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Volume 57(12): 1149–1158, 2009. Journal of ... Maho Kodama, Katsura Tsukamoto, Kimiko Yoshida, Kazunori Aoki, Shiro Kanegasaki, ...... J Clin Invest 117:2553–2561 ... Stanger BZ, Tanaka AJ, Melton DA (2007) Organ size is limited by.

Volume 57(12): 1149–1158, 2009 Journal of Histochemistry & Cytochemistry

http://www.jhc.org ARTICLE

The Journal of Histochemistry & Cytochemistry

Embryonic Stem Cell Transplantation Correlates With Endogenous Neurogenin 3 Expression and Pancreas Regeneration in Streptozotocin-injured Mice Maho Kodama, Katsura Tsukamoto, Kimiko Yoshida, Kazunori Aoki, Shiro Kanegasaki, and Gary Quinn Section for Studies on Metastasis (MK,GQ) and Genetics Division (KY,KA), National Cancer Center Research Institute, Tokyo, Japan; Sanwa Kagaku Kenkyusho Co., Ltd., Research Management Department, Mie, Japan (KT); and Central Laboratory, Effector Cell Institute, Inc., Tokyo, Japan (SK,GQ)

S U M M A R Y Pancreatic b cell regeneration remains poorly understood, yet stimulation of adult b cell neogenesis could lead to therapies for type 1 and type 2 diabetes. We studied the effect of embryonic stem (ES) cell transplantation on pancreas regeneration following b cell injury. Female Balb/c nude mice were treated with streptozotocin to induce hyperglycemia and received an ES cell transplant 24 hr later beneath the renal capsule. Transplantation of ES cells prevented hyperglycemia in a subset of mice, maintaining euglycemia and mild glucose tolerance up to 5 weeks. Pancreata of euglycemic mice showed histological evidence of b cell regeneration and expression of pancreas and duodenum transcription factor-1 (PDX-1) and neurogenin 3 (Ngn3) in ductal epithelium. Cell tracing analysis indicated that significant b cell neogenesis from progenitor cells occurred between 2 to 3 weeks following injury in ES cell–transplanted mice but not in sham-transplanted animals. Significantly, whereas pancreas-localized ES cells or their derivatives were adjacent to sites of regeneration, neogenic pancreatic epithelia, including Ngn31 cells, were endogenous. In conclusion, transplanted ES cells can migrate to the injured pancreas. Transplantation is associated with enhanced endogenous regeneration characterized by expression of Ngn3 and increased b cell differentiation from endogenous progenitor cells. This manuscript contains online supplemental material at http://www.jhc.org. Please visit this article online to view these materials. (J Histochem Cytochem 57:1149–1158, 2009)

THERE IS STILL REMARKABLE MYSTERY surrounding the mechanisms that govern adult pancreas regeneration and the promoting/limiting factors (Levine and ItkinAnsari 2008). What is known from studies reported to date is that robust b cell regeneration does not normally manifest in diabetic humans (Menge et al. 2008) or rodents (Bonner-Weir et al. 1981) following injury in the pancreas. However there is evidence for some degree of b cell regeneration in both species, even in old age, during the early diabetic state (Meier et al. 2006; Nir et al. 2007). This might be amenable to safe Correspondence to: Gary Quinn, PhD, Central Laboratory, Effector Cell Institute, Inc., 4-7-7 Aobadai, Meguro-ku, Tokyo 153-0042, Japan. E-mail: [email protected] Received for publication April 27, 2009; accepted August 14, 2009 [DOI: 10.1369/jhc.2009.954206]. C The Histochemical Society, Inc.


KEY WORDS pancreas regeneration diabetes neurogenin 3 PDX-1 pancreatic islets embryonic stem cells streptozotocin islet pancreatic b cells cell transplantation

enhancement using cell or pharmacological therapy, if the mechanisms could be identified. Hence, it may be possible to uncover a therapeutic b cell regenerative responsiveness by intervention in the early phase of b cell loss in diabetes sufferers (Rood et al. 2006). Countering this hope, though, is the possibility that the number of available stem cells in the adult pancreas, unlike the liver, capable of rebuilding lost tissue is limited (Stanger et al. 2007). Some regeneration models suggest that there are substantial facultative stem cells, duct cells, present in the pancreas (Wang et al. 1995), and duct cell metaplasia following specific b cell injury has been observed (Bonner-Weir et al. 1981). Underlying all studies of b cell regeneration is the continuing debate over whether, following death by injury or normal turnover, adult b cells are replenished from preexisting b cells or from specialized 1149

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1150 progenitors, so-called adult stem cells. Recent evidence in this area may suggest that actually both b cell replication and neogenesis from progenitor cells can occur in the adult pancreas (Nir et al. 2007; Teta et al. 2007; Xu et al. 2008). What accounts for the different outcomes in these studies seems to be the variation in the type of injury model employed. Nevertheless, in the normal adult pancreas (Dor et al. 2004) and in the immediate aftermath of b cell ablation (Nir et al. 2007), replication appears to be the predominant route for b cell neogenesis, whereas in the longer term, response to b cell damage (Movassat and Portha 2007; Xu et al. 2008) and in humans in general (Butler et al. 2007), a contribution from duct cell metaplasia to b cell replication appears to be important. Although adult b cell regeneration from ductal epithelium recapitulates aspects of fetal b cell development, one important distinction to date has been the absence until very recently (Xu et al. 2008) of any clear evidence of neurogenin 3 (Ngn3) expression, an essential protein for all fetal islet cell development (Gradwohl et al. 2000), in the adult pancreas (BonnerWeir et al. 2004). Previously, we found that b cell damage can induce transplanted embryonic stem (ES) cells to undergo embryonic-like pancreatic endocrine and exocrine neogenesis in vivo from PDX-11 ductal epithelium between 15 and 28 days after transplantation (Kodama et al. 2008). We also found that transplanted ES cells localized to the injured but not to the normal pancreas (Takeshita et al. 2006). In this study, we show that a subset of renal capsule–transplanted ES cells migrating to the pancreas did not differentiate into b cells by themselves, but improved glycemic control in mice following b cell loss.

Materials and Methods Animals and Streptozotocin (STZ) Treatment

Animal experiments were performed in compliance with the guidelines of the Institute for Laboratory Animal Research at the National Cancer Center Research Institute. Female Balb/c nude mice (CLEA; Tokyo, Japan), aged 7 weeks, were used for all experiments. Blood glucose measurements were performed on whole venous blood collected from the tail vein using the Freestyle Flash Blood monitoring system (Nipro; Tokyo, Japan) according to the manufacturer’s instructions. The basic experimental regimen consisted of a single 300-ml intraperitoneal injection of STZ (Sigma Aldrich; St. Louis, MO) within 15 min of dissolution in freshly prepared 20 mM cold citrate buffer (pH 4.5); 240 mg/kg was used to induce hyperglycemia, and 175–200 mg/kg was used to induce b cell damage with low incidence of overt hyperglycemia. Non-STZ–treated mice received 300 ml citrate buffer alone. Twenty-four hr after STZ

Kodama, Tsukamoto, Yoshida, Aoki, Kanegasaki, Quinn treatment, ES cells were transplanted beneath the left renal capsule as described previously (Kodama et al. 2008). PBS was dispensed under the renal capsule for sham transplantation. At subsequent time points, the animals’ glucose homeostasis was monitored, and mice were sacrificed for immunohistochemical analyses. Intraperitoneal glucose tolerance testing was performed as described by the Animal Models of Diabetes Complications Consortium (www.amdcc.org). Mice were fasted for 7 hr before intraperitoneal injection with 2 mg/kg glucose solution (Sigma). In vivo imaging analysis of transplanted ES cells was conducted in a cryogenically cooled IVIS system (Xenogen; Alameda, CA) as described previously (Takeshita et al. 2006). Tumor growth was monitored weekly by whole-body bioluminescent imaging. Culturing and In Vivo Transplantation of ES Cells

The firefly luciferase–expressing ESJ1 cell line, a J1 cell clone of 129SV male origin, was established previously (Kodama et al. 2008). The SK7 cell line, derived from an Institute of Cancer Research strain, was a gift from Professor Shoen Kume (Kumamoto University). SK7 cells were transfected with the pLucNeo plasmid (Promega; Madison, WI) and a stable, luciferaseexpressing clone was isolated for use in this study. ESJ1 cells were cultured under feeder-free conditions, whereas SK7 cells were cultured on a layer of inactivated mouse embryonic fibroblasts. Both ES cell lines were maintained as described previously (Kodama et al. 2008). For transplantation, ES cells were trypsinized and dispersed to a single-cell suspension, then counted and washed in PBS. Two ml of cell suspension containing 1 3 105 or 1 3 106 ES cells was transplanted beneath the left renal capsule. Pancreas Tissue Extraction and Luciferase Assay

The pancreas tissues were dissected, snap frozen, and pulverized under liquid nitrogen. An equal volume of tissue lysis buffer (125 mM Tris-phosphate, pH 7.8, 10 mM dithiothreitol, 10 mM EDTA, 50% glycerol, 5% Triton-X, 0.2 mM phenylmethylsulfonyl fluoride, 1 mg/ml aprotinin, 1 mg/ml leupeptin) was added, and incubated on ice for 20 min. Five ml of supernatant was mixed with 100 ml of luciferase assay reagent (PicaGene; Toyo, Inc., Tokyo, Japan), and the light unit of the luciferase activity was assayed using a luminometer (MiniLumat LB 9506; EG and G Berthold, Vilvoorde, Belgium). The data were expressed as the relative luciferase activity, which is the chemiluminescence elicited from pancreas tissue extract divided by the background chemiluminescence of the lysis buffer alone. The analyses (carried out in triplicate) were repeated a minimum of two times, and the mean 6 standard deviations were plotted.


ES Cell Transplantation and Pancreas Regeneration

The Journal of Histochemistry & Cytochemistry

Immunofluorescence and Immunohistochemical Analyses

Tissues were preserved in 10% formalin solution, embedded in paraffin wax, and sectioned. Sections were deparaffinized and rehydrated, and antigen retrieval was performed by autoclaving sections for 5 min in antigen recovery buffer (Sigma). Antibody staining was performed as described previously (Kodama et al. 2008). The following primary antibodies and dilutions were used: anti-insulin (rat, 1:500; R and D Systems, Minneapolis, MN; #MAB1417), anti-PDX-1 (rabbit, 1:200; Upstate, Charlottesville, VA; #07-696), anti-PDX-1 [mouse, 1:2000 tyramide signal amplification (TSA); Developmental Studies Hybridoma Bank (DSHB), University of Iowa; #F6A11], anti-Ki67 (rabbit, 1:500; Vector Laboratories, Burlingame, CA; #VPK451), anti-Ngn3 (mouse, 1:2000 TSA; DSHB, University of Iowa; #F25A1B3), and anti-a-amylase (1:500; Sigma, #A8273). For detection of BrdU1 cells, the BrdU In Situ Detection Kit (BD Biosciences; Tokyo, Japan) was used, and fluorescence detection was performed by TSA using the NEN TSA fluorescence systems kit according to the manufacturer’s guidelines (PerkinElmer; Wellesley, MA). For staining with mouse primary antibodies, the M.O.M. kit from Vector laboratories was used, followed by TSA. Fluorescence microscopy was performed using a Nikon Eclipse E1000 epifluorescence microscope and 203 and 403 objectives, equipped with a QICAM FAST1394 CCD camera. Original images with original fluorescent colors were acquired using MetaMorph V6.1r4 software (Universal Imaging Corp.; Sunnyvale, CA) and overlaid using Adobe Photoshop CS2 V9.0. For measurement of tissue section area, and histomorphometry, ImageJ software (V 1.31v) (available from http://rsb.info.nih. gov/ij/download.html) was used. For morphometric analyses of BrdU1 b cells, sections were costained for insulin and BrdU. The number of BrdU1 b cells was expressed as a percentage of the total number of b cells for each tissue section. For each animal, 3 3 3-mm-thick whole pancreas tissue sections, each separated by 30 mm, were examined. Fluorescence In Situ Hybridization (FISH)

The digoxigenin (DIG)-labeled Y-chromosome probe was purchased from Chromosome Science Lab, Inc. (Sapporo, Japan), and FISH was performed on 3-mm tissue sections according to the probe manufacturer’s guidelines. Briefly, sections were deparafinized and rehydrated, then denatured for 10 min in 2 3 saline sodium citrate (SSC) in a microwave oven. Sections were partially digested using pepsin (Sigma) dissolved in 0.1 N HCl for 20 min at 37C, then dehydrated before applying the probe solution. After denaturation at 90C for 10 min, hybridization was performed in a humidified chamber at 37C for 16 hr. Washing was performed

using 50% formamide/2 3 SSC. Immunological detection was performed using anti-DIG primary antibody (Dako; Glostrup, Denmark) and AlexaFluro 488 secondary antibody. Sections were counterstained with 4′,6-diamidino-2-phenylindole. Tracing of b Cell Replication

To ascertain the relative contribution of cell replication vs progenitor differentiation, mice were given BrdU (BD Biosciences) by a single intraperitoneal injection on two consecutive days (days 2 and 3) following injury and ES cell or sham transplantation. Then on days 5, 12, and 21, the mice were sacrificed (n53 per group at each time point), and pancreata were examined histomorphometrically for BrdU1/insulin1 double-positive cells as described above. The total number of b cells, as well as the number of BrdU1/ insulin1 cells on each section, were counted and averaged for each mouse. Statistical Analysis

Two-tailed Student’s t-tests were performed, with p,0.05 or p,0.01 considered to be significant. Values are given as mean 6 SD or 6 SEM.

Results ES Cell Transplantation Maintains Long-term Euglycemia in STZ-treated Mice

A single high dose of 240 mg/kg STZ caused hyperglycemia within 1 week (blood glucose concentration .200 mg/dl) in 7-week-old female Balb/c nude mice, confirming previous findings (Kodama et al. 2008). When mice (n516) received 1 3 105 undifferentiated ESJ1 cells by transplantation beneath the left renal capsule 24 hr after STZ treatment, one third (6/16) of the ESJ1-transplanted animals remained euglycemic (blood glucose concentration ,200 mg/dl) from day 3 (when glucose measurements were first made) until sacrifice on day 35 (Figure 1A). During the experimental period, the euglycemic mice remained healthy, with positive weight gain (data not shown) and absence of polyuria, and they maintained mild glucose intolerance (Figures 1B and 1C). The remaining ES cell–transplanted mice progressed to severe hyperglycemia, as did the sham-transplanted (PBS instead of ES cells) group. However, the progression to severe hyperglycemia (.400 mg/dl) was significantly (p,0.05) delayed in the ES cell transplant group (Figure 1D). At sacrifice, pancreata of euglycemic animals appeared normal, with no macroscopic ES tumors, unlike results seen previously following intraperitoneal ES cell transplantation (Takeshita et al. 2006). Prompted by earlier results indicating ES cell homing to the injured pancreas (Takeshita et al. 2006), we examined pancreata for evidence of ES cell migration

The Journal of Histochemistry & Cytochemistry


Kodama, Tsukamoto, Yoshida, Aoki, Kanegasaki, Quinn

Figure 1 Embryonic stem (ES) cell transplantation prevents hyperglycemia in streptozotocin (STZ)-treated mice. (A) ES cell–transplanted mice (6/16) were euglycemic (white bar) 7 days after STZ treatment (STZ 1 ESJ1 Tx) and remained so until day 35, whereas 0/18 sham-transplanted animals (STZ 1 Sham Tx) were euglycemic. (B,C) Euglycemic mice maintained a consistently mild glucose intolerance between day 7 (B) and day 21 (C), compared with control mice. (D) Non-fasting blood glucose levels in sham- and ES-transplanted mice (hyperglycemic and euglycemic) up to day 25. Until day 15, the average blood glucose concentration was lower in the ES-transplanted hyperglycemic group, compared with the sham group (glucometer detection limit, 500 mg/dl). Triangles, STZ 1 ESJ1 transplant (euglycemic); circles, STZ 1 ESJ1 transplant (hyperglycemic); diamonds, STZ 1 sham transplant, *p,0.05. (E) Luciferase activity was significantly increased in pancreatic protein extracts from STZ (175 mg/kg; all mice remained euglycemic) 1 ES cell–transplanted mice (STZ 1 ESJ1), compared with non-injured ES cell–transplanted mice (ESJ1), *p,0.05.

from the renal capsule. Luciferase enzymatic activity was detected in tissue from STZ-injured but not STZuntreated transplant recipients 4 weeks after luciferasetransgenic ES cell transplantation (Figure 1E). A similar result was also found with an allogenic luciferasetransgenic ES cell line, SK7 (data not shown). These data indicated that STZ injury promotes ES cell migration from the renal capsule into the pancreas, which seems to be associated with recovery from high-dose STZ following ES cell transplantation. Pancreas Regeneration in Euglycemic Mice From PDX-11 Ductal Epithelium

We compared ES cell–transplanted and sham-transplanted pancreata immunohistochemically. Among the recov-

ered animals, evidence of regeneration was identified, involving both large intralobular ductal epithelium, as well as smaller ductal cells, and characterized by expression of the mature b cell and pancreatic stem cell marker PDX-1 (Figure 2). PDX-1 expression in adult rodent pancreas ductal epithelium is commonly associated with endocrine regeneration (Bonner-Weir et al. 2004). Some ducts displayed near-uniform expression of PDX-1 throughout the duct wall (Figures 2B and 3A). Although the number of b cells in the vicinity of these PDX-11 ducts was comparably low, most were individual cells or small clusters of cells found in the connective tissue surrounding these ducts, i.e., not embedded in islets or embedded in exocrine parenchyma, suggesting that they were derived from adjacent

The Journal of Histochemistry & Cytochemistry

ES Cell Transplantation and Pancreas Regeneration


Figure 2 Neurogenin 3 (Ngn3) and pancreas and duodenum transcription factor-1 (PDX-1) expression in STZ-treated ES cell–transplanted mice. Nuclear-localized PDX-1 expression was detected in the duct cells of STZ 1 ES cell–transplanted mice (A–D,I,J) but not in duct cells of STZ 1 sham transplant (E,F) or normal control (PDX-1) animals. A population of Ngn3 nuclear-localized duct cells was also identified in the same regions partially overlapping with PDX-1 expression. (A,C) Ngn3 expression in regenerating pancreas. (B,D) PDX-1 expression in pancreas merged with A and C, respectively. Small boxed areas are magnified in lower right corner to aid visualization of individual positive cells. (I,J) Ngn3 expression adjacent to a pancreatic islet and in smaller ducts, respectively. (E,G) Ngn3 expression was not detected in sham-transplanted and control pancreas, respectively. (F,H) PDX-1 expression merged with E and G, respectively. Pancreatic islets (broken line) are indicated by “i.” Nuclear counterstain is 4′,6-diamidino-2-phenylindole (DAPI) (dark blue). Bars: A,B 5 20 mm; C–J 5 10 mm.

PDX-11 duct cells (Figure 3). These b cells were nondividing (ki672; data not shown). A key distinction between adult pancreas regeneration and fetal pancreas development to date has been the absence and absolute requirement, respectively, of Ngn3 protein expression. In the present study, however, we detected Ngn3 protein expression in isolated

cells within the wall of large ducts coexpressing PDX-1 (Figures 2A and 2B) and also as clusters, both PDX-11 and PDX-12, within smaller ducts. In addition, Ngn31 duct cells were found associated with established islets (Figure 2I). There was an overlap with PDX-1 expression among the Ngn31 cells, a pattern resembling the secondary transition stage of fetal endocrine pancreas


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The Journal of Histochemistry & Cytochemistry

Figure 3 b cells at sites of regeneration. Adjacent to PDX-1- and Ngn3expressing ductal epithelium, isolated and small clusters of insulin1 cells were found in STZ 1 ES–transplanted mice. Amylase staining was weak, other than in ductal lumen (L), compared with other areas of the pancreas (inset). A and B are near-adjacent sections. (A) PDX-1 (nuclear green) and Ngn3 (nuclear red); (B) a-amylase (cytoplasmic green) and insulin (cytoplasmic red). Nuclear counterstain is DAPI (dark blue). Bar 5 10 mm.

development (Pictet and Rutter 1972). The presence of Ngn31 cells 5 weeks after STZ treatment is an indication of prolonged b cell neogenesis. No overlap between Ngn3 and insulin expression was detected (data not shown). Neogenic Endocrine Progenitor Tissue Is Derived From an Endogenous Source

Identification of the fate of ES cell derivatives following migration to the pancreas is a key step toward understanding their role in the regenerative response to b cell injury. To distinguish male (ES-derived) from female (endogenous) cells and to identify the origin of PDX-1/Ngn3–expressing ductal epithelial cells, Ychromosome–specific interphase FISH was performed on tissue sections from regenerating pancreata. Typically, the pancreas-localized ES cells existed as small clusters adherent to the interstitium and adjacent to sites of neogenesis. Contrary to our expectations based on former data (Kodama et al. 2008), the ES cells themselves were never identified as components of normal ductal, exocrine, or endocrine tissue within the pancreas. However, areas of regeneration (PDX-11/ Ngn31 ductal tissue) and ES cell–derived cells were closely associated, a typical example of which is shown in Figure 4. The data suggest that the exogenous cells have a role to play in stimulating the endogenous response. Hence, sex-specific FISH analysis demonstrated that infiltrating ES cell derivatives do not comprise the metaplastic pancreatic epithelium. Importantly, in mice that became hyperglycemic following STZ treatment and ES cell transplantation, ES cell infiltration into the pancreas was rare or not detected (data not shown), whereas all of the recovered mice analyzed had readily detectable ES cell clusters in the pancreas. Together this suggests that ES cell homing from the renal capsule to the pancreas indirectly stimulates b cell regeneration after high-dose STZ. ES Cell Transplantation Induces b Cell Neogenesis From Endogenous Progenitors

We devised an experiment to estimate the relative contribution of the processes of b cell replication vs pro-

genitor cell differentiation toward the injury response in ES cell–transplanted and sham-transplanted mice. STZ was administered at 200 mg/kg, which in our hands typically induces overt hyperglycemia: .200 mg/dl blood glucose concentration for three consecutive readings in ,50% of animals (Kodama et al. 2008). Hence, most of the animals were euglycemic at the time of sacrifice (see supplementary Table 1). The immunohistochemically detectable nucleotide analog, BrdU, was administered shortly after STZ injury/ES cell transplantation, and then the numbers of b cells that were BrdU1 at an early (day 5) and two later time points (days 12 and day 21) were counted. We made two rational assumptions: (a) Any given b cell could only divide once during a 3-week experimental period; and (b) the b cell replication component of BrdU1/Ins1 cells would be the same during all three time point analyses, because b cell progenitors are stimulated to divide within a 1-week period after injury (Xu et al. 2008). On the basis of these assumptions, we postulated that any increase in the number of BrdU1 b cells on day 5, day 12, and day 21 would indicate b cell neogenesis by progenitor cell differentiation. The results of this experiment were quite clear. A slight increase, on average, in the rate of b cell replication in the ES cell–transplanted vs sham-transplanted animals was found at day 5 (Figure 5), although it was not statistically significant. At day 12, there was no detectable difference in the overall number of BrdU1 cells compared with day 5, although the marginal difference between ES cell–transplanted and sham-transplanted animals remained. However at day 21, we detected the first indication of progenitor-derived b cell neogenesis: a significant (p,0.01) rise in the fraction BrdU1 b cells in the ES cell–transplanted group compared with this group at day 5 and day 12. There was also a significant (p,0.01) difference between ES cell–transplanted and sham-transplanted animals at day 21 (Figure 5). The data are compatible with a wave of b cell differentiation from progenitor cells in the ES cell–transplanted group that were stimulated to divide during the first week following pancreas injury. Although this experiment was ended at day 21, it is likely that the margin of progenitor-derived cells would have increased further,

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ES Cell Transplantation and Pancreas Regeneration


Figure 4 Neogenic pancreatic endocrine tissue is derived from an endogenous source. Y-chromosome–specific interphase fluorescence in situ hybridization was used to distinguish donor (male, Y1; green nuclear signal) from host (female, Y2) pancreatic tissue. PDX-11 and Ngn31 ductal epithelium and all endocrine cells that were examined (.8000) were Y-chromosome–negative. However, sites of regeneration were in proximity to transplanted cells. (A–C) A series of demagnified images showing a cluster of ES cell derivatives (Y1 cells, arrow) in the context of the pancreas, adjacent to a large region of endogenous (Y2) cell regeneration within an intralobular duct (d). Arrows indicate clusters of ES-derived Y1 cells. (D) A section near-adjacent to C showing expression of PDX-1 and Ngn3 in endogenous cells within the duct (d) shown in C. (E–G) Examples of small clusters of ES cell derivatives (arrowheads) within the pancreas of different STZ 1 ES cell– transplanted mice, and localized to the interstitium. (H) Positive control male pancreas tissue section centered on an islet (broken line, i). Nuclear counterstain is DAPI (dark blue). Bars: C,D 5 20 mm; B,E–H 510 mm; A 5 3 mm.

based on immunohistochemical detection of Ngn1/ PDX-11 duct cells at day 35 following pancreas injury (Figure 2).

Discussion In this study, we report that in a subset of STZ-treated mice that received renal capsule–transplanted ES cells, Ngn3 expression, enhanced b cell neogenesis, and resistance to hyperglycemia were observed. These regenerative responses were associated with migration of transplanted cells to the injured pancreas.

The islets of the adult pancreas are fully developed shortly after birth. Thereafter, adjustments to the pool of b cells in response to fluctuations in metabolic demand appear to arise from self-duplication (Dor et al. 2004). However, this mechanism does not wholly account for pathological b cell death, in which there appear to be alternative routes for b cell replenishment that are collectively understood to be weak (Rood et al. 2006). Pancreatic b cell regeneration appears to include a rapid response (Levine and Itkin-Ansari 2008), which is likely to be b cell replication, and a delayed response that involves duct cell metaplasia

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Figure 5 Lineage analysis of b cell neogenesis. A single moderate dose (200 mg/kg) of STZ was given to mice on day 0, and transplantation (ES cell or sham) was performed on day 1. BrdU was administered to mice from three different groups [STZ 1 ES cell transplant (black bars, n59), STZ 1 sham transplant (hatched bars, n59), and non-treated controls (white bars, n53)] on days 2 and 3 of the study. BrdU/insulin double-positive cells were counted and expressed as a percentage of total insulin1 cells in tissues from STZtreated animals sacrificed at days 5, 12, and 21. For non-treated controls, morphometry was performed on day 21 only. Day 5 values represent the rate of b cell neogenesis by mitosis of preexisting b cells. Day 12 and day 21 values represent the combined b neogenesis from preexisting b cells and that from progenitor differentiation. A significant rise in the rate of BrdU1 b cells in the STZ 1 ES transplant group at day 21, compared with days 5 and 12 and compared with sham-transplanted mice, is compatible with the onset of a wave of b cell differentiation in this group between days 12 and 21, from progenitor cells that were stimulated to divide shortly after injury. Data are given as mean 6 SEM, *p,0.01.

(Bonner-Weir et al. 1981; Xu et al. 2008). STZ-induced b cell death resembles the specific b cell loss during diabetes and typically results in a characteristically weak regenerative response with incomplete recovery of lost cells. To our knowledge, this response to STZ has never previously been associated with expression of Ngn3. Caution, however, is warranted in the use of rodent models, because STZ may additionally damage the surviving cells, hampering the regenerative response, at least its cell replication component, leading to an underestimation of the importance of cell duplication in diabetic individuals (Nir et al. 2007). In comparison with the delayed response seen in a previous study involving STZ-treated rodents (BonnerWeir et al. 1981), our present findings suggest that ES cell transplantation caused an amplification or prolongation of the endogenous response to b cell damage, exposing sites of regeneration and indirectly causing or enhancing Ngn3 protein expression at these sites. Conversely, in the absence of transplanted ES cells, Ngn3 protein expression was not detectable, indicat-

Kodama, Tsukamoto, Yoshida, Aoki, Kanegasaki, Quinn ing that it may otherwise be very transient, at undetectable levels, or perhaps may not be expressed at all. Unlike previously, when ES cells were administered intraperitoneally (Takeshita et al. 2006), ES cells that localized to the pancreas in the present study did not form noticeable tumors; instead, these cells existed as small clusters (see Figure 4). The absence of tumors may indicate that they are in some differentiated state, although it is still possible that some of these pancreaslocalized ES-derived cells are tumorigenic. It will be important to characterize the subsets of ES-derived cells that congregate within the pancreas. This will be addressed in a future study. What we can determine to date is that these cells appear to be Ngn32 and PDX2, as well as negative for differentiated islet cell markers such as insulin and glucagon, while many of them are proliferating (Ki671). Identification of these cells may help to explain their role, if any, in pancreas regeneration following STZ-induced injury. In any case, insulin1 cells were not detected among the ES cell derivatives in the pancreas. Although the role of the ES cells remains unclear, a plausible speculation might be that they secrete mitogenic factors. In both b cell replication and neogenesis from progenitor cells, exogenous growth factors could amplify the response. The pancreatic duct cells are known to express receptors for mitogenic factors, which, following activation, would lead to an expanded pool of precursor cells susceptible to endogenous b cell differentiation. Keratinocyte growth factor (Movassat et al. 2003), GLP-1/exendin-4 (Tourrel et al. 2001), ghrelin (Irako et al. 2006), gastrin (Rooman et al. 2001), epidermal growth factor (Rooman and Bouwens 2004), and activin 1 betacellulin (Li et al. 2004) all appear to promote regeneration in this manner. However, a more complicated involvement of the ES cells in endocrine cell specification and differentiation cannot be ruled out. Lineage tracing of b cell replication has been applied to the problem of cell ontogeny in the adult pancreas (Dor et al. 2004; Teta et al. 2007). In these studies, it was demonstrated that cell replication is the primary if not only method of b cell neogenesis in the normal and acutely injured adult pancreas. In the present study, we applied a cell tracing approach to distinguish b cell replication and progenitor cell differentiation in our STZ injury/ES cell transplantation model. For the latter route, following progenitor cell expansion, an undefined time lag for b cell differentiation is required. This b cell differentiation time period was estimated to lie between day 12 and day 21 following initial b cell injury, due to the detection of a significant rise in neogenic b cells at day 21 compared with earlier time points in the ES cell–transplanted group. In the case of b cell neogenesis through replication of preexisting b cells, it would appear that only a single round of replication occurred for any b cell during


The Journal of Histochemistry & Cytochemistry

ES Cell Transplantation and Pancreas Regeneration

the experimental period based on the findings of Teta et al. (2007), namely, that .1 month refractory period follows adult b cell replication. Hence, although further analysis is required to show a clear effect of ES cell transplantation on b cell replication, the data presented here indicate a significant effect of ES cell transplantation on b cell differentiation from progenitor cells at day 21. In the sham-transplanted animals, no significant b cell neogenesis was observed during the course of the experiment. The apparent absence of b cell differentiation from progenitor cells in shamtransplanted mice is in line with the results of Teta et al. (2007), who demonstrated that acute pancreas injury does not ordinarily induce progenitor-derived neogenic b cells. A question raised is whether ES cell transplantation can reverse established hyperglycemia. When we transplanted ES cells into mice with established hyperglycemia 15 days following high-dose STZ (n57), no reversal of hyperglycemia was observed up to day 35, despite a 10-fold higher cell number (1 3 106) than used at 24 hr after STZ (see supplementary Figure 1). This demonstrates that ES cells do not have an independent glucose-lowering ability and that the effect on glycemic control seen in animals following day 1 transplantation appears to require interaction with host cells. For type 1 diabetes mellitus, replacement of a patient’s islets of Langerhans is the only treatment that achieves an insulin-independent, constant normoglycemic state and avoidance of hypoglycemic episodes (Pileggi et al. 2006). In the future, in vitro–differentiated ES cells may provide an unlimited supply of b cells for transplantation. Before that is possible however, a key test of function for ES-derived b cells is their ability to improve glucose regulation in diabetic animals, often STZ-injured athymic Balb/c nude mice. In light of the results of our study, contaminating ES cells or their derivatives (Shi et al. 2005) need to be considered, not only as a teratoma risk, but also as a potential stimulator of endogenous regeneration, an effect that could be attributed to the transplanted b-like cells. Collectively, the data presented here indicate that ES cell transplantation is associated with a regenerative response to b cell death by STZ. However, although we believe it unlikely, we cannot rule out the possibility that the results seen are an effect of STZ. The pattern and time scale (around 3 weeks) of the onset of endogenous b cell neogenesis from progenitors are similar to those of ES cell–derived b cell neogenesis in vivo (Takeshita et al. 2006; Kodama et al. 2008). In both cases, endocrine pancreas fetal development is comparable: PDX-11 ductal epithelium acquires Ngn3 expression and finally leads to endocrine gene expression. Hence, interaction between the injured pancreas and the ES cells may promote reciprocal b cell neogenesis. A relationship akin to this has been reported

following neuronal stem cell transplantation into the hypoxia-ischemia–injured mouse brain (Park et al. 2002). In summary, we describe for the first time evidence of Ngn3-mediated b cell neogenesis in the adult pancreas following STZ treatment causing resistance to hyperglycemia. This response is correlated with ES cell transplantation, and resembles the developmental ontogeny of fetal endocrine b cells. Further studies are warranted to identify the signaling pathways involved in this regeneration, which will have direct relevance to diabetes therapy. Acknowledgments We thank Dr. Takahiro Ochiya for making available to us his laboratory facilities at the Section for Studies on Metastasis (NCCRI), Dr. Fumitaka Takeshita for assistance with animal care, Dr. Yoshinori Ikarashi (National Cancer Center Research Institute) for helpful discussion during the study, and Prof. Shoen Kume (Kumamoto University) for the gift of the SK7 ES cell line. The Ngn3 and PDX-1 monoclonal antibodies were developed by Dr. O.D. Madsen and were obtained from the Developmental Studies Hybridoma Bank developed under the auspices of the National Institute of Child Health and Human Development and maintained by the University of Iowa, Department of Biological Sciences, Iowa City, IA.

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The Journal of Histochemistry & Cytochemistry

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