Generation of Islets from Pancreatic Progenitor Cells 22

Diabetes, whether autoimmune type 1 or type 2, results from an inadequate supply of insulin- producing β cells. Transplantation of islets iso- lated from cadaver pancreata has become more successful with new protocols and immunosup- pressive drugs.

However, new sources of insulin-producing cells must become available to extend β-cell replacement therapy to more of the thousands of patients with diabetes. Because the β cell is the cell lacking in diabetes, it is gen- erally assumed that the replacement of just the β cell may be sufficient, but clear evidence is still lacking.

As late as two decades ago, it was still com- monly thought that one was born with all the β cells that one ever had; however, it has become clear that the mass of pancreatic β cells is dynamic and is regulated in an effort to maintain euglycemia.

A number of in vivo models have provided evidence that there is a cell renewal process (both replication and neo- genesis) that occurs at low levels in normal adult rodents and can be stimulated greatly by experimental conditions. This latter case is what should be considered regeneration; it is likely, but still needs rigorous evidence, that normal growth and regeneration in response to injury or to increased stimulation involve the same progenitor cells and the same path- ways. Understanding the regulation of the nor- mal process may lead to new therapies for diabetes that involve generation of new islets either in vitro for transplantation or in vivo by

stimulation of the endogenous pancreas. We will use replication to indicate the mitotic divi- sion of a cell already with a β cell phenotype and neogenesis to indicate the formation of new β cells from either progenitor or stem cells.

Normal Growth

Islet mass increases more than 20-fold from the newborn to the adult, but the volume of islet tis- sue relative to that of the pancreas decreases from birth to adulthood.

The relation of β cell mass and age through life in the Sprague- Dawley rat can be represented by a simple mass model equation.

The β cell mass is linearly cor- related with body weight from 4 weeks to 6 months of age in male mice.

The slope of the linear regression varies with mouse strain. In our compilation of data from different aged Sprague-Dawley rats, it was clear that β cell mass increased with age in the adult rat because of increases in cell number. At any one time, the number of β cells is determined by the balance of cell renewal and cell loss.

The mechanisms to achieve a dynamic state of β cell mass include changes in new cell forma- tion both by replication and by differentiation of new islets (neogenesis), changes in individual cell size/volume (atrophy to hypertrophy), and changes in cell loss or death rates.

All of these

22

Generation of Islets from Pancreatic Progenitor Cells

Susan Bonner-Weir, Tandy Aye, Akari Inada, Elena Toschi, and Arun Sharma

309

mechanisms have been shown in adult rodents.

When there is an increase in β cell mass, the cell renewal rate must be greater than that of cell loss. Loss of β cells has been shown to occur in normal conditions both in vitro and in vivo dur- ing the involution of the β cell mass in the post- partum pancreas

and in the normal neonatal rat.

In the neonatal period in rodents, there is a remodeling of the endocrine pancreas that is characterized by very active β-cell replication and neogenesis and an increase in β-cell apopto- sis near the time of weaning.

In a longitudinal study,

the β cell mass increased throughout the life of the male Lewis rat: Initially by an increase in cell number, and then from 15 months onward by an increase in cell size or hypertro- phy of the β cell. In addition, β-cell hypertrophy has been reported in rats during glucose infu- sions,

pregnancy,

and partial pancreatectomy (Px).

Neogenesis is responsible for most of the islet growth during the fetal stage, with replica- tion being high in the perinatal period. In rats, we have found two waves of neogenesis during the neonatal period: One immediately after birth and the second around weaning. Using data from our longitudinal study of the β cell mass and its determinants,

we estimate that more than 30% of the β cells seen at day 31 could not be accounted for by replication of preexisting β cells. However, with the natural turnover of cells, a gradual but complete replacement of the β-cell population should occur over time. Thus, the endocrine pancreas should be considered a slowly renewed tissue.

Experimental evidence from rodents shows that there is a substantial compensatory effort by the β cells to maintain normal glycemic levels in the face of obesity and insulin resistance. Two types of compensation can occur: A functional one in which each β cell secretes more insulin, and a second in which there is an increase in β cell mass. Functional adaptations include changes in threshold for glucose-induced insulin secretion that occur during pregnancy

and glucose-induced increases in glucokinase activity.

Nonetheless, the β cell mass itself is the major factor that determines the amount of insulin that can be secreted. Mice with geneti- cally induced peripheral insulin resistance have an up to 30-fold larger β cell mass, compared with normal mice.

There is also evidence from human autop- sied pancreata to suggest a compensatory growth of the β cell mass in obesity. A small but

careful study of autopsied pancreata from patients with type 2 diabetes and nondiabetic subjects showed that the β cell mass of obese subjects was approximately 40% larger than that of lean subjects.

This compensatory growth of the human β cell mass with obesity was recently confirmed using many more pan- creata.

What Is the Origin of New β Cells?

Although it is clear that new β cells are formed in the adult pancreas, the source of these new β cells continues to be a matter of debate.

Suggestions for the origin of new β cells include:

1) Replication of preexisting β cells

; 2) differ- entiation from another islet cell or an intraislet stem cell

; 3) transdifferentiation from acinar cells; and 4) differentiation from a cell within the duct epithelium

(Figure 22.1). More than one mechanism may be involved in new β-cell for- mation after birth, and normal growth and regeneration or response to injury may occur through different mechanisms. For example, replication of hepatocytes accounts for most of liver growth and regeneration, but the oval cells retain a bipotential capacity to form either new hepatocytes or bile duct epithelium. In aiming to expand the β cell mass either in vivo or in vitro for therapeutic purposes, the main crite- rion is that the cells of origin have the capacity to expand the functional β cell number.

Replication

Many scientists were mistakenly led to think

that β cells did not replicate because of the low

observed replication rate of β cells and the con-

cept that terminally differentiated cells do not

replicate. Yet the enlarged islets and increased

β cell mass seen in obesity and insulin resistance

supported replication. Replication was easily

verified by incorporation of labeled thymidine

or its analog bromodeoxyuridine (BrdU) into

new DNA, immunostaining of cell cycle-specific

proteins, or the presence of mitotic figures. In

adult mice, β cell compensation as the result of

pregnancy or response to insulin resistance

occurs predominantly by replication, with little

neogenesis.

Using various morphometric

analyses, the increase in β cell mass in these

studies was shown to result predominantly from an increase in islet size, not islet number.

A recent study using lineage-tracing techniques confirmed the major role of replication in the normal adult mouse.

Replication is also a major response during the regeneration after partial Px. At 3 and 7 days after a 90% Px in the adult rat, both the β cells and exocrine cells have mitotic indices three- to fourfold higher than that of the sham animals.

At 14 days, the exocrine cells have only a slightly increased mitotic index, whereas that of the β cells remains double that of the sham animals even at 3 weeks. Because glucose is a powerful mitogenic stimulus for β cells, the continued enhancement of β-cell replication probably results from the mild hyperglycemia seen in these animals. Replication makes a significant contribution to the increase in β cell mass in these Px animals by increasing the number of β cells in the islets. Islets of equivalent size from 4-week Px and sham-operated animals have similar amounts of RNA, but the proportion of β cells is increased in the Px islets.

Intraislet Stem or Progenitor Cells

Some studies have made a case for progenitor cells residing within the islets. In streptozotocin- treated rats, cells that stained for both insulin and somatostatin were suggested as intraislet

progenitor or stem cells that regenerated into β cells.

Treatment with betacellulin after strep- tozotocin led to an increase in the number of insulin-positive cells, as well as BrdU-labeled somatostatin-positive cells, providing support for the intraislet progenitor concept.

This hypothesis was also favored in a cell-lineage tracing study which labeled cells expressing neurogenin 3, a transcription factor active in islet development, in adult pancreas.

Twenty- four hours after the labeling pulse, the few labeled cells were found only in islets; the sim- plest explanation was that there were progenitor cells within the islet. Another cell type, the nestin-positive, hormone-negative cell grown from isolated islets, has been suggested as an in vitro islet progenitor cell.

These studies are provocative and potentially important, but more work is needed to understand whether these cells contribute significantly to in vivo β-cell growth or regeneration.

Acinar Cell Transdifferentiation

It has been difficult to prove that differentiated adult pancreatic acinar cells are converted to insulin-producing cells. The term transdifferen- tiation is often used to describe the process.

A likely scenario is that the acinar cells regress to a duct-like phenotype, and these cells then function as progenitors. Duct-like “tubular

Figure 22.1. Possible sources of cells that can give rise to new insulin-producing β cells. New β cells are found in the pancreas after birth. The origin

of new β cells may include: 1) Replication of preexisting β cells; 2) differentiation from another islet cell or an intraislet stem cell; 3) transdifferentia-

tion from acinar cells; and 4) differentiation from a cell within the duct epithelium.

complexes” have been reported in various dis- eases such as chronic pancreatitis, pancreatic adenocarcinoma and cystic fibrosis,

pancre- atitis after ductal ligation,

and in regenera- tion of exocrine pancreas after experimentally induced acute pancreatitis.

The origin of these structures has been debated, with sugges- tions ranging from proliferation or reduplica- tion of intralobular ducts

to dedifferentiation of acinar tissue into duct-like structures.

In ductal ligation studies, it is clear that there is replication of ductal cells, increased number of hormone-positive cells budding from ducts, and initially increased β cell mass; however, it is unclear if these replicating ducts originate from acinar or duct tissue.

A rat duct ligation model in which gastrin was infused produced changes consistent with transdifferentiation of acinar cells to duct cells and subsequent β-cell neogenesis.

A similar pathway leading to β- cell neogenesis has been postulated in rats receiving glucose infusions.

Interestingly, these tubular complexes resem- ble the focal regions of regeneration seen in the Px model. Data from this latter model sup- ports a regenerative origin of these focal regions:

1) The BrdU data show expansion of the ductal tree and then further proliferation of branching ductules; 2) the coincidence of the appearance and disappearance of the focal regions with the marked growth of the pancreatic remnant; and 3) the continuum of differentiation from ductal epithelium to islets and acini.

Neogenesis

Replication is clearly a pathway for new β cells in adult rodents, but neogenesis is also seen. The process of neogenesis in the adult pancreas closely resembles embryonic new islet forma- tion [Figure 22.2 (see color section)].

In both, hormone-containing cells are seen first within the basement membrane of the duct epithelium and then the islet cells delaminate and coalesce.

Adult duct tissue retains the ability to differen- tiate into islet cells. When adult ductal epithe- lium was wrapped in fetal mesenchyme and implanted in nude mice, approximately 20% of the grafts resulted in budding of islet-like clus- ters that had islet hormone immunostaining.

Whereas replication is easy to verify, measurement of neogenesis has been less direct

or definitive. There are no good markers for it yet, and increased hormone staining within the ducts has been considered indicative of neogen- esis. In normal adult rodent pancreas, occa- sional (1 in 1000) cells within the ductal epithelium immunostain for an islet hormone,

but neogenesis is seen experimentally with dietary treatment with soybean trypsin inhibitors,

overexpression of interferon (IFN)- γ in the β cells of transgenic mice,

overexpres- sion of transforming growth factor (TGF)- α,

after partial pancreatectomy,

after cellophane wrapping of the head of the pancreas,

after treatment with exendin 4

or betacellulin,

and clinically with recent onset of type 1 dia- betes and severe liver disease.

In humans, neogenesis seems to have a more important role in the compensation of the β cell mass in obe- sity, compared with replication. In the samples from the Butler-Mayo collection

as well as from the more than 100 human organ donations to the Harvard Islet Transplantation program, neogeneic regions of ductal profiles and hor- mone-positive cells budding from these ducts are more common than enlarged islets that would be seen with enhanced replication. The dominance of the replicative pathway in adult mice may reflect their long telomeres such that telomere shortening is not limiting,

whereas in humans, in whom telomere shortening is limit- ing and the rate of β-cell replication appears very low,

neogenesis may be more important.

Models of Neogenesis

In rodents, there is considerable evidence of neogenesis in a number of models, but no line- age-tracing evidence is available as yet. Dor et al.

recently found no evidence of neogenesis in mice after a 70% partial Px using lineage trac- ing. However, one must realize that it is always difficult to show the absence of an event.

Additionally, this work raised a number of experimental concerns, particularly regarding the possibility that the sampling was inade- quate.

In contrast, other studies show strong, albeit circumstantial evidence, of islet neogene- sis in the adult.

The ductal ligation models mentioned above

showed increases of ductal replication, islet neo-

genesis, and β cell mass.

An important

unanswered question is whether the acinar tis-

sue mainly dies and the ductal component

proliferates or, alternatively, the acinar pheno- type is lost (transdifferentiates or dedifferenti- ates) to become a ductal phenotype. In either case, the budding of islet tissue from ductal structures is well documented.

In the RIP-IFN- γ transgenic mice, newly formed islets were often seen bulging within the lumen of the ducts, suggesting their origin from ducts.

Intriguingly, occasional cells that express intestinal or hepatocyte markers were also seen budding from the ducts, implying plas- ticity. Initially, it was thought that the insulitis seen in this model may have triggered the neo- genesis, but similar duct proliferation and islet neogenesis were observed with this transgene on an immunocompromised background. In the RIP-IFN- γ transgenic mice, PDX1 protein, a transcription factor important for both pancreas development and β-cell function, is seen in pro- liferating ducts from which there is budding of new islets; additional transcription factors known to be involved in the differentiation of islets were expressed in these areas,

again suggesting active new islet formation.

The cellophane-wrapped pancreas of Syrian golden hamster resulting in partial duct obstruction has been studied by the groups of Rosenberg and Vinik.

This model differs from the ductal ligation model because there is no obvious acinar degeneration reported. In the original study, half of the streptozotocin- induced diabetic animals became euglycemic after cellophane wrapping, with the restoration thought to be the result of new islet formation rather than replication.

Even though the early studies defining the time course of euglycemia restoration after the surgery are incomplete, and the model is only reported in the hamster, this model has led to the identification of a putative factor that initiates islet neogenesis, INGAP (islet neogenesis associated protein), a member of the pancreatitis-associated peptide family.

More work is needed to determine if this protein can stimulate neogenesis in other models and species.

In the 90% Px rat model, a well-defined rem- nant of 10% of the weight and of islet mass left in a young adult rat regenerates to 27% of the weight of the sham-operated pancreas with 45%

of the sham islet mass by 4 weeks after sur- gery.

The regeneration occurs in this model both by replication of preexisting differ- entiated acinar and β cells and by neogenesis, resulting in the rapid formation of whole new

lobes.

At 72 hours after surgery, small clumps of branching ductal structures project 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 days after Px, most of these regions have differ- entiated into new pancreatic lobes with a normal composition of exocrine and endocrine cells.

Oval Cells as Possible Pancreatic Islet Progenitors

Because neogenesis implies the formation of new islets from the ducts, one must consider which cells are the actual progenitors. By ultra- structural analysis in both embryonic

and postnatal pancreas, hormone-positive cells are seen within the basement membrane of the duc- tal epithelium, therefore circulating or stromal cells can be ruled out as progenitors for the bulk of new islet formation. Another possibility may be the pancreatic equivalent of “oval cells”

found in the liver.

After liver injury from car- cinogenic agents, there seems to be proliferation of small (7 µm), oval cells just at the periductu- lar junction; these cells have the potential to dif- ferentiate into bile duct epithelium or hepatocytes.

In this schema, pancreatic oval cells would proliferate, delaminate from the epithelium with disruption of the plasma mem- brane, and coalesce into islets. Ultrastructural analysis of the common pancreatic duct of adult rats shows rare (1 in 100–200 cells) basal cells that are small and ovoid with fairly undifferenti- ated cytoplasm; these cells resemble liver oval cells.

When Px rats were given colchicine in order to arrest mitosis at 20 hours after surgery, there was no evidence of an amplification of a small population of putative stem cells/oval cells, with all the arrested cells being columnar epithelial cells. Even so, in the liver, the oval cells are not the usual mechanism for regeneration but rather a backup one.

Additionally, estimates of the number of pro-

genitor cells needed for formation of a new islet

are not consistent with a progenitor role for

these “oval cells” in the Px model. After partial

Px in rats, fully formed new islets can be seen by

72 hours after surgery, each consisting of

1000–1500 cells. No increased replication in the

pancreas is seen about 24 hours after surgery, so

the islets must form within 48 hours. With cell- cycle times in rodents being 10–20 hours, 16 starting cells with a doubling time of 10 hours (five doublings over 48 hours) would be required to make 1000 cells or one islet. If the doubling time were 12 hours, then 64 cells would be required. Because there are so few nonduct cells found ultrastructurally within the basement membrane of the duct epithelium, these calcula- tions favor the possibility that the duct epithelial cells themselves are the progenitor cells.

Ductal Epithelial Cells as Pancreatic Progenitors

Another schema would have the ductal epithe- lial cells regress to a less differentiated stage after replication, and then in a dedifferentiated state function as progenitor cells.

This hypoth- esis allows for both a lifetime supply and a large number of potential progenitor cells. External signals can then direct differentiation of these multipotent cells to endocrine, acinar, or mature duct phenotypes. This hypothesis is consistent with data from many models and the estimated number of cells needed for each new islet described above. Lineage tracing experiments using a duct promoter are in process and should address this question directly.

Data from the regenerating rat pancreas after partial Px favor this hypothesis. After partial Px, ductal cells rapidly replicate and dedifferentiate with a marked increase in PDX1 protein. PDX1 protein is normally expressed in pancreatic ducts until shortly before birth; others have sug- gested it as a marker for the adult progenitor cells.

The return to a more embryonic phe- notype precedes the formation of whole new lobes of pancreas.

A wave of DNA synthesis (as seen by BrdU incorporation) passes through the ductal tree. Whereas little to no PDX1 pro- tein was detected in the epithelium of the quies- cent common pancreatic duct from unoperated or sham-operated animals 24 hours after Px, most of the ductal epithelial cells were BrdU

PDX1

or BrdU

PDX1

in the Px animals. Then by 3 days after Px, few cells of the common pan- creatic duct epithelium were BrdU

but most cells still expressed PDX1 protein. Addition of PDX1 protein, which has a protein transduction domain that allows the protein to enter cells, to cultured ductal cells induced mRNA of PDX1,

insulin, and several other β-cell genes, support- ing the origin of the new islets from duct cells themselves.

The MT-TGF- α transgenic mouse supports the concept of ductal cells as the islet progenitor cells.

In this mouse, the pancreas has uncontrolled ductular proliferation resulting in metaplastic ductules, increased fibrosity, and interstitial cel- lularity.

PDX1 expression was seen in the meta- plastic ductal epithelium, as well as focally expressed PAX6.

The data from both MT-TGF- α and RIP-IFN-γ mice on the expression of PDX1 protein after replication of the ductal cells are consistent with regression of the duct cells to a more embryonic state. In the MT-TGF- α trans- genic mice, 6% of the epithelial cells in the meta- plastic ducts expressed low levels of insulin, with 1% showing strong staining and presence of insulin granules,

again suggesting the transition from duct to islet phenotype.

The marked change in composition of porcine neonatal pancreatic cell clusters trans- planted under the kidney capsule of immunode- ficient mice adds even further support.

When transplanted, the majority of cells can be stained with the duct marker cytokeratin 7, but, after several months, 94% of the cells were β cells.

During this engraftment, as well as in intact neonatal pancreas, cells costained for both insulin and cytokeratin 7 are found, suggesting residual expression of duct markers as duct cells become β cells. This finding is extended by gene profiling with microarrays of new versus mature islets 7 days after partial Px in rats.

The β cells of new islets, which can be identified in the new lobes, express a variety of duct markers at both the mRNA and protein level, also supporting the duct progenitor hypothesis.

Recent Evidence for Pancreatic Progenitor Cells with Limited Clonal Expansion

Two recent articles

reported isolation of cells

from whole mouse pancreas that can be expanded

clonally and express low levels of insulin and

other pancreatic markers. These cells were rare

(one in several thousand) and not yet localized

within the pancreas. Although it is not clear yet

that these cells can differentiate to fully

functional islet cells, their potential is very excit-

ing, and further studies on such cells are eagerly awaited.

Implications of In Vivo Data for Formation of New β Cells for Therapy

Understanding the regulation of the normal process may lead to new therapies for diabetes that involve generation of new islets either in vitro for transplantation or in vivo by stimu- lation of the endogenous pancreas. β cells are formed after birth from several different origins or pathways. Data support occurrence of β-cell replication, and differentiation from a pancre- atic stem cell/progenitor within the islet or within the duct, or from a duct-like structure that was previously a mature duct or acinar cell, or a combination of these pathways. In each model, more than one pathway may be involved.

In fact, the data suggest that different states (injury versus normal growth) or different species favor one or another pathway. An important issue is how this knowledge can be used to generate new islets for therapeutic replacement.

In vitro replication of β cells has been studied for many years.

It is clear from these studies that the conditions used in vitro do not yet match those found in vivo because it has not been possible to even double the number of β cells in vitro. Under conditions favoring sub- stantial expansion, using growth factors and matrices or oncogene expression, the β-cell phe- notype is lost

but there has been question whether the proliferation of contaminant ductal cells diluted out the β cells.

Reaggregation of the expanded cells did lead to some reexpression of islet mRNAs, but more work is needed to improve the expansion of primary β cells.

Studies manipulating the culture further may eventually yield conditions for expansion and then redifferentiation of β cells; this is an area of great importance.

Several groups have been studying the poten- tial of non–islet pancreatic tissue for in vitro gen- eration of islets. Human duct-cell-rich fractions remaining after islet isolation and purification have been cultured and shown to form new islets after exposure to growth factors and matrix.

Over 3–4 weeks of culture, the insulin content per flask increased 10- to 15-fold as the DNA content increased up to 7-fold. The cultivated

human islet buds, shown by immunofluores- cence to consist of cytokeratin 19-positive duct cells and hormone-positive islet cells, were glu- cose responsive. With semiquantitative multi- plex reverse transcriptase-polymerase chain reaction, transcription factors known to be involved in islet development, such as Ngn3 and Pax4, which were not detected in initial cells, adult islets, or acinar fractions, were evident in final differentiated cultures. The finding that transcription factors important to embryonic islet development are involved in islet differen- tiation from adult human pancreatic cells sug- gests an active differentiation process. The efficiency of differentiation is at present inade- quate to render this process clinically useful.

Work continues to increase the new islet forma- tion in vitro. The cells that give rise to the new islets in this heterogeneous cell population have not been identified as yet, although they have been shown to be initially in the epithelial com- ponent and thought to be ductal.

Their ductal origin is supported by other studies that have manipulated in culture the human pancreatic duct PANC1 cell line,

or cells expanded from mouse pancreatic duct cells to give islet hor- mone-expressing cells.

In the latter report, the cells were able to reverse diabetes in mice, but concerns have been raised about compatibility of these results with the very low insulin content of the cells.

However, Bouwens and col- leagues

have shown that both human and rat acinar tissue in culture can become duct-like or even hepatocyte-like, depending on the culture conditions, suggesting that at least some of the cells from the human cultures may have been acinar cells previously. Some acinar cells clearly die when cultured but some may lose their aci- nar phenotype and regress to a duct-like pro- genitor as we have suggested for the mature ducts.

Other approaches to enhance islet neogenesis

are being investigated using signaling mole-

cules that have been reported to stimulate β cell

growth or differentiation. These molecules,

including exendin 4/GLP-1,

betacel-

lulin,

EGF/gastrin,

tungstate,

INGAP,

and conophylline,

are being tested in vivo as

well as in culture. Identification of the progeni-

tor cells by lineage tracing from cells with acinar

or ductal phenotype, or localization of cells that

have clonal expansion and differentiative

capacities, will also be critical to this field. The

importance of identifying these cells is further

highlighted by recent results suggesting that in humans neogenesis may have a more important role in increasing β cell mass than replication.

Thus, for the purpose of therapeutically expand- ing the human β cell mass, the main criterion should be to identify progenitor cells that have the capacity to expand, either in vitro or in vivo, the functional β cell number. Hence, to achieve the therapeutic goal, a continued inter- est in the process of neogenesis of β cells from progenitors is necessary and will require many different avenues to study the regulation of its normal process. This, we suggest, would lead to new therapies for diabetes that involve genera- tion of new islet tissue, either in vivo by stimula- tion of the endogenous pancreas or in vitro for transplantation.

References

1. Shapiro AM, Lakey JR, Ryan EA, et al. Islet transplan- tation in seven patients with type 1 diabetes mellitus using a glucocorticoid-free immunosuppressive regi- men. N Engl J Med 2000;27:230–238.

2. Ryan EA, Lakey JR, Paty BW, et al. Successful islet trans- plantation: continued insulin reserve provides long- term glycemic control. Diabetes 2002;51:2148–2157.

3. Gu D, Sarvetnick N. A transgenic model for study- ing islet development. Recent Prog Horm Res 1994;49:

161–165.

4. Bonner-Weir S. Life and death of the pancreatic beta cells. Trends Endocrinol Metab 2000;11:375–378.

5. Bonner-Weir S, Sharma A. Pancreatic stem cells.

J Pathol 2002;197:519–526.

6. Kloppel G, Lohr M, Habich K, Oberholzer M, Heitz PU. Islet pathology and the pathogenesis of type 1 and type 2 diabetes mellitus revisited. Surv Synth Pathol Res 1985;4:110–125.

7. Finegood DT, Scaglia L, Bonner-Weir S. Dynamics of β-cell mass in the growing rat pancreas: estimation with a simple mathematical model. Diabetes 1995;44:

249–256.

8. Bonner-Weir S. Islet growth and development in the adult. J Mol Endocrinol 2000;24:297–302.

9. Scaglia L, Smith FE, Bonner-Weir S. Apoptosis contributes to the involution of β cell mass in the post partum rat pancreas. Endocrinology 1995;136:

5461–468.

10. Scaglia L, Cahill CJ, Finegood DT, Bonner-Weir S.

Apoptosis participates in the remodeling of the endocrine pancreas in the neonatal rat. Endocrinology 1997;138:1736–1741.

11. Montanya E, Nacher V, Biarnes M, Soler J. Linear correlation between beta cell mass and body weight throughout life in Lewis rats: role of beta cell hyperplasia and hypertrophy. Diabetes 2000;49:

1341–1346.

12. Bonner-Weir S, Deery D, Leahy JL, Weir GC.

Compensatory growth of pancreatic B-cells in adult

rats after short-term glucose infusion. Diabetes 1989;38:49–53.

13. Jonas J-C, Sharma A, Hasenkamp W, et al. Chronic hyperglycemia triggers loss of pancreatic β cell differ- entiation in an animal model of diabetes. J Biol Chem 1999;274:14112–14121.

14. Parsons JA, Brelje TC, Sorenson RL. Adaptation of islets to pregnancy: increased islet cell proliferation and insulin secretion correlates with the onset of placental lactogen secretion. Endocrinology 1992;130:1459–1466.

15. Chen C, Hosokawa H, Bumbalo LM, Leahy JL.

Regulatory effects of glucose on the catalytic activity and cellular content of glucokinase in the pancreatic B cell. J Clin Invest 1994;94:1616–1620.

16. Bruning JC, Winnay J, Bonner-Weir S, Taylor SI, Accili D, Kahn CR. Development of a novel polygenic model of NIDDM in mice heterozygous for IR and IRS-1 null alleles. Cell 1999;88:561–572.

17. Butler AE, Janson J, Bonner-Weir S, Ritzel R, Rizza RA, Butler PC. Beta-cell deficit and increased beta-cell apoptosis in humans with type 2 diabetes. Diabetes 2003;52:102–110.

18. Yoon KH, Ko SH, Cho JH, et al. Selective β-cell loss and α-cell expansion in patients with type 2 dia- betes mellitus in Korea. J Clin Endocrinol Metab 2003;88:2300–2308.

19. Dor Y, Brown J, Martinez OI, Melton DA. Adult pan- creatic beta-cells are formed by self-duplication rather than stem-cell differentiation. Nature 2004;429:41–46.

20. Fernandes A, King LC, Guz Y, Stein R, Wright CV, Teitelman G. Differentiation of new insulin-producing cells is induced by injury in adult pancreatic islets.

Endocrinology 1997;138:1750–1762.

21. Guz Y, Nasir I, Teitelman G. Regeneration of pancre- atic beta cells from intra-islet precursor cells in an experimental model of diabetes. Endocrinology 2001;142:4956–4968.

22. Sharma A, Zangen DH, Reitz P, et al. The home- odomain protein IDX-1 increases after an early burst of proliferation during pancreatic regeneration.

Diabetes 1999;48:507–513.

23. Parsons JA, Bartke A, Sorenson RL. Number and size of islets of Langerhans in pregnant human growth hormone-expressing transgenic, and pituitary dwarf mice: effect of lactogenic hormones. Endocrinology 1995;136:2013–2021.

24. Bock T, Pakkenberg B, Buschard K. Increased islet volume but unchanged islet number in ob/ob mice.

Diabetes 2003;52:1716–1722.

25. Brockenbrough JS, Weir GC, Bonner-Weir S.

Discordance of exocrine and endocrine growth after 90% pancreatectomy in rats. Diabetes 1988;37:232–236.

26. Zangen DH, Bonner-Weir S, Lee CH, et al. Reduced insulin, GLUT2, and IDX-1 in B-cells after partial pan- createctomy. Diabetes 1997;46:258–264.

27. Li L, Seno M, Yamada H, Kojima I. Betacellulin improves glucose metabolism by promoting conver- sion of intraislet precursor cells to beta-cells in strep- tozotocin-treated mice. Am J Physiol Endocrinol Metab 2003;285:E577–583.

28. Gu G, Brown JR, Melton DA. Direct lineage tracing reveals the ontogeny of pancreatic cell fates during mouse embryogenesis. Mech Dev 2003;120:35–43.

29. Zulewski H, Abraham EJ, Gerlach MJ, et al.

Multipotential nestin-positive stem cells isolated from

adult pancreatic islets differentiate ex vivo into pan-

creatic endocrine, exocrine, and hepatic phenotypes.

Diabetes 2001;50:521–533.

30. Bouwens L. Transdifferentiation versus stem cell hypothesis for the regeneration of islet beta-cells in the pancreas. Microsc Res Tech 1998;43:332–336.

31. Bockman DE, Black O Jr, Mills LR, Webster PD. Origin of tubular complexes developing during induction of pancreatic complexes during induction of pancreatic adenocarcinoma by 7,12-dimethylbenz(a) antracene.

Am J Physiol 1978;90:645–651.

32. Porta EM, Stein AA, Patterson P. Ultrastructural changes of the pancreas and liver in cystic fibrosis.

Am J Clin Pathol 1964;42:451–465.

33. Willemer S, Elsasser HP, Kern HF, Adler G. Tubular complexes in cerulein- and oleic acid-induced pancre- atitis in guts: glycoconjugate pattern, immunocyto- chemical and ultrastructural findings. Pancreas 1987;2:669–675.

34. DeLisle RC, Grendel JH, Williams JA. Growing pan- creas acinar cells (post pancreatitis and fetal) express a ductal antigen. Pancreas 1990;5:381–388.

35. Dreiling DA, Bordalo OR, Noronha M, Greenstein R.

Update: big duct, little duct or toxic metabolic patho- genesis of pancreatitis. Am J Gastroenterol 1979;71:

424–426.

36. Edstrom C. Further quantitative structural studies of the pancreatic islet parenchyma in rats with duct liga- tion. Acta Soc Med Ups 1971;76:127–138.

37. Hultquist GT, Karlsson U, Hallner ACH. The regener- ative capacity of the pancreas in duct-ligated rats. Exp Pathol 1979;17:44–52.

38. Rooman I, Lardon J, Bouwens L. Gastrin stimulates beta-cell neogenesis and increases islet mass from transdifferentiated but not from normal exocrine pan- creas tissue. Diabetes 2002;51:686–690.

39. Lipsett M, Finegood DT. Beta-cell neogenesis during prolonged hyperglycemia in rats. Diabetes 2002;51:

1834–1841.

40. Pictet R, Rutter WJ. The endocrine pancreas. In:

Steiner D, Freinkel N, eds. Handbook of Physiology.

Baltimore: Williams & Wilkins; 1972:25–66.

41. Dudek RW, Lawrence IE Jr. Morphologic evidence of interactions between adult ductal epithelium of pancreas and fetal foregut mesenchyme. Diabetes 1988;37:891–900.

42. Dudek RW, Lawrence IE Jr, Hill RS, Johnson RC.

Induction of islet cytodifferentiation by fetal mes- enchyme in adult pancreatic ductal epithelium.

Diabetes 1991;40:1041–1048.

43. Madden ME, Sarras MP Jr. The pancreatic ductal sys- tem of the rat: cell diversity, ultrastructure, and inner- vation. Pancreas 1989;4:472–485.

44. Weaver CV, Sorenson RL, Kaung HC. Immuno- cytochemical localization of insulin-immunoreactive cells in the ducts of rats treated with trypsin inhibitor.

Diabetologia 1985;28:781–785.

45. Gu D, Sarvetnick N. Epithelial cell proliferation and islet neogenesis in IFN- γ transgenic mice.

Development 1993;118:33–46.

46. Wang TC, Bonner-Weir S, Oates PS, et al. Pancreatic gastrin stimulates islet differentiation of transforming growth factor α-induced ductular precursor cells.

J Clin Invest 1993;92:1349–1356.

47. Bonner-Weir S, Baxter LA, Schuppin GT, Smith FE.

A second pathway for regeneration of the adult exocrine and endocrine pancreas: a possible recapit-

ulation of embryonic development. Diabetes 1993;42:

1715–1720.

48. Rosenberg L, Vinik AI, Pittenger GL, Rafaeloff R, Duguid WP. Islet-cell regeneration in the diabetic hamster pancreas with restoration of normoglycemia can be induced by local growth factors. Diabetologia 1996;39:256–262.

49. Xu G, Stoffers DA, Habener JF, Bonner-Weir S.

Exendin-4 stimulates both β-cell replication and neo- genesis, resulting in increased β-cell mass and improved glucose tolerance in diabetic rats. Diabetes 1999;48:2270–2276.

50. Stoffers DA, Kieffer TJ, Hussain MA, et al.

Insulinotropic glucagon-like peptide 1 agonists stimu- late expression of homeodomain protein IDX-1 and increase islet size in mouse pancreas. Diabetes 2000;49:741–748.

51. Yamamoto K, Miyagawa J, Waguri M, et al.

Recombinant human betacellulin promotes the neogenesis of beta-cells and ameliorates glucose intol- erance in mice with diabetes induced by selective alloxan perfusion. Diabetes 2000;49:2021–2027.

52. Gepts W. Contribution a l’etude morphologique des ilots de Langerhans au cours du diabete. Ann Soc R Sci Med Nat Brux 1957;10:105–108.

53. Gepts W. Pathological anatomy of the pancreas in juvenile diabetes. Diabetes 1965;14:619–633.

54. Forsyth NR, Wright WE, Shay JW. Telomerase and differentiation in multicellular organisms: turn it off, turn it on, and turn it off again. Differentiation 2002;69:188–197.

55. Tyrberg B, Ustinov J, Otonkoski T, Andersson A.

Stimulated endocrine cell proliferation and differenti- ation in transplanted human pancreatic islets: effects of the ob gene and compensatory growth of the implantation organ. Diabetes 2001;50:301–307.

56. Bonner-Weir S, Toschi E, Inada A, et al. The pancre- atic duct epithelium serves as a potential pool of pro- genitor cells. Pediatr Diabetes 2004;5(Suppl 2):16–22.

57. Rooman I, Lardon J, Bouwens L. Gastrin stimulates beta-cell neogenesis and increases islet mass from transdifferentiated but not from normal exocrine pan- creas tissue. Diabetes 2002;51:686–690.

58. Sarvetnick NE, Gu D. Regeneration of pancreatic endocrine cells in interferon-gamma transgenic mice.

Adv Exp Med Biol 1992;321:85–89.

59. Kritzik MR, Jones E, Chen Z, et al. PDX-1 and Msx-2 expression in the regenerating and developing pan- creas. J Endocrinol 1999;163:523–530.

60. Kritzik MR, Krahl T, Good A, et al. Transcription fac- tor expression during pancreatic islet regeneration.

Mol Cell Endocrinol 2000;164:99–107.

61. Rafaeloff R, Pittenger GL, Barlow SW, et al. Cloning and sequencing of the pancreatic islet neogenesis associated protein (INGAP) gene and its expression in islet neogenesis in hamsters. J Clin Invest 1997;99:

2100–2109.

62. Rosenberg L, Duguid WP, Vinik AI. The effect of cel- lophane wrapping of the pancreas in the Syrian golden hamster: autoradiographic observations. Pancreas 1989;4:31–37.

63. Rosenberg L, Vinik AI. Trophic stimulation of the

ductular-islet cell axis: a new approach to the

treatment of diabetes. Adv Exp Med Biol

1992;321:95–104.

64. Rosenberg LA, Brown RA, Duguid WP. A new approach to the induction of duct epithelial hyperpla- sia and nesidioblastosis by cellophane wrapping of the hamster pancreas. J Surg Res 1983;35:63–72.

65. Bonner-Weir S, Trent DF, Weir GC. Partial pancreatec- tomy in the rat and subsequent defect in glucose- induced insulin release. J Clin Invest 1983;71:1544–1553.

66. Bonner-Weir S, Stubbs M, Reitz P, Taneja M, Smith FE. Partial pancreatectomy as a model of pancreatic regeneration. In: Sarvetnick N, ed. Pancreatic Growth and Regeneration. Basel, Switzerland: Karger Landes Systems; 1997:138–153.

67. Farber E. The multistep nature of cancer develop- ment. Cancer Res 1984;44:4217–4223.

68. Evarts RP, Nagy P, Marsden E, Thorgeirsson SS.

A precursor-product relationship exists between oval cells and hepatocytes in rat liver. Carcinogenesis 1987;8:1737–1740.

69. Germain L, Blouen MJ, Marceau N. Biliary epithelial and hepatocyte cell lineaged relationships in embry- onic rat liver. Cancer Res 1988;48:4909–4918.

70. Bouwens L, De Blay E. Islet morphogenesis and stem cell markers in rat pancreas. J Histochem Cytochem 1996;44:947–951.

71. Song SY, Gannon M, Washington MK, et al.

Expansion of Pdx1-expressing pancreatic epithelium and islet neogenesis in transgenic mice overexpress- ing transforming growth factor α. Gastroenterology 1999;117:1416–1426.

72. Noguchi H, Kaneto H, Weir GC, Bonner-Weir S. PDX- 1 protein containing its own antennapedia-like pro- tein transduction domain can transduce pancreatic duct and islet cells. Diabetes 2003;52:1732–1737.

73. Jhappan C, Stahle C, Harkins RN, Fausto N, Smith GH, Merlino GT. TGF α overexpression in transgenic mice induces liver neoplasia and abnormal development of the mammary gland and pancreas. Cell 1990;61:

1137–1146.

74. Yoon K-H, Quickel RR, Tatarkiewicz K, et al.

Differentiation and expansion of beta cell mass in porcine neonatal pancreatic cell clusters transplanted into nude mice. Cell Transplant 1999;8:673–689.

75. Toschi E, Aye T, Fuller A, et al. Gene expression pro- filing of new β cells: evidence for differentiating β cells passing through a ductal phenotype. Diabetes 2004;53(suppl 2):1613.

76. Suzuki A, Nakauchi H, Taniguchi H. Prospective isola- tion of multipotent pancreatic progenitors using flow- cytometric cell sorting. Diabetes 2004;53:2143–2152.

77. Seaberg RM, Smukler SR, Kieffer TJ, et al. Clonal iden- tification of multipotent precursors from adult mouse pancreas that generate neural and pancreatic lineages.

Nat Biotechnol 2004;22:1115–1124.

78. Weir GC, Bonner-Weir S. Beta-cell precursors: a work in progress. Nat Biotechnol 2004;22:1095–1096.

79. Hellerstrom C, Swenne I, Andersson A. Islet cell repli- cation and diabetes. In: Lefebvre PJ, Pipeleers DG, eds.

The Pathology of the Endocrine Pancreas in Diabetes.

Heidelberg: Springer-Verlag; 1988:141–170.

80. Chick WL. Beta cell replication in rat pancreatic monolayer cultures: effects of glucose, tolbutamide, glucocorticoid, growth hormone and glucagon.

Diabetes 1973;22:687–693.

81. Brelje TC, Scharp DW, Lacy PE, et al. Effect of homol- ogous placental lactogens, prolactins, and growth hor- mones on islet B-cell division and insulin secretion in

rat, mouse and human islets: implication for placental lactogen regulation of islet function during preg- nancy. Endocrinology 1993;132:879–887.

82. Halvorsen TL, Beattie GM, Lopez AD, Hayek A, Levine F. Accelerated telomere shortening and senescence in human pancreatic islet cells stimulated to divide in vitro. J Endocrinol 2000;166:103–109.

83. Beattie GM, Cirulli V, Lopez AD, Hayek A. Ex vivo expansion of human pancreatic endocrine cells. J Clin Endocrinol Metab 1997;82:1852–1856.

84. Beattie GM, Itkin-Ansari P, Cirulli V, et al. Sustained proliferation of PDX-1

cells derived from human islets. Diabetes 1999;48:1013–1019.

85. Lefebvre VH, Otonkoski T, Ustinov J, Huotari MA, Pipeleers DG, Bouwens L. Culture of adult human islet preparations with hepatocyte growth factor and 804G matrix is mitogenic for duct cells but not for beta- cells. Diabetes 1998;47:134–137.

86. Bonner-Weir S, Taneja M, Weir GC, et al. In vitro cul- tivation of human islets from expanded ductal tissue.

Proc Natl Acad Sci USA 2000;97:7999–8004.

87. Gao R, Ustinov J, Pulkkinen MA, Lundin K, Korsgren O, Otonkoski T. Characterization of endocrine pro- genitor cells and critical factors for their differentia- tion in human adult pancreatic cell culture. Diabetes 2003;52:2007–2015.

88. Hardikar AA, Marcus-Samuels B, Geras-Raaka E, Raaka BM, Gershengorn MC. Human pancreatic pre- cursor cells secrete FGF2 to stimulate clustering into hormone-expressing islet-like cell aggregates. Proc Natl Acad Sci USA 2003;100:7117–7122.

89. Ramiya VK, Marraist M, Arfors KE, Schatz DA, Peck AB, Cornelius JC. Reversal of insulin dependent dia- betes using islets generated in vitro from pancreatic stem cells. Nat Med 2000;6:278–282.

90. Sachs DH, Bonner-Weir S. New islets from old. Nature 2000;6:278–282.

91. Rooman I, Heremans Y, Heimberg H, Bouwens L.

Modulation of rat pancreatic acinoductal transdiffer- entiation and expression of PDX-1 in vitro.

Diabetologia 2000;43:907–914.

92. Heimberg H, Bouwens L, Heremans Y, Van De Casteele M, Lefebvre V, Pipeleers D. Adult human pancreatic duct and islet cells exhibit similarities in expression and differences in phosphorylation and complex formation of the homeodomain protein Ipf- 1. Diabetes 2000;49:571–579.

93. Lardon J, De Breuck S, Rooman I, et al. Plasticity in the adult rat pancreas: transdifferentiation of exocrine to hepatocyte-like cells in primary culture. Hepatology 2004;39:1499–1507.

94. Lardon J, Huyens N, Rooman I, Bouwens L. Exocrine cell transdifferentiation in dexamethasone-treated rat pancreas. Virchows Arch 2004;444:61–65.

95. Wang Y, Perfetti R, Greig N, et al. Glucagon-like pep- tide-1 can reverse the age-related decline in glucose tolerance in rats. J Clin Invest 1997;99:2883–2889.

96. Zhou J, Wang X, Pineyro MA, Egan JM. Glucagon-like peptide 1 and exendin-4 convert pancreatic AR42J cells into glucagon- and insulin-producing cells. Diabetes 1999;48:2358–2366.

97. Brand SJ, Tagerud S, Lambert P, et al. Pharmacological

treatment of chronic diabetes by stimulating pancre-

atic beta-cell regeneration with systemic co-adminis-

tration of EGF and gastrin. Pharmacol Toxicol

2002;91:414–420.

98. Rooman I, Bouwens L. Combined gastrin and epider- mal growth factor treatment induces islet regenera- tion and restores normoglycaemia in C57Bl6/J mice treated with alloxan. Diabetologia 2004;47:259–265.

99. Rooman I, Lardon J, Flamez D, Schuit F, Bouwens L.

Mitogenic effect of gastrin and expression of gas- trin receptors in duct-like cells of rat pancreas.

Gastroenterology 2001;121:940–949.

100. Fernandez-Alvarez J, Barbera A, Nadal B, et al. Stable and functional regeneration of pancreatic beta-cell population in nSTZ-rats treated with tungstate.

Diabetologia 2004;47:470–477.

101. Ogata T, Li L, Yamada S, et al. Promotion of β-cell dif-

ferentiation by conophylline in fetal and neonatal rat

pancreas. Diabetes 2004;53:2596–2602.