the most outstanding feature of cells is that they produce a large amount of insulin , the only hormone that lowers blood glucose , and secrete it continuously in response to changes in the extracellular glucose concentration . insulin secretion from pancreatic cells also is modified by a variety of nutrients , hormones and neuronal signals in the maintenance of systemic glucose homeostasis1 . for treatment of diabetes with absolute insulin deficiency normal pancreatic cells adjust insulin secretion continually in response to varying blood glucose levels ; exogenous insulin administration can not maintain blood glucose levels within the narrow physiological range that protects from development of various diabetic complications . although transplantation of the pancreas or pancreatic islets has been an efficient therapeutic option for cure of patients with insulindeficient diabetes , the chronic shortage of donor organs limits widespread application of such transplantation . thus , regenerative medicine , including generation of pancreatic cells , pancreatic islets or whole pancreas , is an intriguing approach to the development of future therapy for diabetes2 . in the present review , we discuss functional differentiation of native pancreatic cells as the scientific basis of regenerative medicine in the field of diabetes , and introduce approaches to regeneration of cells ( figure  1 ) . new pancreatic cells could be generated by induction of selfreplication ( proliferation ) of preexisting islet cells or differentiation / transdifferentiation of adult stem / progenitor cells ( neogenesis ) in vivo . in addition , the new cells could also be obtained by forced proliferation of isolated preexisting cells or differentiation / transdifferentiation of embryonic stem cells ( escs)/inducedpluripotent stem cells ( ipscs ) or adult stem / progenitor cells in vitro . pancreatic cells secrete adequate amounts of insulin in response to extracellular glucose concentration so that blood glucose levels are controlled within a narrow physiological range ; severe hypoglycemia or hyperglycemia seldom occurs in healthy subjects . this is because the cell has properties of : ( i ) insulin biosynthesis ; ( ii ) glucose sensing ; ( iii ) metabolismsecretion coupling ; and ( iv ) regulated exocytosis ( figure  2 ) . glucose entering cells through glucose transporters is metabolized , leading to an increase in the adenosine triphosphate ( atp ) concentration , closure of the atpsensitive k ( katp ) channels , depolarization of the cell membrane and opening of the voltagedependent ca channels , which allows ca influx . the resultant rise in intracellular ca concentration triggers soluble nethylmaleimidesensitive factor attachment protein receptor ( snare)dependent exocytosis of insulin granules . cells thus must possess functions of : ( i ) insulin biosynthesis ; ( ii ) glucose sensing ; ( iii ) metabolismsecretion coupling ; and ( iv ) regulated exocytosis . pc1/3 , prohormone convertase 1/3 ; pc2 , prohormone convertase 2 ; snap25 , synaptosomalassociated protein 25 ; stx , syntaxin ; vamp2 , vesicleassociated membrane protein 2 . although our knowledge of the development of wellregulated insulin secretion is limited , analyses of developing and differentiating pancreatic cells provide clues . it is known that fetal and neonatal pancreatic cells lack glucose responsiveness3 . by using rat models , it was found that fetal pancreatic cells show lower facilitation of glucose oxidation than adult cells4 . as forced depolarization can stimulate insulin secretion similarly in both fetal and adult cells4 , fetal cells seem to show insulin biosynthesis , granule formation and expression of soluble nethylmaleimidesensitive factor attachment protein receptor ( snare ) proteins . thus , immaturity in glucose metabolism might account for the lack of glucose responsiveness in fetal pancreatic cells . in fact , expression levels of key enzymes [ mitochondrial glycerol3phosphate dehydrogenase ( mgpdh ) and mitochondrial malate dehydrogenase ] in nadh ( the reduced form of nicotinamide adenine dinucleotide ) shuttles , which have an important role in adenosine triphosphate ( atp ) production , are significantly lower in fetal than in adult cells , and forced expression of mgpdh improves glucoseinduced insulin secretion in fetal pancreatic islets6 . immaturity of glucose metabolism in fetal pancreatic cells is also supported by a study using human and porcine fetuses7 , and by a recent dna microarray study in fetal rat cells8 . however , the mechanism of cell acquisition of glucoseresponsiveness during growth is still largely unknown . it has been reported that addition of glucagonlike peptide1 ( glp1 ) , an incretin hormone , induces both first and second phase glucoseinduced insulin secretion in human fetal isletslike cell clusters ( immature islets)9 . although this finding reflects an acute effect of glp1 on the function of immature cells , glp1 also has roles in differentiation of pancreatic cells . a study has shown by using fetal pig islets that glp1 facilitates differentiation of the immature precursor of pancreatic cells to mature cells10 . because glp1 begins to secret from lcells on the gut tube by intake of foods after delivery , it is possible that glp1 has physiological roles in functional differentiation of pancreatic cells . in contrast , it is known that terminal differentiation of pancreatic cells requires expression of the transcription factor vmaf musculoaponeurotic fibrosarcoma oncogene homolog a ( mafa ) , which regulates expression of genes participating in glucoseinduced insulin secretion including glut2 , glucokinase , sur1 and kir6.211 . a recent report showed the possibility that mafa is involved in the acquisition of glucose responsiveness in neonatal rat pancreatic cells12 , an important role in functional maturation of the cells . although the relationship between mafa and incretins is unknown , as mafa increases the expression of the glp1 receptor13 , their interaction or cooperation in differentiation of pancreatic cells seems likely most studies on regeneration of pancreatic cells in vivo have been carried out in rodents using pancreatic injury models . nicotinamide , an inhibitor of poly(adenosine diphosphateribose ) synthethase / polymerase , prevents the development of diabetes in experimental animals after administration of the cell toxins , streptozotocin and alloxan14 . in vitro studies have shown that the agent has beneficial effects on proliferation and differentiation of pancreatic endocrine cells15 , but the mechanism is not known . exendin4 , an analog of glp1 , has been reported to enhance both proliferation and neogenesis of pancreatic cells in rats with 90% pancreatectomy17 . betacellulin , a growth factor belonging to the epidermal growth factor ( egf ) family , has been shown to promote neogenesis of cells and ameliorate glucose intolerance in mice with selective alloxan perfusion18 , and is also reported to enhance proliferation of cells in 90% pancreatectomized rats19 . the reg gene , which is induced in regenerating pancreatic islets , has been identified20 . there are several lines of studies suggesting the cell origin of regenerated pancreatic cells . in transgenic mice expressing interferongamma specifically in pancreatic cells , a dramatic proliferation of pancreatic ductal cells , and the appearance of primitive endocrine cells and their subsequent differentiation into endocrine cells has been reported21 . during regeneration , transitional intermediate cells expressing both carbonic anhydrase ii and amylase22 , and bearing both endocrine and exocrine granules23 appear . the authors speculate from these findings that pancreatic duct cells represent facultative progenitors in adult pancreas . however , their results also suggest that pancreatic acinar cells give rise to intermediate cells that have characteristics of pancreatic duct cells , and then differentiate into endocrine cells . it has been reported that overexpression of transforming growth factor induces expansion of pancreatic and duodenal homeobox 1 ( pdx1)expressing ductal epithelium in the pancreas , and that focal areas of islet neogenesis can be observed24 . as pancreatic acinar cells isolated from transforming growth factor transgenic mice convert into ductal cells25 , the expanded pancreatic ductal cells expressing pdx1 in these mice might well be derived from pancreatic acinar cells . in addition , some pancreatic injury models have been shown to exhibit pancreas regeneration . after ligation of the pancreatic duct in rats , replacement of exocrine acini by ductlike structures is observed27 . this acinoductal metaplasia has been thought to be at least in part the result of transdifferentiation of amylasepositive pancreatic acinar cells into amylasenegative and cytokeratinpositive ductlike cells28 . by treating the rats with dexamethasone to inhibit loss of amylase expression , transitional cells coexpressing amylase and cytokeratin 20 were detected28 , supporting the notion of acinartoductal transdifferentiation . furthermore , insulinpositive cells that also express amylase have been found , indicating acinartoendocrine transdifferentiation . although histological analysis has shown that neogenesis or regeneration of pancreatic cells occurs in certain conditions , the cellular origin of the new cells has not been shown . recent studies using genetic cell lineage tracing or other cell labeling methods suggest that adult pancreatic cells are not derived from noncells29 . using genetic cell lineage tracing , dor et  al.29 showed that adult pancreatic cells in mice are maintained predominantly by selfreplication of preexisting cells . they labeled pancreatic cells selectively with human alkaline phosphatase by creloxpbased conditional recombination in adult pancreas , and chased the fate of preexisting cells . in this system , when new cells in an adult pancreas are generated from noncells , such as stem / progenitor cells , the frequency of the labeled cells should decrease after a chase period . the results showed that the labeling frequency of the cells remained unchanged , indicating that the new cells were generated primarily from preexisting cells . they concluded that terminally differentiated cells retain proliferative capacity , and cast doubt on a significant role for adult stem cells in cell replenishment29 . georgia and bhushan30 reported that during neonatal development , cyclin d2 expression in the endocrine pancreas coincided with the replication of endocrine cells and a massive increase in islet mass . cyclin d2 is not required for exocrine and ductal cell proliferation , but is required for replication of endocrine cells . in cyclin d2 mice , pancreatic islets are much smaller and cell mass thus , cyclin d2 plays a key role in cell replication , which might be the primary mechanism for maintaining postnatal cell mass30 . furthermore , teta et  al.31 showed by using a dna analogbased lineagetracing technique that unlike gastrointestinal and skin epithelia , specialized progenitors do not contribute to adult cell mass , not even during acute cell regeneration . instead , adult cells exhibit equal proliferation potential , and expand from within a vast and uniform pool of mature cells32 . thus , it is likely that pancreatic cell mass is maintained primarily by selfreplication of preexisting cells in adult mice , although the existence of pancreatic tissuespecific stem / progenitor cells can not be excluded . the turnover rate of pancreatic cells in adult is generally low , expansion of the cells being detected only in several conditions including neonate , pregnancy and obesity . the mass of pancreatic cells increases in the neonatal period as the body and organs grow . however , the increases in cell number and the weight of the whole pancreas are not a coincidence33 , suggesting different mechanisms in the regulation of the mass of pancreatic endocrine and exocrine cells . we have recently shown that noncells contribute to an increase in mass expansion of pancreatic cells from neonate through to youth34 . although the signals leading to proliferation of pancreatic cells are not yet well characterized , a recent study has shown that egfreceptor signaling is required for expansion of cells in mice under highfat diet conditions and pregnancy35 . in addition , during pregnancy , an increase in insulin resistance in pregnant mothers maintains the nutrient flow to the growing fetus . prolactin and placental lactogen counterbalance this resistance , and prevent maternal hyperglycemia by driving expansion of the maternal population of insulinproducing cells . it should be noted that hepatic activation of extracellular regulated kinase signaling induces proliferation of pancreatic cells through a neuronalmediated relay of metabolic signals36 . in addition , there are several reports showing identification of functional pancreatic adult stem / progenitor cells in vivo . it has been shown that progenitors for cells are activated in injured adult mouse pancreas , and are located in the ductal lining . differentiation of the adult progenitors is ngn3 dependent , and gives rise to all islet cell types both in situ and cultured in embryonic pancreas explants37 . that study strongly suggests that adult cells can be generated not only from preexisting cells , but also from noncells . however , because such progenitors can be detected only when the cells begin to express ngn3 , their precise origin and properties are not ascertained . although inada et  al.38 reported that by using lineage tracing utilizing human carbonic anhydrase ii ( caii ) promoter a subset of adult cells are generated from pancreatic duct cells , solar et  al.39 showed that the duct cells ( hnf 1 as the marker ) lose potential for differentiation into cells after birth . thus , the origin of newly formed cells in the adult pancreas remains to be identified . in vitro expansion of pancreatic cells represents an attractive strategy for obtaining a large amount of cells for transplantation . indeed , human cells possess proliferation capacity when cultured on extracellular matrices with growth factors and hormones40 . however , the capacity is very limited while preserving the cell phenotype43 , expansion of cells often occurs along with loss of the cell phenotype ( i.e. , expression and secretion of insulin)44 . emt was originally defined in the context of developmental stages : a biological process that allows a polarized epithelial cell to undergo multiple biochemical changes that enable it to assume a mesenchymal cell phenotype45 . the first report that referred phenotypic changes of pancreatic cells to the emt was carried out by gershengorn et  al.46 , and a similar phenomenon was observed by others47 . however , other studies using lineage tracing showed that murine cells do not undergo emt48 . they found that most proliferative mesenchymal cells migrating out from pancreatic islets in vitro were not derived from cells , and suggested that these cells do not represent a useful source for the generation of physiologically competent cells for treatment of diabetes48 . at that time , the journal , diabetes , stated that  emt participation in mechanism of cell expansion has been preempted51 . nevertheless , we and others have shown direct evidence of emtlike phenotypic changes in mouse and human pancreatic cells52 . we have established a culture system using fetal pancreatic cells as feeder cells that induce dedifferentiation of adult cells , and we have developed a method whereby preexisting pancreatic cells can be traced throughout the culture process , even when the cells lose insulin expression53 . we showed that pancreatic cells of adult mice undergo dedifferentiation into mesenchymelike fibroblastoid cells ( i.e. , emt ) , and that the process is associated associated with the progression of the cell cycle . human pancreatic cells were also reported to have the potential to proliferate on emt and redifferentiate into islet progenitorlike cells54 . these studies suggest the feasibility of the generation of a large amount of insulinsecreting cells by in vitro expansion of pancreatic cells . the first report of the in vitro generation of like cells from mouse es cells was by lumelsky et  al.55 in 2001 . however , as these insulinpositive cells showed low insulin production capacity and poor insulin secretory response , the cells could not be identified as cells . in fact , later studies showed that es cellderived insulinpositive cells can be generated as a result of uptake of exogenous insulin57 . in 2006 , a new protocol was developed to generate insulinsecreting cells , in which differentiation processes mimic pancreatic organogenesis by directing cells through stages resembling definitive endoderm , guttube endoderm , pancreatic endoderm and endocrine precursor59 . however , the insulinsecreting cells made by this method showed poor glucose responsiveness and appeared immature . a later study made by the same group showed that glucoseresponsive insulinsecreting cells can be generated by transplantation of the cells at the stage of pancreatic endoderm60 . insulinsecreting cells have also been generated from inducedpluripotent stem ( ips ) cells61 , but precise analysis of the insulin secretion has not been carried out . cmet , cmet positive ; ckit , ckit negative ; cd45 , cd45 negative ; dna , deoxyribonucleic acid ; egf , epidermal growth factor ; es , embryonic stem ; facs , fluorescenceactivated cell sorting ; gck , glucokinase ; glp1 , glucagonlike peptide1 ; glut , glucose transporter ; iapp , islet amyloid polypeptide ; ipc , isletproducing stem cells ; ips , iduced pluripotent ; kgf , keratinocyte growth factor ; kir , inwardrectifier potassium ion channel ; lif , leukemia inhibitory factor ; mafa , musculoaponeurotic fibrosarcoma oncogene homolog a ; mrna , messenger ribonucleic acid ; na , not applicable ; neurod , neurogenic differentiation ; nkx2.2 , nk2 homeobox 2 ; nod , nonobese diabetic ; pdx1 , pancreatic and duodenal homeobox 1 ; sur1 , sulphonylurea receptor 1 ; ter119 , ter119 negative . calculation based on our own measurement of insulin content of normal islet : 250  g / mg protein [ 75  ng / islet , 3  g ( 500 pmol)/g dna ] . insulin secretion was measured as accumulation for 24  h. several studies have attempted to identify stem / progenitor cells in the adult pancreas . zulewski et  al.16 showed that cells expressing the neural stem cell marker nestin occur in human and rat pancreatic islets , and that these cells can be isolated and cultured for a long time . it has been shown that cultured nestinpositive cells can be differentiated into insulinproducing cells16 , and that such cells from human fetal pancreas when transplanted can be expanded and differentiated into isletlike cell clusters , which can reverse hyperglycemia in diabetic mice65 . these candidate progenitor cells proliferate in the serumfree conditions used for neural stem cell ( nsc ) culture and form spherical cell clusters like nscs by floating culture . interestingly , the cells in the spherical clusters show characteristics of both pancreatic and neural precursors . furthermore , the potential progenitors give rise to multiple types of neural cells , including neurons and glial cells , and also differentiate into insulinproducing (  ) cells , glucagonproducing (  ) cells , and somatostatinproducing (  ) cells . the insulinproducing cells derived from these progenitors are glucosecompetent in terms of ca responsiveness and insulin secretion . suzuki et  al.67 reported isolation of pancreatic progenitor cells from adult mouse by using fluorescenceactivated cell sorting . the isolated cells express cmet , the receptors for hepatocyte growth factor , but do not express hematopoietic and vascular endothelial antigens , such as cd45 , ter119 , ckit and flk1 . thus , hepatocyte growth factor / cmet signaling might play an important role in the maintenance of these progenitorlike cells . the cells formed clonal colonies in vitro , and differentiated into multiple pancreatic lineages from single cells . however , no functional analysis has been carried out for the differentiated pancreatic cells induced from these candidate progenitors . taken together , these findings suggest that although stem / progenitorlike cells might be obtained from the adult pancreas , it is not yet clear that such isolated stem / progenitorlike cells have the full potential to differentiate into native pancreatic cells and function as stem / progenitors in the pancreas in vivo . peck et  al.68 isolated and cultured pancreatic ductal cells from prediabetic nonobese diabetic mice , and induced isletlike cell clusters containing insulinproducing cells . although the insulin secretory capacity of the induced cells was extremely low , transplantation of the cells to diabetic mice could lead to normalization of blood glucose levels . it is possible that these insulinproducing cells further differentiate in vivo , but no relevant functional analyses after transplantation have been carried out . the ductal cells were expanded as a monolayer , and the cells were then overlaid with a thin layer of matrigel . the cells formed 3d structures of ductal cysts from which isletlike clusters budded . however , they reported the possibility in a later study that contaminated cells in the starting material underwent dedifferentiation and redifferentiation during the culture72 . in contrast , bouwens et  al.73 have claimed the importance of transdifferentiation of pancreatic exocrine cells in regeneration of pancreatic cells . they reported that pancreatic exocrine cells can be converted into insulinproducing cells by culturing with egf and leukemia inhibitory factor74 . they isolated pancreatic acinar cells from adult rats , and cultured the cells in suspension without adding any growth factor or cytokines . insulinpositive cells were detected at the periphery of the spherical cell clusters derived from the acinar cells76 . although these studies suggest transdifferentiation of pancreatic acinar cells into insulinproducing cells , neither direct evidence of the origin of these cells nor their precise insulin secretory properties were identified . at the same time , we also reported transdifferentiation of pancreatic acinar cells , and utilized the method of cell lineage tracing based on the creloxp system to determine the origin of the newlymade insulinsecreting cells77 . cell adhesion in pancreatic acinar cells induces dedifferentiation , and the subsequent activation of the phosphoinositide 3kinase / akt pathway recovers the cell cell adhesion , which induces redifferentiation into endocrine or ductal cells ( figure  3)78 . this explains the rarity of in vivo transdifferentiation of acinar cells into endocrine cells , as even in severe cases of pancreatitis or cancer , cell  cell contacts are not completely destroyed . the acinar cellderived insulinsecreting cells secreted insulin in response to a variety of stimuli , including glucose and glp1 . however , insulin production of the cells was lower than that of native islets . as the newlymade cells expressed genes , such as hnf6 and pgp9.5 , which are not expressed in native cells , but did not express mafa , which regulates the expression of genes that participate in glucoseinduced insulin secretion77 , the cells were thought to be in an immature state . cell adhesion , which causes dedifferentiation of the acinar cells . meanwhile , epidermal growth factor receptors are activated , which is followed by activation of the phosphoinositide 3kinase pi3k / akt pathway . within a few days of culture , cadherinmediated cell cell adhesion is recovered by the enhanced expression of ecadherin , which is essential for redifferentiation of the dedifferentiated cells into insulinsecreting cells . thorel et  al.83 reported that cells transdifferentiate into cells under conditions of severe loss of cells . they generated mice carrying a transgene containing the diphtheria toxin receptor under the control of the insulin promoter ( ripdtr ) . in male ripdtr mice , more than 99% of the cells in the adult pancreas can be destroyed by injection of diphtheria toxin . when such mice were kept alive for up to 10  months with insulin treatment during the initial 5  months , the cell mass increased to 10% of normal animals on average , and glycemic control improved in some of the mice . by using a conditional cell lineage tracing method , the origin of the regenerated cells was shown not to be surviving preexisting cells , but rather glucagonexpressing cells . although their findings suggest the usefulness of cells as a source of new cells , the conversion of cells to cells decreases the cell population , which might cause the loss of glucagon action in the maintenance of glucose homeostasis . however , they later reported that neartotal ablation of cells did not affect the insulin counterregulatory response and glucose homeostasis in mice84 . talchai et  al.85 recently showed that pancreatic cell dedifferentiation rather than cell death is responsible for diabetic cell failure in several different diabetic mouse models . in their experiments , adult mouse cells underwent dedifferentiation and reverted to progenitorlike cells expressing ngn3 , oct4 , nanog and lmyc under conditions of physiological stress , such as cellspecific disruption of foxo1 . it has also been shown that ectopic expression of certain transcription factors can change the fate between  and cells86 . these studies suggest an attractive future therapeutic strategy for diabetes in which pancreatic cells can be generated from cells by inducing their transdifferentiation in vivo . there are many different approaches towards the development of cell replacement therapy for insulindeficient diabetes mellitus . however , in vivo regeneration of cells in humans is not realistic at present from both efficacy and safety points of view , and most newly generated insulinsecreting cells in vitro are not fully differentiated cells , as assessed by both insulin secretory properties and gene expression profile . another unique and intriguing strategy of regenerative medicine for diabetes has recently been proposed by kobayashi et  al.87 , who made use of the technique of  blastocyst complementation  , which enables in vivo generation of organs derived from pluripotent stem cells ( pscs : es cells or ips cells ) , and generated a rat pancreas in a mouse by injecting wildtype rat pscs into a pdx1 ( pancreatogenesisdisabled ) mouse blastocyst . thus , a human pancreas might be generated from human pscs in a pig for use in organ transplantation , although many issues of concern must be addressed to bring this principle into clinical use . human pancreatic cells have recently been shown to express glp1 and prohormone convertase 1/3 ( pc1/3 ) , as well as glucagon and pc288 . in addition , a recent study showed ectopic expression of glucosedependent insulinotropic polypeptide ( gip ) in pancreatic cells in mice with the complete absence of proglucagonderived peptide89 . we must expand our knowledge of the physiological processes of differentiation , proliferation and regeneration of pancreatic cells as an essential step on the path to cell therapy to cure patients with absolute insulin deficiency . this work was supported by a crest grant from the japan science and technology agency , and grantinaid for scientific research and grants for the global center of excellence ( coe ) programs  global center of excellence for education and research on signal transduction medicine in the coming generation ,  global center for education and research in integrative membrane biology , and kobe translational research cluster , the knowledge cluster initiative from the ministry of education , culture , sports , science and technology , japan .