- Email: [email protected]
Generating new pancreas from old
Review
TRENDS in Endocrinology and Metabolism
Vol.15 No.5 July 2004
Generating new pancreas from oldq Anandwardhan A. Hardikar National Institute of Diabetes and Digestive and Kidney Diseases, Bldg 50/Room 4128, National Institutes of Health, Bethesda, MD 20892, USA
Pancreas regeneration after tissue damage is a key response to pancreatic injury, involving pancreatic duct progenitor cells and intra-islet precursor cells. Surgical removal of the pancreas, duct obstruction by cellophane wrapping and bone marrow-derived stem cell transplantation act as inductive stimuli, leading to pancreas regeneration. The exact role of growth and differentiation factors regulating pancreatic b-cell mass remains unknown. Here, I will attempt to integrate recent findings and speculate on the factors that trigger this fascinating response, wherein the pancreas responds to a deficit in cell mass and undergoes new islet formation, leading to restoration of normal b-cell mass. I will also discuss recent advances in regenerating endocrine pancreatic cells, which could affect stem cell-based approaches to treating diabetes mellitus. The pancreas is a unique organ because the endocrine pancreas, composed of the hormone-producing cells, is located within the exocrine part of the tissue. The endocrine pancreas is organized into small clusters of cells, the islets of Langerhans, which comprise at least four types of cells: (i) the glucagon-secreting a cells; (ii) the insulin-secreting b cells; (iii) the somatostatin-secreting d cells and (iv) the pancreatic polypeptide-secreting PP cells. Islets are highly vascularized, with a network of capillaries that remain in close contact with the hormoneproducing cells, and thereby enable continuous monitoring of blood glucose and secretion of the regulatory hormones – insulin, glucagon and somatostatin. These clusters of endocrine cells are scattered within the exocrine portion of the pancreas, comprising acinar cells, which secrete different digestive enzymes – such as pancreatic amylase (carbohydrate digestion); trypsin, chymotrypsin and carboxypeptidase (protein digestion); and pancreatic lipase (fat digestion) – into the duodenum via the pancreatic duct. The pancreas has a remarkable capacity to regenerate and repair tissue damage. After surgical resection of nondiabetic adult rodent pancreas, regenerative stimuli induce the pancreatic precursor cells to undergo a phase of cell proliferation and differentiation that leads to the restoration of the lost organ. Further work has shown that the adult mammalian pancreas can regenerate in response to different insults such as physical ligation of the pancreatic duct, chemical toxins and virus-induced diabetes [1 – 3]. Inductive signals that influence pancreatic regeneration can also be achieved in a chemically induced q Supplementary data associated with this article can be found at doi: 10.1016/j.tem.2004.05.001 Corresponding author: A.A. Hardikar ([email protected]).
model of diabetes [4,5]. This process of pancreas regeneration is similar to endocrine pancreas development. In both these processes, multipotent precursor cells proliferate and differentiate to form pancreatic islets. Among the numerous genes that orchestrate their expression during different phases of pancreas development, Pdx1 (also known as Ipf1), a homeobox gene necessary for pancreas and b-cell development [6], is detected early in the pancreatic domain of the embryonic gut tube. Later on, most Pdx1 expression is seen in the islet b cells. During pancreatic regeneration, islets are often seen to bud off from the pancreatic ducts and these buds produce increased levels of PDX1 protein, accompanied by a smaller increase in gene transcription [7]. These findings indicate that the initiation of developmental pathways forms an essential step during adult pancreatic regeneration. This review aims to describe the most recent advances in our understanding of the regenerative biology of the endocrine pancreas and the potential applicability of these findings to the treatment of type 1 diabetes in humans. Pancreas development and differentiation of precursor cells into islet b cells Shortly after gastrulation, the embryo contains three major groups of cells: (i) ectoderm, which forms the skin and the central nervous system; (ii) mesoderm, which forms blood, bone and muscle; and (iii) endoderm, which gives rise to the respiratory and digestive systems. The endodermal layer transforms into a primitive gut tube [8], which eventually develops out-pouches of pancreatic buds (Figure 1). The dorsal and ventral pancreatic buds then undergo branching to give rise to both; ductal and acinar components of the adult pancreas. In a process that is now beginning to be understood, pancreatic precursors migrate in response to various paracrine and autocrine signals [9,10], and come together to form islet-like cell clusters. In response to gradients of different growth and developmental factors (outlined in supplementary material Table 1; http://archive.bmn.com/supp/tem/ Hardikar_Table1.doc) and cell – cell contact, cells in these clusters differentiate into hormone-producing cells of the pancreatic islet. During the early stages of pancreas development, the number of extracellular matrix and cell adhesion molecules is high in the pancreatic precursors (Pdx1-expressing cells) and adult islets, but low in the endocrine precursor cells (supplementary material Table 1; http://archive.bmn. com/supp/tem/Hardikar_Table1.doc). The differences in time of production of various adhesion markers, such as claudin 1 and 3 and clusterin, indicate the
www.sciencedirect.com 1043-2760/$ - see front matter. Published by Elsevier Ltd. doi:10.1016/j.tem.2004.05.001
Review
TRENDS in Endocrinology and Metabolism
199
Vol.15 No.5 July 2004
Islet Endocrine precursors
Duct
Acini
Endoderm
Pancreas bud
Branching morphogenesis
Developed pancreas TRENDS in Endocrinology & Metabolism
Figure 1. Early stages in the development of dorsal pancreas in rodents. The endoderm shows out-pouching of pancreatic buds (only the dorsal bud shown here), which then undergo branching morphogenesis to form the duct and acinar structure of the developed pancreas. Islet cell precursors migrate under the influence of various growth and differentiation factors and cluster to form hormone-expressing islets.
importance of cell – matrix and cell – cell interactions in the early phase of development of the pancreas. During pancreas development, two mesodermally derived structures – the notochord and the dorsal aorta – send permissive signals to the prospective pancreatic endoderm, leading to the expression of pancreatic genes, such as Pdx1 [11 – 14]. At embryonic day (E) 9.0 in the mouse, Pdx1 expression marks dorsal and ventral domains of the developing pancreas. As pancreatic buds expand and branch, signals from the adjacent mesenchyme direct cells towards an endocrine or exocrine fate [15] and neurogenin-3 (Ngn3)-expressing endocrine progenitor cells are detected. Ngn3 expression marks the progenitors that will give rise to all the cells of the adult islet [16,17]. These and other studies have defined several important stages of endocrine cell development. The genetic programs initiated at these stages of development are now beginning to be identified. Of these, Pdx1 is necessary for early pancreas specification, whereas Ngn3 expression marks early endocrine differentiation. Several other transcription factors, such as Nkx2.2 and Pax4 [18,19], are also important during pancreatic stem cell differentiation [20]. Recently, more thorough analyses have been possible, enabling the simultaneous quantitative analysis of mRNA of thousands of genes [21]. Databases such as the Endocrine Pancreas Consortium database (EPConDB; http://www.cbil. upenn.edu/EPConDB/index.html) enable us to look at different genes according to their homology, function and/or chromosome location. The EPConDB presents data from human and mouse pancreatic array experiments and also provides annotated information (obtained from sequenced mouse cDNA libraries) about genes expressed in the endocrine pancreas. Although these methodologies now enable relative gene expression to be assessed, the major issue in these analyses is the abundance of RNA. Because endocrine pancreatic cells represent , 2 – 3% of the entire adult pancreas, it is not surprising that the high-abundance transcripts are preferentially detected www.sciencedirect.com
during microarray analyses. Therefore, better assays on distinct populations of endocrine precursors need to be used in chip analyses [22] or, alternatively, the low abundance genes detected in microarray experiments must be confirmed with more sensitive techniques, such as quantitative real time RT– PCR. Genes involved in endocrine differentiation and islet function Adult islets express genes coding for many transcription factors that are also expressed during embryonic pancreas development. As mentioned above, Pdx1, which is expressed in the entire embryonic pancreas, is restricted to b cells in mature islets. PDX1 mutations in humans (in maturity onset diabetes of young; MODY4) and mice (targeted Pdx1) demonstrate that loss of one functional copy of Pdx1 results in islet defects, ranging from impaired glucose regulation to insulin-dependent diabetes [23]. Pdx1 regulates the expression of several genes in the islets, including those encoding insulin, islet amyloid polypeptide, glucokinase and GLUT-2 [24– 27], and a 50% reduction in the expression of the Pdx1 gene was shown to reduce greatly the capacity of islet b cells to regulate blood glucose [28]. Pdx1 has also been implicated in b-cell maintenance [7] and an impaired ability to generate new b cells might result in diabetes. Although regenerative processes are thought to play a major role in the response to acute decreases in islet cell mass, islet b cells have a limited ability to proliferate [29]. Regeneration is a widely occurring, well known phenomenon and pancreas regeneration has been studied in several animal models. Pancreas regeneration Under normal conditions, the pancreas is thought to sense the mass of functional b cells and institute regenerative processes, which lead to restoration of the b-cell mass. The pancreas shows compensatory mechanisms whereby b cells undergo increased proliferation during pregnancy, under the influence of hormones (particularly placental lactogens), or show hypertrophy during progression to obese conditions [30]. Regeneration, which is similar to
200
Review
TRENDS in Endocrinology and Metabolism
such compensatory mechanisms, is seen under conditions that involve an insult to the pancreas, such as partial resection of the pancreas. Partial pancreatectomy Pancreatic regeneration following pancreatectomy has been demonstrated in several animal models [1– 4,31] and in humans [32]. Pancreatectomy has been used as a model of diabetes and substantial regeneration of both endocrine and exocrine pancreas has been shown in rodents after 90% pancreatectomy [33]. After pancreatectomy, several genes including those encoding clusterin [34], insulin-like growth factor I and the pancreas regeneration protein (Reg) have been shown to be upregulated [35] in proliferating ductules and capillary endothelial cells and these studies have led to the speculation that pancreatic duct epithelia harbors islet precursor cells [36,37]. In vitro systems have shown that cells obtained from primary cultures of enriched ducts [38] or from a cell line that produces duct cell markers (Cytokeratin 7 and 19 [10]) can differentiate into insulin-expressing islet-like cell clusters. However, in none of these studies has the pancreatic duct been seen to undergo asymmetric cell division – a property of true stem cells – and neither have the cells been used in lineage tracing experiments to demonstrate that the same cell(s) would differentiate into insulin-producing cells. Nonetheless, these studies [10,38] show that islet precursor cells are present in the adult pancreas and that these could be induced to proliferate and differentiate into hormone-producing cells. It has been demonstrated in an animal model [4] that pancreatic regeneration can occur in hyperglycemic animals, in which most of the insulinproducing b cells were selectively killed by a toxin (Figure 2). Apart from this model of pancreatic regeneration, several other animal models have demonstrated the regenerative capacity of the adult pancreas.
Figure 2. Pancreas from BALB/c mice of (a) control (sham operated) group and (b) streptozotocin-induced diabetic (200 mg kg21) and sham operated group. (c) At 15 days after the resection, islets were seen budding off the pancreatic ducts in animals from streptozotocin diabetic and pancreatectomized groups. (d) A dithiazone-stained islet isolated from the regenerated pancreas of mice shown in (c). Scale bars, 10 mm. Reproduced with permission from the Society for Endocrinology [4]. www.sciencedirect.com
Vol.15 No.5 July 2004
Insulinoma-bearing NEDH rats When insulinoma cells were transplanted into NEDH (New England Deaconess Hospital) rats, the islet cells of these rats became increasingly atrophic and reduced their insulin production [39,40]. However, removal of the tumor resulted in transient (24–48 h) hypoinsulinemic hyperglycemia, followed by attainment of normoglycemia [40,41]. Diminution in pancreatic b-cell mass caused by subcutaneous implantation of an insulinoma is associated with reduced Reg gene expression [40]. This model demonstrates the presence of delicate intrinsic regulators of islet cell mass, which is governed by implantation of extrinsic insulin-producing cells. Thus, in the absence of factors that lead to b-cell destruction, pancreatic b cells can regulate their mass to account for any increase or decrease in the mass of insulin-producing b cells. The partial pancreatectomy model demonstrates pancreatic regeneration after a major insult to the pancreatic mass. However, the NEDH rat model shows that fine regulators of pancreatic cell mass exist and can respond to an increase or decrease in the mass of islet b cells. Cellophane wrapping of hamster pancreatic ducts Partial duct obstruction of hamster pancreas [1,42] was shown to induce islet cell regeneration in streptozotocininduced diabetes in at least 50% of the animals studied. In this model, the new islet cells appear to originate from precursor cells associated with the ductal epithelium. Cellophane wrapping of the hamster pancreas induced cell proliferation in the pancreatic ducts and development of new endocrine tissue in the pancreas of adult hamsters. Electron microscopic and immunocytochemical studies of these newly developed islets revealed cells containing insulin, glucagon and somatostatin [43]. Similarly, incorporation of 3H-thymidine by duct epithelial cells and islet cells was increased after wrapping of the pancreas [43]. It was also shown that tissue extracts from cellophanewrapped pancreas contain protein(s) that induce nesidioblastosis or new islet formation [44]. The active component in this extract (ilotropin) was shown to be effective in reversal of streptozotocin-induced diabetes [44]. Ilotropin, a heat and acid stable protein with a molecular mass of , 20 – 45 kDa, can stimulate the proliferation of isolated duct cells in culture. With the use of mRNA and a differential display technique, 20 genes were shown to be expressed in the partially obstructed (regenerating), but not the non-obstructed (non-regenerating), pancreas [45]. One of these, the gene encoding islet neogenesis-associated peptide was found to be unique to the cellophane-wrapped pancreas and was capable of stimulating the proliferation of ductal cells in culture [46]. These studies demonstrate that, in response to cellophane wrapping of the pancreatic duct, nesidioblastotic factors are secreted by the pancreas and can induce islet regeneration. IFN-g transgenic mice The interferon-g (INF-g) transgenic mouse model was developed by Sarvetnick and Gu [47] to demonstrate pancreatic islet regeneration after immunodestruction of islet b cells in transgenic mice expressing the gene encoding IFN-g (Ifng). In these mice, Ifng is placed
Review
TRENDS in Endocrinology and Metabolism
under the regulation of the human insulin (INS) promoter. The transgenic mice show pancreatic inflammation and progressive loss of islets 6 –8 weeks after their birth [48] and the destruction of the islets resembles that seen in type 1 diabetes mellitus. However, the islets show a rapid regenerative process involving replication of duct cells and subsequent differentiation into islet b cells [49]. The regenerative process closely resembles pancreatic islet development and offers a model for studying the cell lineage relationships of islet cells [50]. Interestingly, the duct cells retain their ability to proliferate and to differentiate into islets, which is not seen under normal conditions. Recent studies have shown that the pathway for IFN-g-mediated pancreatic regeneration in the INS – Ifng transgenic mice requires participation of the transcription factors Pax4, Pax6, Pdx-1 and islet-1 (Isl-1), which control initial pancreatic development [50,51]. Epidermal growth factor (EGF) and its receptor are also upregulated and EGF receptors were seen in the differentiating duct-like structures. These ducts have been shown to proliferate and contribute to the regeneration of pancreatic islets [52]. Recent studies [22,53] have identified pathways of transcription factors that are sequentially involved in pancreatic development. Although the major abnormalities in pancreas development result from deficiencies in molecules such as PDX-1, transcription factors such as Isl-1 and Pax-6 are also thought to be important for obtaining a b-cell phenotype. Further studies with this model have demonstrated the role of various transcription factors including PDX-1 and hepatic nuclear factor 3b, the PAX proteins, and Isl-1 during the regenerative process, showing that synthesis of these transcription factors in the pancreatic ducts of IFN-g transgenic mice is a necessary and ongoing process after islet destruction [50]. Future directions and stem cell-based approaches A reduction in the mass of functional b cells is a major contributing factor in type 1 and type 2 diabetes. The conventional treatment for type 1 diabetes has been to transplant exogenous insulin-producing islet cells, although this has been limited by the scarcity of donor pancreas available and the lack of efficient and efficacious protocols to isolate and transplant the donor islets. However, the recent demonstration of islet transplantation [54] indicates that considerable success in islet grafts can be achieved, although the scarcity of donor islets still remains a limitation. Several laboratories have approached this problem by attempting to use embryonic stem (ES) cells [55 – 57], but the efficiency of differentiation of ES cells to insulin-producing cells has been very low. Furthermore, the potential use of ES cells in humans is fraught with ethical issues. A recent study showed that oval cells from the liver could transdifferentiate into insulin-producing cells in longterm culture [58]. Putative precursor (or progenitor) cells have also been derived from adult humans and rodent pancreas (for review, see Refs [59,60]) after manipulation in vitro to differentiate them into hormone-producing cells. In view of the scarcity of donor islets and the ethical issues involved in transplantation of stem cells, mechanisms that www.sciencedirect.com
Vol.15 No.5 July 2004
201
would induce endogenous precursor and stem cells to differentiate into insulin-producing cells hold promise for the future. Pancreatic islets have been shown to regenerate after direct insult to the pancreas or islet b cells. Recently, transplantation of adult bone marrow-derived cells expressing Kit was shown to reduce hyperglycemia in mice [61] that were rendered diabetic with streptozotocin. In this study, the investigators showed that most transplanted bone marrow cells were localized to ductal and islet structures and that their presence was accompanied by a proliferation of recipient pancreatic cells, resulting in increased insulin production. Although diabetes reversal per se was not demonstrated, these data demonstrate the capacity of bone marrow-derived stem cells to initiate endogenous pancreatic tissue regeneration by a previously unrecognized mechanism. It has been shown that stem cells in the adult bone marrow have the potential to differentiate into different cell types [62,63], including insulin-producing cells [64]. Although pancreatic islet progenitors in the bone marrow are still to be isolated and characterized, these studies demonstrate that pools of b-cell progenitors exist in the bone marrow and could be mobilized to serve as precursors for tissue regeneration. Another recent demonstration in NOD mice has shown that islet regeneration can lead to ‘permanent reversal’ of diabetes [65]. Live male splenocytes injected into female diabetic NOD mice provided CD45- mesenchymal precursor cells for the reconstitution of functional islets. The donor splenocytes also contribute to the reversal of autoimmunity, possibly by ‘re-educating’ naive T cells through presentation of matched major histocompatibility complex class I molecules and self-antigens, yielding islets almost free of any signs of autoimmunity. This study again shows that adult diabetic NOD mice contain endogenous precursor cells capable of giving rise to new functional islets after the underlying autoimmune disease is eliminated. The ability of an endogenous population of NOD mouse stem cells to give rise to new islets suggests that therapies to reverse autoimmune diabetes need not incorporate transplantation of exogenous adult islets. The use of fresh splenocytes eliminates the need for cell culture manipulations that transform stem cells of fetal or adult origin into malignant precursors or fusion hybrids with an abnormal DNA content. Because the cell donors and hosts are adults this system precludes any ethical issues associated with the use of ES cells. These recent findings [58,61– 65] suggest that pancreatic progenitor cells might not be limited to the pancreas, but that cells from other tissues could be mobilized and induced to differentiate and contribute to the regenerative process. Stimuli such as surgical removal of a part of the pancreas, cellophane wrapping and transplantation of bone marrow cells can induce pancreatic regeneration to different extents. The NEDH rat model has shown that, in some way, the pancreas can ‘sense’ the total mass of insulin-producing cells and regulate it when grafted with an excess of insulin-producing cells and when this graft is subsequently removed. If such regulators really exist, the entire process could be visualized as a balance between regenerative and destructive factors and identifying and
202
Review
TRENDS in Endocrinology and Metabolism
enhancing the regenerative processes might greatly influence the outcome of diabetes. In light of the scarcity of donor islets available for transplantation to diabetic individuals, further studies to understand the mechanisms of adult human pancreatic regeneration would help us in exploiting this inexhaustible source of pancreatic progenitor cells for cell replacement therapy and better management of diabetes. Acknowledgements I would like to thank Elizabeth Geras-Raaka, NIDDK, National Institutes of Health, Bethesda, MD for critical discussion and suggestions in the preparation of this review and the editorial office of Journal of Endocrinology for permission to publish graphics from a previously published article. I apologize to colleagues whose work I was unable to cite in the text or reference section owing to space limitations.
References 1 Rafaeloff, R. et al. (1997) Cloning and sequencing of the pancreatic islet neogenesis associated protein (INGAP) gene and its expression in islet neogenesis in hamsters. J. Clin. Invest. 99, 2100– 2109 2 Yoon, J.W. and Notkins, A.L. (1983) Virus-induced diabetes in mice. Metabolism 32, 37 – 40 3 Fernandes, A. et al. (1997) Differentiation of new insulin-producing cells is induced by injury in adult pancreatic islets. Endocrinology 138, 1750 – 1762 4 Hardikar, A.A. et al. (1999) Effect of partial pancreatectomy on diabetic status in BALB/c mice. J. Endocrinol. 162, 189– 195 5 Finegood, D.T. et al. (1999) Prior streptozotocin treatment does not inhibit pancreas regeneration after 90% pancreatectomy in rats. Am. J. Physiol. 276, E822– E827 6 Stoffers, D.A. et al. (1997) Pancreatic agenesis attributable to a single nucleotide deletion in the human IPF1 gene coding sequence. Nat. Genet. 15, 106 – 110 7 Sharma, A. et al. (1999) The homeodomain protein IDX-1 increases after an early burst of proliferation during pancreatic regeneration. Diabetes 48, 507 – 513 8 Wells, J.M. and Melton, D.A. (1999) Vertebrate endoderm development. Annu. Rev. Cell Dev. Biol. 15, 393 – 410 9 Lammert, E. et al. (2003) Role of endothelial cells in early pancreas and liver development. Mech. Dev. 120, 59 – 64 10 Hardikar, A.A. et al. (2003) Human pancreatic precursor cells secrete FGF2 to stimulate clustering into hormone-expressing islet-like cell aggregates. Proc. Natl. Acad. Sci. U. S. A. 100, 7117 – 7122 11 Hebrok, M. et al. (2000) Regulation of pancreas development by hedgehog signaling. Development 127, 4905– 4913 12 Kim, S.K. et al. (2000) Activin receptor patterning of foregut organogenesis. Genes Dev. 14, 1866 – 1871 13 Kim, S.K. et al. (1997) Notochord to endoderm signaling is required for pancreas development. Development 124, 4243 – 4252 14 Lammert, E. et al. (2001) Induction of pancreatic differentiation by signals from blood vessels. Science 294, 564– 567 15 Miralles, F. et al. (1998) Follistatin regulates the relative proportions of endocrine versus exocrine tissue during pancreatic development. Development 125, 1017– 1024 16 Gradwohl, G. et al. (2000) Neurogenin3 is required for the development of the four endocrine cell lineages of the pancreas. Proc. Natl. Acad. Sci. U. S. A. 97, 1607 – 1611 17 Gu, G. et al. (2002) Direct evidence for the pancreatic lineage: NGN3 þ cells are islet progenitors and are distinct from duct progenitors. Development 129, 2447– 2457 18 Sussel, L. et al. (1998) Mice lacking the homeodomain transcription factor Nkx2.2 have diabetes due to arrested differentiation of pancreatic b cells. Development 125, 2213 – 2221 19 Sosa-Pineda, B. et al. (1997) The Pax4 gene is essential for differentiation of insulin-producing b cells in the mammalian pancreas. Nature 386, 399 – 402 20 Hardikar, A. and Gershengorn, M. Chapter 51: stem cells. In Diabetes Mellitus: A Fundamental and Clinical Text (3rd ed), (LeRoith, D., et al. eds), (in press) www.sciencedirect.com
Vol.15 No.5 July 2004
21 Lander, E.S. and Weinberg, R.A. (2000) Genomics: journey to the center of biology. Science 287, 1777– 1782 22 Chiang, M.K. and Melton, D.A. (2003) Single-cell transcript analysis of pancreas development. Dev. Cell 4, 383– 393 23 Dutta, S. et al. (1998) Regulatory factor linked to late-onset diabetes? Nature 392, 560 24 Christmanson, L. et al. (1990) The human islet amyloid polypeptide (IAPP) gene. Organization, chromosomal localization and functional identification of a promoter region. FEBS Lett. 267, 160 – 166 25 Ohlsson, H. et al. (1993) IPF1, a homeodomain-containing transactivator of the insulin gene. EMBO J. 12, 4251– 4259 26 Waeber, G. et al. (1996) Transcriptional activation of the GLUT2 gene by the IPF-1/STF-1/IDX-1 homeobox factor. Mol. Endocrinol. 10, 1327– 1334 27 Watada, H. et al. (1996) PDX-1 induces insulin and glucokinase gene expressions in a TC1 clone 6 cells in the presence of bcellulin. Diabetes 45, 1826 – 1831 28 Brissova, M. et al. (2002) Reduction in pancreatic transcription factor PDX-1 impairs glucose-stimulated insulin secretion. J. Biol. Chem. 277, 11225– 11232 29 Yamaoka, T. (2002) Regeneration therapy of pancreatic b cells: towards a cure for diabetes? Biochem. Biophys. Res. Commun. 296, 1039– 1043 30 Bernard-Kargar, C. and Ktorza, A. (2001) Endocrine pancreas plasticity under physiological and pathological conditions. Diabetes 50, S30– S35 31 Morisset, J. et al. (2000) Pancreatic inflammation, apoptosis, and growth: sequential events after partial pancreatectomy in pigs. Pancreas 21, 321 – 324 32 Schlegel, R.D. et al. (2000) Is there pancreatic regeneration? Morphological and functional certification after a corporocaudal splenopancreatectomy. Acta Gastroenterol. Latinoam. 30, 107– 113 33 Brockenbrough, J.S. et al. (1988) Discordance of exocrine and endocrine growth after 90% pancreatectomy in rats. Diabetes 37, 232– 236 34 Min, B.H. et al. (2003) Clusterin expression in the early process of pancreas regeneration in the pancreatectomized rat. J. Histochem. Cytochem. 51, 1355 – 1365 35 Smith, F.E. et al. (1991) Enhanced insulin-like growth factor I gene expression in regenerating rat pancreas. Proc. Natl. Acad. Sci. U. S. A. 88, 6152 – 6156 36 Bonner-Weir, S. et al. (1993) A second pathway for regeneration of adult exocrine and endocrine pancreas. A possible recapitulation of embryonic development. Diabetes 42, 1715 – 1720 37 Hayashi, K. et al. (1999) Regional differences in the cellular proliferation activity of the regenerating rat pancreas after partial pancreatectomy. Arch. Histol. Cytol. 62, 337 – 346 38 Bonner-Weir, S. et al. (2000) In vitro cultivation of human islet from expanded ductal tissue. Proc. Natl. Acad. Sci. U. S. A. 97, 7999– 8004 39 Bedoya, F.J. et al. (1986) Differential regulation of glucokinase activity in pancreatic islets and liver of the rat. J. Biol. Chem. 261, 10760 – 10764 40 Miyaura, C. et al. (1991) Expression of reg/PSP, a pancreatic exocrine gene: relationship to changes in islet b-cell mass. Mol. Endocrinol. 5, 226– 234 41 Clas, D. et al. (1988) Islet cell differentiation and proliferation induced by a pancreatic cytosol extract. Surg. Forum 39, 164 – 166 42 Rosenberg, L. et al. (1990) Trophic stimulation of the ductal/islet cell axis: a new approach to the treatment of diabetes. Surgery 108, 191– 197 43 Rosenberg, L. et al. (1989) The effect of cellophane wrapping of the pancreas in the Syrian golden hamster: autoradiographic observations. Pancreas 4, 31 – 37 44 Rosenberg, L. (1998) Induction of islet cell neogenesis in the adult pancreas: the partial duct obstruction model. Microsc. Res. Tech. 43, 337– 346 45 Rafaeloff, R. et al. (1997) Cloning and sequencing of the pancreatic islet neogenesis associated protein (INGAP) gene and its expression in islet neogenesis in hamsters. J. Clin. Invest. 99, 2100 – 2109 46 Rafaeloff, R. et al. (1996) Identification of differentially expressed genes induced in pancreatic islet neogenesis. FEBS Lett. 378, 219 – 223 47 Sarvetnick, N.E. and Gu, D. (1992) Regeneration of pancreatic
Review
48 49
50 51
52 53
54
55
56
TRENDS in Endocrinology and Metabolism
endocrine cells in interferon-g transgenic mice. Adv. Exp. Med. Biol. 321, 85 – 89 Gu, D. and Sarvetnick, N. (1993) Epithelial cell proliferation and islet neogenesis in IFN-g transgenic mice. Development 118, 33– 46 Kayali, A.G. et al. (2003) The stromal cell-derived factor-1a/CXCR4 ligand – receptor axis is critical for progenitor survival and migration in the pancreas. J. Cell Biol. 163, 859– 869 Kritzika, M.R. et al. (2000) Transcription factor expression during pancreatic islet regeneration. Mol. Cell. Endocrinol. 164, 99 – 107 Krakowski, M. et al. (2000) IFN-g overexpression within the pancreas is not sufficient to rescue Pax4, Pax6,and Pdx-1 mutant mice from death. Pancreas 21, 399 – 406 Arnush, M. et al. (1996) Growth factors in the regenerating pancreas of g-interferon transgenic mice. Lab. Invest. 74, 985 – 990 Schwitzgebel, V.M. et al. (2000) Expression of neurogenin3 reveals an islet cell precursor population in the pancreas. Development 127, 3533 – 3542 Shapiro, A.M. et al. (2000) Islet transplantation in seven patients with type 1 diabetes mellitus using a glucocorticoid-free immunosuppressive regimen. N. Engl. J. Med. 343, 230 – 238 Soria, B. et al. (2000) Insulin-secreting cells derived from embryonic stem cells normalize glycemia in streptozotocin-induced diabetic mice. Diabetes 49, 157 – 162 Lumelsky, N. et al. (2001) Differentiation of embryonic stem cells to
57 58
59
60 61 62 63 64
65
Vol.15 No.5 July 2004
203
insulin-secreting structures similar to pancreatic islets. Science 292, 1389– 1394 Assady, S. et al. (2001) Insulin production by human embryonic stem cells. Diabetes 50, 1691– 1697 Yang, L. et al. (2002) In vitrotrans-differentiation of adult hepatic stem cells into pancreatic endocrine hormone-producing cells. Proc. Natl. Acad. Sci. U. S. A. 99, 8078 – 8083 Lechner, A. and Habener, J.F. (2003) Stem/progenitor cells derived from adult tissues: potential for the treatment of diabetes mellitus. Am. J. Physiol. Endocrinol. Metab. 284, E259– E266 Bonner-Weir, S. and Sharma, A. (2002) Pancreatic stem cells. J. Pathol. 197, 519 – 526 Hess, D. et al. (2003) Bone marrow-derived stem cells initiate pancreatic regeneration. Nat. Biotechnol. 21, 763 – 770 Jiang, Y. et al. (2002) Pluripotency of mesenchymal stem cells derived from adult marrow. Nature 418, 41 – 49 Reyes, M. et al. (2002) Origin of endothelial progenitors in human postnatal bone marrow. J. Clin. Invest. 109, 337 – 346 Ianus, A. et al. (2003) In vivoderivation of glucose-competent pancreatic endocrine cells from bone marrow without evidence of cell fusion. J. Clin. Invest. 111, 843– 850 Kodama, S. et al. (2003) Islet regeneration during the reversal of autoimmune diabetes in NOD mice. Science 302, 1223– 1227
Elsevier.com – Dynamic New Site Links Scientists to New Research & Thinking Elsevier.com has had a makeover, inside and out. Designed for scientists’ information needs, the new site, launched in January, is powered by the latest technology with customer-focused navigation and an intuitive architecture for an improved user experience and greater productivity. Elsevier.com’s easy-to-use navigational tools and structure connect scientists with vital information – all from one entry point. Users can perform rapid and precise searches with our advanced search functionality, using the FAST technology of Scirus.com, the free science search engine. For example, users can define their searches by any number of criteria to pinpoint information and resources. Search by a specific author or editor, book publication date, subject area – life sciences, health sciences, physical sciences and social sciences – or by product type. Elsevier’s portfolio includes more than 1800 Elsevier journals, 2200 new books per year, and a range of innovative electronic products. In addition, tailored content for authors, editors and librarians provides up-to-the-minute news, updates on functionality and new products, e-alerts and services, as well as relevant events. Elsevier is proud to be a partner with the scientific and medical community. Find out more about who we are in the About section: our mission and values and how we support the STM community worldwide through partnerships with libraries and other publishers, and grant awards from The Elsevier Foundation. As a world-leading publisher of scientific, technical and health information, Elsevier is dedicated to linking researchers and professionals to the best thinking in their fields. We offer the widest and deepest coverage in a range of media types to enhance cross-pollination of information, breakthroughs in research and discovery, and the sharing and preservation of knowledge. Visit us at Elsevier.com.
Elsevier. Building Insights. Breaking Boundaries. www.sciencedirect.com
TRENDS in Endocrinology and Metabolism
Vol.15 No.5 July 2004
Generating new pancreas from oldq Anandwardhan A. Hardikar National Institute of Diabetes and Digestive and Kidney Diseases, Bldg 50/Room 4128, National Institutes of Health, Bethesda, MD 20892, USA
Pancreas regeneration after tissue damage is a key response to pancreatic injury, involving pancreatic duct progenitor cells and intra-islet precursor cells. Surgical removal of the pancreas, duct obstruction by cellophane wrapping and bone marrow-derived stem cell transplantation act as inductive stimuli, leading to pancreas regeneration. The exact role of growth and differentiation factors regulating pancreatic b-cell mass remains unknown. Here, I will attempt to integrate recent findings and speculate on the factors that trigger this fascinating response, wherein the pancreas responds to a deficit in cell mass and undergoes new islet formation, leading to restoration of normal b-cell mass. I will also discuss recent advances in regenerating endocrine pancreatic cells, which could affect stem cell-based approaches to treating diabetes mellitus. The pancreas is a unique organ because the endocrine pancreas, composed of the hormone-producing cells, is located within the exocrine part of the tissue. The endocrine pancreas is organized into small clusters of cells, the islets of Langerhans, which comprise at least four types of cells: (i) the glucagon-secreting a cells; (ii) the insulin-secreting b cells; (iii) the somatostatin-secreting d cells and (iv) the pancreatic polypeptide-secreting PP cells. Islets are highly vascularized, with a network of capillaries that remain in close contact with the hormoneproducing cells, and thereby enable continuous monitoring of blood glucose and secretion of the regulatory hormones – insulin, glucagon and somatostatin. These clusters of endocrine cells are scattered within the exocrine portion of the pancreas, comprising acinar cells, which secrete different digestive enzymes – such as pancreatic amylase (carbohydrate digestion); trypsin, chymotrypsin and carboxypeptidase (protein digestion); and pancreatic lipase (fat digestion) – into the duodenum via the pancreatic duct. The pancreas has a remarkable capacity to regenerate and repair tissue damage. After surgical resection of nondiabetic adult rodent pancreas, regenerative stimuli induce the pancreatic precursor cells to undergo a phase of cell proliferation and differentiation that leads to the restoration of the lost organ. Further work has shown that the adult mammalian pancreas can regenerate in response to different insults such as physical ligation of the pancreatic duct, chemical toxins and virus-induced diabetes [1 – 3]. Inductive signals that influence pancreatic regeneration can also be achieved in a chemically induced q Supplementary data associated with this article can be found at doi: 10.1016/j.tem.2004.05.001 Corresponding author: A.A. Hardikar ([email protected]).
model of diabetes [4,5]. This process of pancreas regeneration is similar to endocrine pancreas development. In both these processes, multipotent precursor cells proliferate and differentiate to form pancreatic islets. Among the numerous genes that orchestrate their expression during different phases of pancreas development, Pdx1 (also known as Ipf1), a homeobox gene necessary for pancreas and b-cell development [6], is detected early in the pancreatic domain of the embryonic gut tube. Later on, most Pdx1 expression is seen in the islet b cells. During pancreatic regeneration, islets are often seen to bud off from the pancreatic ducts and these buds produce increased levels of PDX1 protein, accompanied by a smaller increase in gene transcription [7]. These findings indicate that the initiation of developmental pathways forms an essential step during adult pancreatic regeneration. This review aims to describe the most recent advances in our understanding of the regenerative biology of the endocrine pancreas and the potential applicability of these findings to the treatment of type 1 diabetes in humans. Pancreas development and differentiation of precursor cells into islet b cells Shortly after gastrulation, the embryo contains three major groups of cells: (i) ectoderm, which forms the skin and the central nervous system; (ii) mesoderm, which forms blood, bone and muscle; and (iii) endoderm, which gives rise to the respiratory and digestive systems. The endodermal layer transforms into a primitive gut tube [8], which eventually develops out-pouches of pancreatic buds (Figure 1). The dorsal and ventral pancreatic buds then undergo branching to give rise to both; ductal and acinar components of the adult pancreas. In a process that is now beginning to be understood, pancreatic precursors migrate in response to various paracrine and autocrine signals [9,10], and come together to form islet-like cell clusters. In response to gradients of different growth and developmental factors (outlined in supplementary material Table 1; http://archive.bmn.com/supp/tem/ Hardikar_Table1.doc) and cell – cell contact, cells in these clusters differentiate into hormone-producing cells of the pancreatic islet. During the early stages of pancreas development, the number of extracellular matrix and cell adhesion molecules is high in the pancreatic precursors (Pdx1-expressing cells) and adult islets, but low in the endocrine precursor cells (supplementary material Table 1; http://archive.bmn. com/supp/tem/Hardikar_Table1.doc). The differences in time of production of various adhesion markers, such as claudin 1 and 3 and clusterin, indicate the
www.sciencedirect.com 1043-2760/$ - see front matter. Published by Elsevier Ltd. doi:10.1016/j.tem.2004.05.001
Review
TRENDS in Endocrinology and Metabolism
199
Vol.15 No.5 July 2004
Islet Endocrine precursors
Duct
Acini
Endoderm
Pancreas bud
Branching morphogenesis
Developed pancreas TRENDS in Endocrinology & Metabolism
Figure 1. Early stages in the development of dorsal pancreas in rodents. The endoderm shows out-pouching of pancreatic buds (only the dorsal bud shown here), which then undergo branching morphogenesis to form the duct and acinar structure of the developed pancreas. Islet cell precursors migrate under the influence of various growth and differentiation factors and cluster to form hormone-expressing islets.
importance of cell – matrix and cell – cell interactions in the early phase of development of the pancreas. During pancreas development, two mesodermally derived structures – the notochord and the dorsal aorta – send permissive signals to the prospective pancreatic endoderm, leading to the expression of pancreatic genes, such as Pdx1 [11 – 14]. At embryonic day (E) 9.0 in the mouse, Pdx1 expression marks dorsal and ventral domains of the developing pancreas. As pancreatic buds expand and branch, signals from the adjacent mesenchyme direct cells towards an endocrine or exocrine fate [15] and neurogenin-3 (Ngn3)-expressing endocrine progenitor cells are detected. Ngn3 expression marks the progenitors that will give rise to all the cells of the adult islet [16,17]. These and other studies have defined several important stages of endocrine cell development. The genetic programs initiated at these stages of development are now beginning to be identified. Of these, Pdx1 is necessary for early pancreas specification, whereas Ngn3 expression marks early endocrine differentiation. Several other transcription factors, such as Nkx2.2 and Pax4 [18,19], are also important during pancreatic stem cell differentiation [20]. Recently, more thorough analyses have been possible, enabling the simultaneous quantitative analysis of mRNA of thousands of genes [21]. Databases such as the Endocrine Pancreas Consortium database (EPConDB; http://www.cbil. upenn.edu/EPConDB/index.html) enable us to look at different genes according to their homology, function and/or chromosome location. The EPConDB presents data from human and mouse pancreatic array experiments and also provides annotated information (obtained from sequenced mouse cDNA libraries) about genes expressed in the endocrine pancreas. Although these methodologies now enable relative gene expression to be assessed, the major issue in these analyses is the abundance of RNA. Because endocrine pancreatic cells represent , 2 – 3% of the entire adult pancreas, it is not surprising that the high-abundance transcripts are preferentially detected www.sciencedirect.com
during microarray analyses. Therefore, better assays on distinct populations of endocrine precursors need to be used in chip analyses [22] or, alternatively, the low abundance genes detected in microarray experiments must be confirmed with more sensitive techniques, such as quantitative real time RT– PCR. Genes involved in endocrine differentiation and islet function Adult islets express genes coding for many transcription factors that are also expressed during embryonic pancreas development. As mentioned above, Pdx1, which is expressed in the entire embryonic pancreas, is restricted to b cells in mature islets. PDX1 mutations in humans (in maturity onset diabetes of young; MODY4) and mice (targeted Pdx1) demonstrate that loss of one functional copy of Pdx1 results in islet defects, ranging from impaired glucose regulation to insulin-dependent diabetes [23]. Pdx1 regulates the expression of several genes in the islets, including those encoding insulin, islet amyloid polypeptide, glucokinase and GLUT-2 [24– 27], and a 50% reduction in the expression of the Pdx1 gene was shown to reduce greatly the capacity of islet b cells to regulate blood glucose [28]. Pdx1 has also been implicated in b-cell maintenance [7] and an impaired ability to generate new b cells might result in diabetes. Although regenerative processes are thought to play a major role in the response to acute decreases in islet cell mass, islet b cells have a limited ability to proliferate [29]. Regeneration is a widely occurring, well known phenomenon and pancreas regeneration has been studied in several animal models. Pancreas regeneration Under normal conditions, the pancreas is thought to sense the mass of functional b cells and institute regenerative processes, which lead to restoration of the b-cell mass. The pancreas shows compensatory mechanisms whereby b cells undergo increased proliferation during pregnancy, under the influence of hormones (particularly placental lactogens), or show hypertrophy during progression to obese conditions [30]. Regeneration, which is similar to
200
Review
TRENDS in Endocrinology and Metabolism
such compensatory mechanisms, is seen under conditions that involve an insult to the pancreas, such as partial resection of the pancreas. Partial pancreatectomy Pancreatic regeneration following pancreatectomy has been demonstrated in several animal models [1– 4,31] and in humans [32]. Pancreatectomy has been used as a model of diabetes and substantial regeneration of both endocrine and exocrine pancreas has been shown in rodents after 90% pancreatectomy [33]. After pancreatectomy, several genes including those encoding clusterin [34], insulin-like growth factor I and the pancreas regeneration protein (Reg) have been shown to be upregulated [35] in proliferating ductules and capillary endothelial cells and these studies have led to the speculation that pancreatic duct epithelia harbors islet precursor cells [36,37]. In vitro systems have shown that cells obtained from primary cultures of enriched ducts [38] or from a cell line that produces duct cell markers (Cytokeratin 7 and 19 [10]) can differentiate into insulin-expressing islet-like cell clusters. However, in none of these studies has the pancreatic duct been seen to undergo asymmetric cell division – a property of true stem cells – and neither have the cells been used in lineage tracing experiments to demonstrate that the same cell(s) would differentiate into insulin-producing cells. Nonetheless, these studies [10,38] show that islet precursor cells are present in the adult pancreas and that these could be induced to proliferate and differentiate into hormone-producing cells. It has been demonstrated in an animal model [4] that pancreatic regeneration can occur in hyperglycemic animals, in which most of the insulinproducing b cells were selectively killed by a toxin (Figure 2). Apart from this model of pancreatic regeneration, several other animal models have demonstrated the regenerative capacity of the adult pancreas.
Figure 2. Pancreas from BALB/c mice of (a) control (sham operated) group and (b) streptozotocin-induced diabetic (200 mg kg21) and sham operated group. (c) At 15 days after the resection, islets were seen budding off the pancreatic ducts in animals from streptozotocin diabetic and pancreatectomized groups. (d) A dithiazone-stained islet isolated from the regenerated pancreas of mice shown in (c). Scale bars, 10 mm. Reproduced with permission from the Society for Endocrinology [4]. www.sciencedirect.com
Vol.15 No.5 July 2004
Insulinoma-bearing NEDH rats When insulinoma cells were transplanted into NEDH (New England Deaconess Hospital) rats, the islet cells of these rats became increasingly atrophic and reduced their insulin production [39,40]. However, removal of the tumor resulted in transient (24–48 h) hypoinsulinemic hyperglycemia, followed by attainment of normoglycemia [40,41]. Diminution in pancreatic b-cell mass caused by subcutaneous implantation of an insulinoma is associated with reduced Reg gene expression [40]. This model demonstrates the presence of delicate intrinsic regulators of islet cell mass, which is governed by implantation of extrinsic insulin-producing cells. Thus, in the absence of factors that lead to b-cell destruction, pancreatic b cells can regulate their mass to account for any increase or decrease in the mass of insulin-producing b cells. The partial pancreatectomy model demonstrates pancreatic regeneration after a major insult to the pancreatic mass. However, the NEDH rat model shows that fine regulators of pancreatic cell mass exist and can respond to an increase or decrease in the mass of islet b cells. Cellophane wrapping of hamster pancreatic ducts Partial duct obstruction of hamster pancreas [1,42] was shown to induce islet cell regeneration in streptozotocininduced diabetes in at least 50% of the animals studied. In this model, the new islet cells appear to originate from precursor cells associated with the ductal epithelium. Cellophane wrapping of the hamster pancreas induced cell proliferation in the pancreatic ducts and development of new endocrine tissue in the pancreas of adult hamsters. Electron microscopic and immunocytochemical studies of these newly developed islets revealed cells containing insulin, glucagon and somatostatin [43]. Similarly, incorporation of 3H-thymidine by duct epithelial cells and islet cells was increased after wrapping of the pancreas [43]. It was also shown that tissue extracts from cellophanewrapped pancreas contain protein(s) that induce nesidioblastosis or new islet formation [44]. The active component in this extract (ilotropin) was shown to be effective in reversal of streptozotocin-induced diabetes [44]. Ilotropin, a heat and acid stable protein with a molecular mass of , 20 – 45 kDa, can stimulate the proliferation of isolated duct cells in culture. With the use of mRNA and a differential display technique, 20 genes were shown to be expressed in the partially obstructed (regenerating), but not the non-obstructed (non-regenerating), pancreas [45]. One of these, the gene encoding islet neogenesis-associated peptide was found to be unique to the cellophane-wrapped pancreas and was capable of stimulating the proliferation of ductal cells in culture [46]. These studies demonstrate that, in response to cellophane wrapping of the pancreatic duct, nesidioblastotic factors are secreted by the pancreas and can induce islet regeneration. IFN-g transgenic mice The interferon-g (INF-g) transgenic mouse model was developed by Sarvetnick and Gu [47] to demonstrate pancreatic islet regeneration after immunodestruction of islet b cells in transgenic mice expressing the gene encoding IFN-g (Ifng). In these mice, Ifng is placed
Review
TRENDS in Endocrinology and Metabolism
under the regulation of the human insulin (INS) promoter. The transgenic mice show pancreatic inflammation and progressive loss of islets 6 –8 weeks after their birth [48] and the destruction of the islets resembles that seen in type 1 diabetes mellitus. However, the islets show a rapid regenerative process involving replication of duct cells and subsequent differentiation into islet b cells [49]. The regenerative process closely resembles pancreatic islet development and offers a model for studying the cell lineage relationships of islet cells [50]. Interestingly, the duct cells retain their ability to proliferate and to differentiate into islets, which is not seen under normal conditions. Recent studies have shown that the pathway for IFN-g-mediated pancreatic regeneration in the INS – Ifng transgenic mice requires participation of the transcription factors Pax4, Pax6, Pdx-1 and islet-1 (Isl-1), which control initial pancreatic development [50,51]. Epidermal growth factor (EGF) and its receptor are also upregulated and EGF receptors were seen in the differentiating duct-like structures. These ducts have been shown to proliferate and contribute to the regeneration of pancreatic islets [52]. Recent studies [22,53] have identified pathways of transcription factors that are sequentially involved in pancreatic development. Although the major abnormalities in pancreas development result from deficiencies in molecules such as PDX-1, transcription factors such as Isl-1 and Pax-6 are also thought to be important for obtaining a b-cell phenotype. Further studies with this model have demonstrated the role of various transcription factors including PDX-1 and hepatic nuclear factor 3b, the PAX proteins, and Isl-1 during the regenerative process, showing that synthesis of these transcription factors in the pancreatic ducts of IFN-g transgenic mice is a necessary and ongoing process after islet destruction [50]. Future directions and stem cell-based approaches A reduction in the mass of functional b cells is a major contributing factor in type 1 and type 2 diabetes. The conventional treatment for type 1 diabetes has been to transplant exogenous insulin-producing islet cells, although this has been limited by the scarcity of donor pancreas available and the lack of efficient and efficacious protocols to isolate and transplant the donor islets. However, the recent demonstration of islet transplantation [54] indicates that considerable success in islet grafts can be achieved, although the scarcity of donor islets still remains a limitation. Several laboratories have approached this problem by attempting to use embryonic stem (ES) cells [55 – 57], but the efficiency of differentiation of ES cells to insulin-producing cells has been very low. Furthermore, the potential use of ES cells in humans is fraught with ethical issues. A recent study showed that oval cells from the liver could transdifferentiate into insulin-producing cells in longterm culture [58]. Putative precursor (or progenitor) cells have also been derived from adult humans and rodent pancreas (for review, see Refs [59,60]) after manipulation in vitro to differentiate them into hormone-producing cells. In view of the scarcity of donor islets and the ethical issues involved in transplantation of stem cells, mechanisms that www.sciencedirect.com
Vol.15 No.5 July 2004
201
would induce endogenous precursor and stem cells to differentiate into insulin-producing cells hold promise for the future. Pancreatic islets have been shown to regenerate after direct insult to the pancreas or islet b cells. Recently, transplantation of adult bone marrow-derived cells expressing Kit was shown to reduce hyperglycemia in mice [61] that were rendered diabetic with streptozotocin. In this study, the investigators showed that most transplanted bone marrow cells were localized to ductal and islet structures and that their presence was accompanied by a proliferation of recipient pancreatic cells, resulting in increased insulin production. Although diabetes reversal per se was not demonstrated, these data demonstrate the capacity of bone marrow-derived stem cells to initiate endogenous pancreatic tissue regeneration by a previously unrecognized mechanism. It has been shown that stem cells in the adult bone marrow have the potential to differentiate into different cell types [62,63], including insulin-producing cells [64]. Although pancreatic islet progenitors in the bone marrow are still to be isolated and characterized, these studies demonstrate that pools of b-cell progenitors exist in the bone marrow and could be mobilized to serve as precursors for tissue regeneration. Another recent demonstration in NOD mice has shown that islet regeneration can lead to ‘permanent reversal’ of diabetes [65]. Live male splenocytes injected into female diabetic NOD mice provided CD45- mesenchymal precursor cells for the reconstitution of functional islets. The donor splenocytes also contribute to the reversal of autoimmunity, possibly by ‘re-educating’ naive T cells through presentation of matched major histocompatibility complex class I molecules and self-antigens, yielding islets almost free of any signs of autoimmunity. This study again shows that adult diabetic NOD mice contain endogenous precursor cells capable of giving rise to new functional islets after the underlying autoimmune disease is eliminated. The ability of an endogenous population of NOD mouse stem cells to give rise to new islets suggests that therapies to reverse autoimmune diabetes need not incorporate transplantation of exogenous adult islets. The use of fresh splenocytes eliminates the need for cell culture manipulations that transform stem cells of fetal or adult origin into malignant precursors or fusion hybrids with an abnormal DNA content. Because the cell donors and hosts are adults this system precludes any ethical issues associated with the use of ES cells. These recent findings [58,61– 65] suggest that pancreatic progenitor cells might not be limited to the pancreas, but that cells from other tissues could be mobilized and induced to differentiate and contribute to the regenerative process. Stimuli such as surgical removal of a part of the pancreas, cellophane wrapping and transplantation of bone marrow cells can induce pancreatic regeneration to different extents. The NEDH rat model has shown that, in some way, the pancreas can ‘sense’ the total mass of insulin-producing cells and regulate it when grafted with an excess of insulin-producing cells and when this graft is subsequently removed. If such regulators really exist, the entire process could be visualized as a balance between regenerative and destructive factors and identifying and
202
Review
TRENDS in Endocrinology and Metabolism
enhancing the regenerative processes might greatly influence the outcome of diabetes. In light of the scarcity of donor islets available for transplantation to diabetic individuals, further studies to understand the mechanisms of adult human pancreatic regeneration would help us in exploiting this inexhaustible source of pancreatic progenitor cells for cell replacement therapy and better management of diabetes. Acknowledgements I would like to thank Elizabeth Geras-Raaka, NIDDK, National Institutes of Health, Bethesda, MD for critical discussion and suggestions in the preparation of this review and the editorial office of Journal of Endocrinology for permission to publish graphics from a previously published article. I apologize to colleagues whose work I was unable to cite in the text or reference section owing to space limitations.
References 1 Rafaeloff, R. et al. (1997) Cloning and sequencing of the pancreatic islet neogenesis associated protein (INGAP) gene and its expression in islet neogenesis in hamsters. J. Clin. Invest. 99, 2100– 2109 2 Yoon, J.W. and Notkins, A.L. (1983) Virus-induced diabetes in mice. Metabolism 32, 37 – 40 3 Fernandes, A. et al. (1997) Differentiation of new insulin-producing cells is induced by injury in adult pancreatic islets. Endocrinology 138, 1750 – 1762 4 Hardikar, A.A. et al. (1999) Effect of partial pancreatectomy on diabetic status in BALB/c mice. J. Endocrinol. 162, 189– 195 5 Finegood, D.T. et al. (1999) Prior streptozotocin treatment does not inhibit pancreas regeneration after 90% pancreatectomy in rats. Am. J. Physiol. 276, E822– E827 6 Stoffers, D.A. et al. (1997) Pancreatic agenesis attributable to a single nucleotide deletion in the human IPF1 gene coding sequence. Nat. Genet. 15, 106 – 110 7 Sharma, A. et al. (1999) The homeodomain protein IDX-1 increases after an early burst of proliferation during pancreatic regeneration. Diabetes 48, 507 – 513 8 Wells, J.M. and Melton, D.A. (1999) Vertebrate endoderm development. Annu. Rev. Cell Dev. Biol. 15, 393 – 410 9 Lammert, E. et al. (2003) Role of endothelial cells in early pancreas and liver development. Mech. Dev. 120, 59 – 64 10 Hardikar, A.A. et al. (2003) Human pancreatic precursor cells secrete FGF2 to stimulate clustering into hormone-expressing islet-like cell aggregates. Proc. Natl. Acad. Sci. U. S. A. 100, 7117 – 7122 11 Hebrok, M. et al. (2000) Regulation of pancreas development by hedgehog signaling. Development 127, 4905– 4913 12 Kim, S.K. et al. (2000) Activin receptor patterning of foregut organogenesis. Genes Dev. 14, 1866 – 1871 13 Kim, S.K. et al. (1997) Notochord to endoderm signaling is required for pancreas development. Development 124, 4243 – 4252 14 Lammert, E. et al. (2001) Induction of pancreatic differentiation by signals from blood vessels. Science 294, 564– 567 15 Miralles, F. et al. (1998) Follistatin regulates the relative proportions of endocrine versus exocrine tissue during pancreatic development. Development 125, 1017– 1024 16 Gradwohl, G. et al. (2000) Neurogenin3 is required for the development of the four endocrine cell lineages of the pancreas. Proc. Natl. Acad. Sci. U. S. A. 97, 1607 – 1611 17 Gu, G. et al. (2002) Direct evidence for the pancreatic lineage: NGN3 þ cells are islet progenitors and are distinct from duct progenitors. Development 129, 2447– 2457 18 Sussel, L. et al. (1998) Mice lacking the homeodomain transcription factor Nkx2.2 have diabetes due to arrested differentiation of pancreatic b cells. Development 125, 2213 – 2221 19 Sosa-Pineda, B. et al. (1997) The Pax4 gene is essential for differentiation of insulin-producing b cells in the mammalian pancreas. Nature 386, 399 – 402 20 Hardikar, A. and Gershengorn, M. Chapter 51: stem cells. In Diabetes Mellitus: A Fundamental and Clinical Text (3rd ed), (LeRoith, D., et al. eds), (in press) www.sciencedirect.com
Vol.15 No.5 July 2004
21 Lander, E.S. and Weinberg, R.A. (2000) Genomics: journey to the center of biology. Science 287, 1777– 1782 22 Chiang, M.K. and Melton, D.A. (2003) Single-cell transcript analysis of pancreas development. Dev. Cell 4, 383– 393 23 Dutta, S. et al. (1998) Regulatory factor linked to late-onset diabetes? Nature 392, 560 24 Christmanson, L. et al. (1990) The human islet amyloid polypeptide (IAPP) gene. Organization, chromosomal localization and functional identification of a promoter region. FEBS Lett. 267, 160 – 166 25 Ohlsson, H. et al. (1993) IPF1, a homeodomain-containing transactivator of the insulin gene. EMBO J. 12, 4251– 4259 26 Waeber, G. et al. (1996) Transcriptional activation of the GLUT2 gene by the IPF-1/STF-1/IDX-1 homeobox factor. Mol. Endocrinol. 10, 1327– 1334 27 Watada, H. et al. (1996) PDX-1 induces insulin and glucokinase gene expressions in a TC1 clone 6 cells in the presence of bcellulin. Diabetes 45, 1826 – 1831 28 Brissova, M. et al. (2002) Reduction in pancreatic transcription factor PDX-1 impairs glucose-stimulated insulin secretion. J. Biol. Chem. 277, 11225– 11232 29 Yamaoka, T. (2002) Regeneration therapy of pancreatic b cells: towards a cure for diabetes? Biochem. Biophys. Res. Commun. 296, 1039– 1043 30 Bernard-Kargar, C. and Ktorza, A. (2001) Endocrine pancreas plasticity under physiological and pathological conditions. Diabetes 50, S30– S35 31 Morisset, J. et al. (2000) Pancreatic inflammation, apoptosis, and growth: sequential events after partial pancreatectomy in pigs. Pancreas 21, 321 – 324 32 Schlegel, R.D. et al. (2000) Is there pancreatic regeneration? Morphological and functional certification after a corporocaudal splenopancreatectomy. Acta Gastroenterol. Latinoam. 30, 107– 113 33 Brockenbrough, J.S. et al. (1988) Discordance of exocrine and endocrine growth after 90% pancreatectomy in rats. Diabetes 37, 232– 236 34 Min, B.H. et al. (2003) Clusterin expression in the early process of pancreas regeneration in the pancreatectomized rat. J. Histochem. Cytochem. 51, 1355 – 1365 35 Smith, F.E. et al. (1991) Enhanced insulin-like growth factor I gene expression in regenerating rat pancreas. Proc. Natl. Acad. Sci. U. S. A. 88, 6152 – 6156 36 Bonner-Weir, S. et al. (1993) A second pathway for regeneration of adult exocrine and endocrine pancreas. A possible recapitulation of embryonic development. Diabetes 42, 1715 – 1720 37 Hayashi, K. et al. (1999) Regional differences in the cellular proliferation activity of the regenerating rat pancreas after partial pancreatectomy. Arch. Histol. Cytol. 62, 337 – 346 38 Bonner-Weir, S. et al. (2000) In vitro cultivation of human islet from expanded ductal tissue. Proc. Natl. Acad. Sci. U. S. A. 97, 7999– 8004 39 Bedoya, F.J. et al. (1986) Differential regulation of glucokinase activity in pancreatic islets and liver of the rat. J. Biol. Chem. 261, 10760 – 10764 40 Miyaura, C. et al. (1991) Expression of reg/PSP, a pancreatic exocrine gene: relationship to changes in islet b-cell mass. Mol. Endocrinol. 5, 226– 234 41 Clas, D. et al. (1988) Islet cell differentiation and proliferation induced by a pancreatic cytosol extract. Surg. Forum 39, 164 – 166 42 Rosenberg, L. et al. (1990) Trophic stimulation of the ductal/islet cell axis: a new approach to the treatment of diabetes. Surgery 108, 191– 197 43 Rosenberg, L. et al. (1989) The effect of cellophane wrapping of the pancreas in the Syrian golden hamster: autoradiographic observations. Pancreas 4, 31 – 37 44 Rosenberg, L. (1998) Induction of islet cell neogenesis in the adult pancreas: the partial duct obstruction model. Microsc. Res. Tech. 43, 337– 346 45 Rafaeloff, R. et al. (1997) Cloning and sequencing of the pancreatic islet neogenesis associated protein (INGAP) gene and its expression in islet neogenesis in hamsters. J. Clin. Invest. 99, 2100 – 2109 46 Rafaeloff, R. et al. (1996) Identification of differentially expressed genes induced in pancreatic islet neogenesis. FEBS Lett. 378, 219 – 223 47 Sarvetnick, N.E. and Gu, D. (1992) Regeneration of pancreatic
Review
48 49
50 51
52 53
54
55
56
TRENDS in Endocrinology and Metabolism
endocrine cells in interferon-g transgenic mice. Adv. Exp. Med. Biol. 321, 85 – 89 Gu, D. and Sarvetnick, N. (1993) Epithelial cell proliferation and islet neogenesis in IFN-g transgenic mice. Development 118, 33– 46 Kayali, A.G. et al. (2003) The stromal cell-derived factor-1a/CXCR4 ligand – receptor axis is critical for progenitor survival and migration in the pancreas. J. Cell Biol. 163, 859– 869 Kritzika, M.R. et al. (2000) Transcription factor expression during pancreatic islet regeneration. Mol. Cell. Endocrinol. 164, 99 – 107 Krakowski, M. et al. (2000) IFN-g overexpression within the pancreas is not sufficient to rescue Pax4, Pax6,and Pdx-1 mutant mice from death. Pancreas 21, 399 – 406 Arnush, M. et al. (1996) Growth factors in the regenerating pancreas of g-interferon transgenic mice. Lab. Invest. 74, 985 – 990 Schwitzgebel, V.M. et al. (2000) Expression of neurogenin3 reveals an islet cell precursor population in the pancreas. Development 127, 3533 – 3542 Shapiro, A.M. et al. (2000) Islet transplantation in seven patients with type 1 diabetes mellitus using a glucocorticoid-free immunosuppressive regimen. N. Engl. J. Med. 343, 230 – 238 Soria, B. et al. (2000) Insulin-secreting cells derived from embryonic stem cells normalize glycemia in streptozotocin-induced diabetic mice. Diabetes 49, 157 – 162 Lumelsky, N. et al. (2001) Differentiation of embryonic stem cells to
57 58
59
60 61 62 63 64
65
Vol.15 No.5 July 2004
203
insulin-secreting structures similar to pancreatic islets. Science 292, 1389– 1394 Assady, S. et al. (2001) Insulin production by human embryonic stem cells. Diabetes 50, 1691– 1697 Yang, L. et al. (2002) In vitrotrans-differentiation of adult hepatic stem cells into pancreatic endocrine hormone-producing cells. Proc. Natl. Acad. Sci. U. S. A. 99, 8078 – 8083 Lechner, A. and Habener, J.F. (2003) Stem/progenitor cells derived from adult tissues: potential for the treatment of diabetes mellitus. Am. J. Physiol. Endocrinol. Metab. 284, E259– E266 Bonner-Weir, S. and Sharma, A. (2002) Pancreatic stem cells. J. Pathol. 197, 519 – 526 Hess, D. et al. (2003) Bone marrow-derived stem cells initiate pancreatic regeneration. Nat. Biotechnol. 21, 763 – 770 Jiang, Y. et al. (2002) Pluripotency of mesenchymal stem cells derived from adult marrow. Nature 418, 41 – 49 Reyes, M. et al. (2002) Origin of endothelial progenitors in human postnatal bone marrow. J. Clin. Invest. 109, 337 – 346 Ianus, A. et al. (2003) In vivoderivation of glucose-competent pancreatic endocrine cells from bone marrow without evidence of cell fusion. J. Clin. Invest. 111, 843– 850 Kodama, S. et al. (2003) Islet regeneration during the reversal of autoimmune diabetes in NOD mice. Science 302, 1223– 1227
Elsevier.com – Dynamic New Site Links Scientists to New Research & Thinking Elsevier.com has had a makeover, inside and out. Designed for scientists’ information needs, the new site, launched in January, is powered by the latest technology with customer-focused navigation and an intuitive architecture for an improved user experience and greater productivity. Elsevier.com’s easy-to-use navigational tools and structure connect scientists with vital information – all from one entry point. Users can perform rapid and precise searches with our advanced search functionality, using the FAST technology of Scirus.com, the free science search engine. For example, users can define their searches by any number of criteria to pinpoint information and resources. Search by a specific author or editor, book publication date, subject area – life sciences, health sciences, physical sciences and social sciences – or by product type. Elsevier’s portfolio includes more than 1800 Elsevier journals, 2200 new books per year, and a range of innovative electronic products. In addition, tailored content for authors, editors and librarians provides up-to-the-minute news, updates on functionality and new products, e-alerts and services, as well as relevant events. Elsevier is proud to be a partner with the scientific and medical community. Find out more about who we are in the About section: our mission and values and how we support the STM community worldwide through partnerships with libraries and other publishers, and grant awards from The Elsevier Foundation. As a world-leading publisher of scientific, technical and health information, Elsevier is dedicated to linking researchers and professionals to the best thinking in their fields. We offer the widest and deepest coverage in a range of media types to enhance cross-pollination of information, breakthroughs in research and discovery, and the sharing and preservation of knowledge. Visit us at Elsevier.com.
Elsevier. Building Insights. Breaking Boundaries. www.sciencedirect.com