The pancreatic ductal epithelium serves as a potential pool of progenitor cells

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Copyright # Blackwell Munksgaard 2004

Pediatric Diabetes 2004: 5: 16—22 Printed in Denmark. All rights reserved

Pediatric Diabetes

Diabetes and stem cells according to: Susan Bonner-Weir et al.

The pancreatic ductal epithelium serves as a potential pool of progenitor cells Bonner-Weir S, Toschi E, Inada A, Reitz P, Fonseca SY, Aye T, Sharma A. The pancreatic ductal epithelium serves as a potential pool of progenitor cells. Pediatric Diabetes 2004: 5: 16—22. # Blackwell Munksgaard, 2004

Abstract: With the increasing success of islet transplantation, b-cell replacement therapy has had renewed interest. To make such a therapy available to more than a few of the thousands of patients with diabetes, new sources of insulin-producing cells must become readily available. The most promising sources are stem cells, whether embryonic or adult stem cells. Clearly identifiable adult pancreatic stem cells have yet to be characterized. Although considerable evidence suggests their possibility, recent lineage-tracing experiments challenge their existence. Even in light of these lineage-tracing experiments, we suggest that evidence for neogenesis or new islet formation after birth remains strong. Our work has suggested that the pancreatic duct epithelium itself serves as a pool for progenitors for both islet and acinar tissues after birth and into adulthood and, thus, that the duct epithelium can be considered ‘facultative stem cells’. We will develop our case for this hypothesis in this perspective.

Postnatal b-cell growth Over the past two decades, we have developed the concept that after birth and into adulthood, the b-cell mass is dynamic and is regulated to maintain glucose homeostasis (1, 2). Recent studies have shown a linear correlation of the b-cell mass and body weight/body mass index in mice, rats, and humans (3—5). Furthermore, b-cell mass is determined by the balance of 1) cell birth/renewal, by replication of pre-existing differentiated b-cells and by neogenesis or differentiation from progenitor cells; 2) cell death, usually in the physiological setting by apoptosis; and 3) cell volume changes, from atrophy to hypertrophy. These determinants differ in importance in their contribution in various models and may differ according to their predominance among species. We will use replication to indicate the mitotic division of a cell already with the phenotype of a

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Susan Bonner-Weir, Elena Toschi, Akari Inada, Petra Reitz, Sonya Y. Fonseca, Tandy Aye and Arun Sharma Department of Medicine, Research Division, Joslin Diabetes Center, Harvard Medical School, Boston, MA, USA Key words: b-cell growth, endocrine pancreas, pancreatic progenitors Corresponding author: Dr Susan Bonner-Weir Joslin Diabetes Center 1 Joslin Place, Boston MA 02215, USA Tel: þ1-617-732-2581 fax: þ1-617-732-2650 e-mail: [email protected]

b-cell and neogenesis to indicate the formation of new b-cells from either progenitor or stem cell.

Replication vs. neogenesis of new b-cells? In adult mice, several studies using morphometric techniques have shown that the b-cell compensation due to pregnancy or response to insulin resistance was predominantly from replication with little neogenesis (6—8). A recent study using lineage-tracing techniques confirmed the predominance of replication in the normal adult mouse (9). With additional experiments, including partial pancreatectomy (Px), the authors concluded that neogenesis does not occur and no pancreatic stem cells exist in the adult (9). Such an interpretation has substantial problems, particularly because it is always difficult to show the absence of some thing or event. The authors themselves state that because of the

Stem cells and diabetes variation in labeling efficiency, inherent to the inducible Crerecombinase-10XP system (CreER system), this strategy may not be able to detect low levels of neogenesis. Another limitation of the CreER system is that when it is expressed using a strong promoter for a prolonged time, the recombinase-dependent excision can occasionally occur in the absence of tamoxifen (10). Such recombinase activity in newly differentiated b-cells may further complicate detection of low levels of neogenesis. By 2 months of age, the level of neogenesis in normal adult rats is low (4), and hence, the conclusion of the Dor et al study is not surprising (9). Performing lineage tracing immediately after birth and around weaning, time points not studied but when neogenesis is more frequent will be critical to test for neogenesis. In addition to the limitation of the inducible CreER system and the time points used in this study, extensive sampling of different islets throughout the pancreas would be needed to confirm their conclusion. From the data given in Dor et al. as total number of islets (greater than 25 mm) per group, the number of islets sampled per animal can be calculated as 50—60. This sampling is inadequate to detect an event of low frequency as 3-month-old mice have between 600 and 1200 islets greater than 25 mm (6). Additionally, to avoid the distortion of scoring the same large islets on different sections, sections must be at least 150 mm apart, but no indications of this precaution are given. The sampling issue is particularly important in the partial Px experiments because new islets would be expected in the new lobes and, yet, no confirmation that such lobes were sampled is given. Our work in the Px rat model has shown clearly that both replication of the pre-existing b-cells and neogenesis of new islets in whole new lobes of pancreas occurred (see Fig. 6 towards the end of this article). Additionally, in multiple models of pancreatic regeneration including Px, hormonestaining cells are present in the ductal epithelium; thus, a lack of such hormone-positive cells in this study further underscores the importance of the sampling issue.

Fig. 1. New islets bud from pancreatic ducts. In the neonatal rat, islets (arrows) are seen to bud from pancreatic ducts, here shown in a 20-d pancreas immunostained for glucagon (black).

are formed after partial Px; these lobes become indistinguishable from the pre-existing lobes (16). In humans, the neogeneic pathway appears to have a more important role in the compensation of b-cell mass with obesity than increased replication. With obesity, there is increased b-cell mass (5, 17). In the samples from these studies as well as from the more than 100 human organ donations to the Harvard Islet Transplantation program, neogeneic regions with many ductal profiles and hormone-positive cells budding from these ducts are seen more commonly than enlarged islets, such as would be seen with enhanced replication (Fig. 3). Additionally, our report of islets generated in vitro from duct-enriched, islet-depleted

9

Estimated β-cell mass without apoptosis

1.6% Measured β-cell mass

8

Contribution from progenitors (30–50%)

Is there a difference in the occurrence of neogenesis among species? Although replication is clearly the major pathway for new b-cells in adult mice, new islets budding from ducts are reported in adult mice, given exendin 4 (11) or betacellulin (12), with overexpression of interferon (IFN)-g (13), or transforming growth factor (TGF)-a (14). Thus, the two pathways for new b-cell formation are likely to exist even in adult mice. In rats, we have found two waves of neogenesis during the neonatal period: one immediately after birth and the other around weaning (Fig. 1). Using data from our longitudinal study of the b-cell mass and its determinants (15), we estimate that over 30% of the new b-cells seen at day 31 were not from replication of pre-existing b-cells (Fig. 2). In the adult rat, new lobes of pancreas Pediatric Diabetes 2004: 5: 16—22

β-cell number × 106

2.8% 7 6 5

2.8%

β-cell mass from replication

4 3 20

24 Days after birth

31

Fig. 2. Estimation of the contribution of new b-cells in the rat around weaning. Using the data on the b-cell determinants in Scaglia et al. (15), the number of b-cells generated by replication and by differentiation from progenitors can be estimated. During the neonatal period, the frequency of apoptosis is higher than in the adult, and the dotted line shows the b-cell mass if there had been no apoptosis during this period. Progenitors contribute about 30% of the b-cells at day 31 after birth in Sprague—Dawley rats.

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Bonner-Weir et al. human pancreatic tissue has been confirmed by Otonkoski’s group (18, 19). Can one conclude from these data that there is a difference among species in the mechanism for adding new b-cells after birth? We think there may be a quantitative difference, rather than a qualitative one; that is to say, we expect both pathways are functional in all species but they may make different contributions in different species. The importance of the replicative pathway in adult mice may reflect their long telomeres such that telomere shortening is not limiting in this species (20). In contrast, in humans, telomere shortening is limiting and, thus, the neogeneic pathway may be more important.

What is the cellular origin of the new islets? Because the ability of neogenesis to occur implies that there is a stem/progenitor cell, estimations of how many progenitors are needed to make a new islet may support the role of a particular cell type as a progenitor. In the rat Px model, we find islets in the proliferating ductule regions that we called ‘foci of regeneration’ at 3 days after surgery, a time that is 48 h after the first enhanced replication within the pancreatic remnant. Each ‘typical islet’ is 150 mm diameter and consists of about 1500 endocrine cells. The work of Jami’s group showed that embryonic islets are polyclonal (21), and we would expect similar polyclonality in the newly formed islets in adult. Without knowing the nature of the progenitor cell, we do not know its doubling time (cellcycle length). However, we can estimate that it is no shorter than 10 or 12 h based on the cell-cycle times for rodent cells, including 14.9 h for b-cells (22, 23), 10—12 h for mouse embryonic stem (ES) cells, 19.7 h for intestinal stem cells (24), and 24—36 h for multipotent adult progenitor cells (25). To make each islet of even 1000

endocrine cells within the 48 h observed in the Px model, between 16 (5 doublings of 10 h each) and 64 (4 doublings of 12 h each) progenitor cells would be needed. As we see the first bromo-deoxyuridine (BrdU) incorporation after Px surgery in the common pancreatic ducts and islets bud from ducts, we need to determine what cells are present in the ducts. Within the basement membrane of the ducts are the ductal epithelial cells and a rare (0.5—1% in larger ducts) small basal cell phenotype that resembles the oval cell of the liver; outside of the basement membrane, there are stromal cells, some of which are nestin positive. Thus, one would expect that one or more of these cells types may serve as the progenitor for islet neogenesis. Three models, as shown in Fig. 4, can be postulated for the cellular origin of islets budding from the ducts. In Model 1, cells outside the basement membrane of the duct epithelium (whether circulating stem cells or cells resident in the stromal population) proliferate, differentiate, and coalesce to form the islet. However, by ultrastructural analysis, in both the embryonic (26) and the postnatal pancreas (Fig. 5), hormone-positive cells are seen within the epithelium of the duct-like structures, in fact, within the basement membrane/ lamina of the ductal epithelium. Therefore, this model can be ruled out for the bulk of new islet formation. In Model 2, progenitor cells within the epithelium, perhaps the oval cell-like ones, proliferate, delaminate Neogenesis Model 1

Progenitor cells outside basement membrane form islets

Fig. 3. Neogenesis in human pancreas. Neogenesis as areas of small ductal profiles with budding hormone-positive cells (insulin, black) are found in many pancreases from obese humans. Such areas have previously been shown in Butler et al. (17).

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Model 2

Progenitor in epithelium proliferate and migrate

Model 3

With replication, duct cells regress to a less differentiated stage and then function as progenitor cells.

Fig. 4. Models for cellular origin of islet progenitor. Three models can be postulated for the cellular origin of islets budding from the ducts. In Model 1, cells outside the basement membrane of the duct epithelium (whether circulating stem cells or cells resident in the stromal population) proliferate, differentiate, and coalesce to form the islet. However, by ultrastructural analysis, in both embryonic (26) and postnatal pancreas (Fig. 5), hormone-positive cells are seen within the basement membrane/lamina of the ductal epithelium. Therefore, this model can be ruled out for the bulk of new islet formation. In Model 2, progenitor cells within the epithelium, perhaps the oval cell-like ones, proliferate, delaminate from the epithelium with disruption of the plasma membrane, and coalesce into islets. Model 2 is unlikely in the pancreatic regeneration after partial pancreatectomy (Px), even though this model could theoretically occur in some situations. In Model 3, with replication the ductal epithelial cells regress to a less differentiated stage and can then function as progenitor cells, which respond to local stimuli to differentiate into islet cells. This model is favored by our data on the regenerating rat pancreas after partial Px. Pediatric Diabetes 2004: 5: 16—22

Stem cells and diabetes Hormone-containing cells form within the ductal epithelium

MT-TGF-α transgenic mouse Fig. 5. Hormone-positive cells form within the ductal epithelium. As illustrated in these micrographs from a zinc-treated metallothionein transforming growth factor-a (MT-TGF-a) mouse (14), insulinpositive cells form within the metaplastic ducts. In upper left, immunostaining for insulin (black) shows 6% of the cells with some insulin immunostaining. Cells (1%) that have intense staining correspond in frequency with the granule-containing cells (arrow) seen in the right lower panel at the ultrastructural level. The hormone-positive cells are within the basement membrane of the epithelium.

from the epithelium with disruption of the plasma membrane, and coalesce into islets. After partial Px, proliferation is initiated in the common pancreatic duct 20 h after surgery and peaks at 24 h. If Px rats were given colchicine to arrest mitosis at 20 h after surgery, all the arrested cells in the common pancreatic duct were of the principal epithelial type, that is, the columnar epithelial cells, with no evidence of an amplification of a small population of putative stem cells/ oval cells. Thus, in the rat regeneration model, Model 2 is unlikely, even though this model could theoretically occur in some situations. In Model 3, with replication the ductal epithelial cells regress to a less differentiated stage and can then function as progenitor cells, which respond to local stimuli to differentiate into islet cells. This model is favored by our data on the regenerating rat pancreas after partial Px.

Support of model 3 in the pancreatic regeneration after partial Px in the adult rat Our model is that of regeneration that follows 90% (partial) Px in the young adult rat (16, 27—30). An anatomically well-defined remnant of 10% weight and 10% islet mass regenerates to 27% of the weight of the sham-operated pancreas, with 45% of the sham islet mass by 4 wk after surgery. A wave of DNA synthesis (as seen by the incorporation of the thymidine analog BrdU) passes through the ductal tree with a threefold increase to 15% BrdUþ nuclei in the common pancreatic duct at 24 h and a return to sham levels by 48 h. Although the islets had PDX-1 immunostaining at all time points, little to no PDX-1 protein was detected in the epithelium of the common pancreatic duct from Pediatric Diabetes 2004: 5: 16—22

unoperated or sham Px animals; these ducts are quiescent with little to no BrdU incorporation. Twenty-four hours after Px, the time of peak proliferation of the common pancreatic duct epithelium, most of the ductal epithelial cells are BrdUþ PDX-1— or BrdUþ PDX-1þ. In contrast, at 3 days after Px, few cells of the common pancreatic duct epithelium are BrdUþ, but most cells express PDX-1 protein. During development, PDX-1 marks the region that will develop into the pancreas, and the loss of this protein results in pancreatic agenesis. PDX-1 protein is expressed in the embryonic pancreatic ducts until shortly before birth; it is the same protein that others have suggested as a marker for the adult progenitor cells (31, 32). In the IFN-g transgenic mice, PDX-1 protein is seen in ducts that are proliferating and from which there is budding of new islets (31). In the metallothionein TGF-a (MT-TGF-a) overexpressing transgenic mice, PDX-1 expression was seen in the metaplastic ductal epithelium and focally there was even Pax-6 expression (32). In the same transgenic mice, we had shown that 6% of the epithelial cells in these metaplastic ducts expressed low levels of insulin, with 1% showing strong staining with actual insulin granules (Fig. 5) (14). The data of both these experimental mice are consistent with our hypothesis that mature duct cells can transiently regain a less differentiated state (PDX-1þ) after replication, and this less restricted phenotype can serve as a multipotent progenitor (28, 29). Our hypothesis allows for both a lifetime supply of and a large number of potential progenitor cells. External stimuli, whether soluble factors or matrix components, can then direct differentiation of these multipotent cells to endocrine, acinar, or mature duct phenotypes. We recently reported that the addition of PDX-1 protein itself to cultured ductal cells induces the mRNA of PDX-1, insulin, and several other b-cell genes, supporting the origin of the new islets from duct cells themselves (33). These regressed or dedifferentiated duct cells should not be considered ‘true stem cells’ but rather as functional or ‘facultative’ stem cells much as the hepatocytes are (34). The regeneration that occurs in this model is by both replication of pre-existing differentiated acinar and bcells and neogenesis, resulting in the rapid formation of whole new lobes (28, 35). At 72 h after surgery, there are small clumps of branching ductal structures projecting from the common pancreatic duct. In sections, these clumps are seen as focal regions of proliferating ductules. These regions comprise 10—15% of the volume of the remnant pancreas, but by 7 d after Px, most of these regions have differentiated into new pancreatic lobes with a normal composition of exocrine and endocrine cells (Fig. 6). The pattern of formation of these new lobes suggests that they form from the ductal epithelium, as in the embryo. Confirmation of this interpretation will be dependent on lineage tracing using marked duct cells; such studies are in progress.

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Implications of the ductal epithelium as the progenitor pool

Fig. 6. New lobes of pancreas form after partial pancreatectomy (Px) in the rat. In this light micrograph of rat pancreas 7 d after 90% Px, clusterin-positive acinar cells (black) are found in one lobe. This same lobe has many cells that were labeled with BrdU that was injected at 2—3 d after surgery. The clusterin staining of acinar and heavy BrdU nuclear labeling mark new lobes that formed in the first few days after partial Px. Although no islet is seen in this field, these new lobes have the normal composition of exocrine and endocrine cells.

With the assumption that islets within these new lobes would be newly formed or neogeneic, we analyzed the gene expression of the new islets compared to the older mature islets in the remnant of the same animal, to develop markers of newly forming b-cells. To identify the new lobes, we used antibodies to clusterin, a glycoprotein of unknown function, expressed in newly differentiated acinar cells but not in newly replicated ones. This marker was identified several years ago by our finding that the acinar tissue of neonatal rats had intense and diffuse clusterin immunostaining, and this staining disappeared within 1—2 wk (Stubbs M and Scaglia L, unpublished data). Additionally, in the regenerating pancreas 1 wk after partial Px, whole lobes showed acinar staining for clusterin; these same lobes were identified as newly formed by the BrdU incorporation in all the cell types. In contrast, daughter cells of preexisting acinar cells in other lobes did not stain for clusterin. Using this marker to identify new lobes, we extracted both new and old islets from the same pancreas by laser-capture microdissection and compared their gene profiles by microarray hybridization (36). The differential expressions of a dozen selected highly expressed genes (particularly cell-surface molecules) were confirmed by reverse transcription-polymerase chain reaction and/or immunostaining. Comparison of gene expression profiles from isolated rat pancreatic ducts, new islets, and adult islets showed 1) the majority of the top candidate markers of new islets were also expressed in ducts and 2) 73 of the 80 most highly expressed genes in ducts were preferentially expressed in new islets, but not in mature islets. The overlapping expression of marker genes in both new b-cells and in pancreatic ducts supports the concept of a ductal origin of the new b-cells.

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One question often asked is ‘If all or most of the ducts are potential progenitors, why is there not islet regeneration in people with type 1 diabetes who receive immunosuppression for a kidney transplant?’ The flip answer is that we actually cannot determine whether there were some neogenesis in such clinical cases because we cannot measure it non-invasively. However, in non-obese diabetic mice, there are a few reports that such regeneration occurs once the autoimmune process is arrested (37, 38) However, none of these studies have shown an increase in b-cell mass nor neogenesis. The improved function is as likely due to residual islets recovering some function after the lymphocyte cytokine production has abated, an increase in b-cells within the residual islets by replication of remaining b-cells, and an increased insulin sensitivity. Although we do not know what triggers ductal replication in vivo, we do know that in the normal adult pancreas, the ducts are quiescent, being restrained by local factors. These restraints are removed if the ducts are isolated from their stroma and put into primary culture. Such findings suggest that local effects of matrix components and soluble factors, with TGF-b being a prime candidate, restrict ductal expansion in vivo (28). However, the local environment probably also provides a second set of signals as morphogens or differentiation factors as neogenesis is often seen when there is in vivo proliferation of the ductal epithelium, as in IFN-g (13), MT-TGF-a transgenic mice (14, 32), Px rats (29), and rodents treated with exendin 4 (11, 39) or keratinocyte growth factor (unpublished observations). Because unlimited islet formation could result in life-threatening hypoglycemia, there may be an evolutionary advantage to suppressed duct proliferation and, thus, a limitation of neogenesis. Understanding the regulation of the process of neogenesis may lead to therapies of islet replacement by stimulation of the endogenous pancreas. Already, several signaling molecules that may trigger ductal proliferation and differentiation, including epidermal growth factor/ gastrin (40, 41), tungstate (42), or islet neogenesisassociated protein (43), have been suggested as therapies. One implication of the pancreatic ducts as a progenitor pool is that the pool may be unlimited in vivo. Duct cells proliferate as well as comprising a substantial proportion of the pancreas, being 10% of the pancreas in rodents and 30—40% in humans (44), compared to 1—2% being islet. Ductal tissue has been expanded in vitro and passaged by many (45, 46). Islet tissue has been generated from both expanded human (18, 19) and mouse (47) ductal tissue as well as human ductal lines (48). Unfortunately, the efficiency of directing duct tissue to differentiate into functional islets is still low, Pediatric Diabetes 2004: 5: 16—22

Stem cells and diabetes but as we learn more about the normal adult process of neogenesis, the in vitro efficiency should improve.

Conclusion Our hypothesis is that mature pancreatic duct cells can transiently regain a less differentiated state (PDX-1þ) after replication, and this less restricted phenotype can serve as a multipotent progenitor. Thus, the pancreatic ductal epithelium serves as a potential pool of progenitor cells. Experiments are in progress to test this hypothesis with lineage tracing. If our hypothesis is correct, the number and availability of potential progenitor cells are promising.

Acknowledgements The authors thank the many other scientists who are or have been in the Section of Islet Transplantation at Joslin for their discussions and insights. The work on which this perspective is based has been funded by grants from the National Institutes of Health (NIDDK-44523, NIDDK-61251, to SBW), the Joslin Diabetes and Endocrinology Research Center cores (NIDDK DK-36836), the Juvenile Diabetes Research Foundation International (SBW and AS), and an important group of private donors. Dr Aye was supported by a Lawson Wilkins Fellowship and Dr Inada was partially supported by the Yamanouchi Foundation and Manpei Suzuki Diabetes Foundation.

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