of progenitor cells: newrole for stellate cells in the pancreas. Biochem J 421: 181–191, 2009.

15. May R, Sureban SM, Hoang N, Riehl TE, Lightfoot SA, RamanujamR, Wyche JH, Anant S, Houchen CW. Doublecortin and CaM kinase-like-1 and leucine-rich-repeat-containing G-protein-coupled receptor markquiescent and cycling intestinal stem cells, respectively. Stem Cells 27:2571–2579, 2009.

16. May R, Sureban SM, Lightfoot SA, Hoskins AB, Brackett DJ, PostierRG, Ramanujam R, Rao CV, Wyche JH, Anant S, Houchen CW.Identification of a novel putative pancreatic stem/progenitor cell markerDCAMKL-1 in normal mouse pancreas. Am J Physiol Gastrointest LiverPhysiol 299: G303–G310, 2010.

17. Minami K, Okuno M, Miyawaki K, Okumachi A, Ishizaki K, OyamaK, Kawaguchi M, Ishizuka N, Iwanaga T, Seino S. Lineage tracing andcharacterization of insulin-secreting cells generated from adult pancreaticacinar cells. Proc Natl Acad Sci USA 102: 15116–15121, 2005.

18. Mizuno N, Bhatia V, Hosoda W, Sawaki A, Hoki N, Hara K, TakagiT, Ko SB, Yatabe Y, Goto H, Yamao K. Histological diagnosis ofautoimmune pancreatitis using EUS-guided trucut biopsy: a comparisonstudy with EUS-FNA. J Gastroenterol 44: 742–750, 2009.

19. Okazaki K, Kawa S, Kamisawa T, Shimosegawa T, Tanaka M.Japanese consensus guidelines for management of autoimmune pancreati-tis: I. Concept and diagnosis of autoimmune pancreatitis. J Gastroenterol45: 249–265, 2010.

20. Oshima Y, Suzuki A, Kawashimo K, Ishikawa M, Ohkohchi N,Taniguchi H. Isolation of mouse pancreatic ductal progenitor cells ex-pressing CD133 and c-Met by flow cytometric cell sorting. Gastroenter-ology 132: 720–732, 2007.

21. Rovira M, Scott SG, Liss AS, Jensen J, Thayer SP, Leach SD. Isolationand characterization of centroacinar/terminal ductal progenitor cells inadult mouse pancreas. Proc Natl Acad Sci USA 107: 75–80, 2010.

22. Sangiorgi E, Capecchi MR. Bmi1 lineage tracing identifies a self-renewing pancreatic acinar cell subpopulation capable of maintainingpancreatic organ homeostasis. Proc Natl Acad Sci USA 106: 7101–7106,2009.

23. Smukler SR, Arntfield ME, Razavi R, Bikopoulos G, Karpowicz P,Seaberg R, Dai F, Lee S, Ahrens R, Fraser PE, Wheeler MB, van derKooy D. The adult mouse and human pancreas contain rare multipotentstem cells that express insulin. Cell Stem Cell 8: 281–293, 2011.

24. Solar M, Cardalda C, Houbracken I, Martin M, Maestro MA, DeMedts N, Xu X, Grau V, Heimberg H, Bouwens L, Ferrer J. Pancreaticexocrine duct cells give rise to insulin-producing beta cells during em-bryogenesis but not after birth. Dev Cell 17: 849–860, 2009.



by 10.220.33.5 on August 28, 2017


ownloaded from


25. Xu X, D’Hoker J, Stange G, Bonne S, De Leu N, Xiao X, Van deCasteele M, Mellitzer G, Ling Z, Pipeleers D, Bouwens L, ScharfmannR, Gradwohl G, Heimberg H. Beta cells can be generated from endog-enous progenitors in injured adult mouse pancreas. Cell 132: 197–207,2008.

26. Yatoh S, Dodge R, Akashi T, Omer A, Sharma A, Weir GC, Bonner-Weir S. Differentiation of affinity-purified human pancreatic duct cells tobeta-cells. Diabetes 56: 1802–1809, 2007.

27. Yin AH, Miraglia S, Zanjani ED, Almeida-Porada G, Ogawa M,Leary AG, Olweus J, Kearney J, Buck DW. AC133, a novel marker for

human hematopoietic stem and progenitor cells. Blood 90: 5002–5012,1997.

28. Zalzman M, Falco G, Sharova LV, Nishiyama A, Thomas M, Lee SL,Stagg CA, Hoang HG, Yang HT, Indig FE, Wersto RP, Ko MS. Zscan4regulates telomere elongation and genomic stability in ES cells. Nature464: 858–863, 2010.

29. Zhu L, Gibson P, Currle DS, Tong Y, Richardson RJ, Bayazitov IT,Poppleton H, Zakharenko S, Ellison DW, Gilbertson RJ. Prominin 1marks intestinal stem cells that are susceptible to neoplastic transforma-tion. Nature 457: 603–607, 2009.



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