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  • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
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  • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
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  • C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
  • C12N2506/22—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from pancreatic cells

Definitions

• the invention relates to a medium for preparing dedifferentiated cells and more particularly to a basal feeding medium for the development, maintenance and expansion of a dedifferentiated cell population with at least bipotentiality, which may be used in an in vi tro method for islet cell expansion.
• Diabetes mellitus has been classified as type I, or insulin-dependent diabetes mellitus (IDDM) and type II, or non-insulin-dependent diabetes mellitus (NIDD ) .
• IDDM insulin-dependent diabetes mellitus
• NIDD non-insulin-dependent diabetes mellitus
• NIDDM patients have been subdivided further into (a) nonobese (possibly IDDM in evolution) , (b) obese, and (c) maturity onset (in young patients) .
• IDDM nonobese
• b obese
• maturity onset in young patients
• Among the population with diabetes mellitus about 20% suffer from IDDM. Diabetes develops either when a diminished insulin output occurs or when a diminished sensitivity to insulin cannot be compensated for by an augmented capacity for insulin secretion.
• Tight glucose control appears to be the key to the prevention of the secondary complications of diabetes.
• DCCT Diabetes Complications and Control Trial
• Strict glucose control was associated with a three-fold increase in incidence of severe hypoglycemia, including episodes of seizure and coma.
• glycosylated hemoglobin levels decreased in the treatment group, only 5% maintained an average level below 6.05% despite the enormous amount of effort and resources allocated to the support of patients on the intensive regime (The Diabetes Control and Complication Trial Research Group, N. Engl . J. Med . , 1993; 29:977-
• a major goal of diabetes research therefore, has been the development of new forms of treatment that endeavor to reproduce more closely the normal physiologic state.
• a closed- loop insulin pump coupled to a glucose sensor, mimicking ⁇ -cell function in which the secretion of insulin is closely regulated has not yet been successful.
• Only total endocrine replacement therapy in the form of a transplant has proven effective in the treatment of diabetes mellitus.
• transplants of insulin- producing tissue are a logical advance over subcutaneous insulin injections, it is still far from clear whether the risks of the intervention and of the associated long-term immunosuppressive treatment are lower those in diabetic patients under conventional treatment .
• pancreas transplantation has limited its wider application and provided impetus for the development of islet transplantation.
• transplantation of islets alone while enabling tight glycemic control, has several potential advantages over whole pancreas transplantation.
• Adequate numbers of isogenetic islets transplanted into a reliable implantation site can only reverse the metabolic abnormalities in diabetic recipients in the short term. In those that were normoglycemic post-transplant, hyperglycemia recurred within 3-12 mo. (Orloff M, et al . , Transplantation 1988; 45:307). The return of the diabetic state that occurs with time has been attributed either to the ectopic location of the islets, to a disruption of the enteroinsular axis, or to the transplantation of an inadequate islet cell mass (Bretzel RG, et al . In: Bretzel RG, (ed) Diabetes mellitus (Berlin: Springer, 1990) p.229) .
• micro- encapsulated islets injected into the peritoneal cavity of the dog fail within 6 months (Soon-Shiong P, et al . , Transplantation 1992; 54: 769), and islets placed into a vascularized biohybrid pancreas also fail, but at about one year.
• histological evaluation of the graft has indicated a substantial loss of islet mass in these devices (Lanza RP, et al . , Diabetes 1992; 41: 1503). No reasons have been advanced for these changes. Therefore maintenance of an effective islet cell mass post-transplantation remains a significant problem.
• One aim of the invention is to provide a platform for the preparation of dedifferentiated intermediate cells derived from post-natal islets of Langerhans, their expansion and the guided induction of islet cell differentiation, leading to insulin- producing cells that can be used for the treatment of diabetes mellitus.
• an in vi tro method for islet cell expansion which comprises the steps of: a) preparing dedifferentiated cells derived from post-natal islets of Langerhans cells; b) expanding the dedifferentiated cells; and c) inducing islet cell differentiation properties of the expanded cells of step b) to become insulin-producing cells.
• step a) and step b) are concurrently effected using a solid matrix, basal feeding medium and appropriate growth factors to permit the development, maintenance and expansion of a dedifferentiated cell population with at least bipotentiality .
• a medium for preparing dedifferentiated cells derived from post-natal islets of Langerhans comprises in a physiologically acceptable culture medium an effective amount of a solid matrix environment for a three-dimensional culture, a matrix protein, and a first and a second factor for developing, maintaining and expanding the dedifferentiated cells.
• the first factor induces a rise in intracellular cAMP
• the second factor is derived from acinar cells.
• the acinar cells must be present in addition to the other three factors in order for the change to occur.
• the first factor may comprise one or more of cholera toxin (CT) , forskolin, high glucose concentrations, a promoter of cAMP, and EGF .
• CT cholera toxin
• the culture medium may comprise DMEM/12 supplemented with an effective amount of fetal calf serum, such as 10%.
• the matrix protein comprises one or more of laminin, collagen type I and MatrigelTM.
• step c) is effected by removing cells from the matrix and resuspended in a basal liquid medium containing soluble matrix proteins and growth factors .
• the basal liquid medium is CMRL
• the basal liquid medium may further comprise glucose concentration of at least 11 mM.
• the basal liquid medium may further comprise inhibitors of known intracellular signaling pathways of apoptosis and/or specific inhibitor of p38.
• an in vi tro method for producing cells with at least bipotentiality comprises the steps of: a) preparing dedifferentiated cells derived from post-natal islets of Langerhans cells from a patient; whereby when the dedifferentiated cells are introduced in si tu in the patient, the cells are expanded and islet cell differentiation properties are induced to become in si tu insulin-producing cells.
• an in vi tro method for stem cell expansion which comprises the steps of: a) preparing dedifferentiated intermediate cells derived from stem cells; b) expanding in vi tro the dedifferentiated intermediate cells; and c) inducing in vi tro stem cell differentiation properties of the expanded cells of step b) to become stem cells.
• the stem cells are selected from the group consisting of muscle, skin, bone, cartilage, lung, liver, bone marrow and hematopoietic cells.
• a method for the treatment of diabetes mellitus in a patient which comprises the steps of a) preparing dedifferentiated cells derived from post-natal islets of Langerhans cells of the patient; and b) introducing the dedifferentiated cells in si tu in the patient, wherein the cells are expanded in si tu and islet cell differentiation properties are induced in si tu to become insulin-producing cells.
• a method for the treatment of diabetes mellitus in a patient which comprises the steps of a) preparing dedifferentiated cells derived from post-natal islets of Langerhans cells of the patient ; b) expanding in vi tro the dedifferentiated cells; c) inducing in vi tro islet cell differentiation properties of the expanded cells of step b) to become insulin-producing cells; and d) introducing the cells of step c) in si tu in the patient, wherein the cells produce insulin in si tu .
• Langerhans is intended to mean islet cells of any origin, such as human, porcine and canine, among others .
• Fig. 1 illustrates cell -type conversion from islet to duct-like structure (human tissues) , (a)
• Islet in the pancreas (b) Islet following isolation and purification, (c) islet in solid matrix beginning to undergo cystic change, (d-f) progressive formation of cystic structure with complete loss of islet morphology.
• Fig. 2 illustrates same progression of changes as in Fig. 1.
• Cells are stained by immunocytochemistry for insulin.
• Fig. 3 illustrates same progression of changes as in Fig. 1.
• Cells stained by immunocytochemistry for glucagon. (a) Islet in pancreas.
• One cell still contains glucagon (arrow) .
• FIG. 4 A-C illustrate demonstration of cell phenotype by CK-19 immunocytochemistry.
• Upper right panel- only the epithelial-like component retains CK-19 immunoreactivity. The solid component has lost its CK- 19 expression, and appears islet-like.
• Fig. 5 A-B illustrate upper panel - Ultrastructural appearance of cells composing the cystic structures in solid matrix. Note the microvilli and loss of endosecretory granules. The cells have the appearance of primitive duct-like cells. Lower panel- ultrastructural appearance of cystic structures removed from the solid matrix and placed in suspension culture. Note the decrease in microvilli and the reappearance of endosecretory granules .
• Fig. 6 A-B illustrate in si tu hybridization for pro-insulin mRNA.
• Upper panel-cystic structures with virtually no cells containing the message.
• Lower panel- cystic structures have been removed from the matrix and placed in suspension culture. Note the appearance now, of both solid and cystic structures.
• the solid structures have an abundant expression of pro-insulin mRNA .
• Fig. 7 illustrates insulin release into the culture medium by the structures seen in the lower panel of Fig. 6. Note that there is no demonstrable insulin secreted from the tissue when in the cystic state (far left column) .
• FN-fibronectin IGF-1-insulin- like growth factor-1
• Glue-glucose Glue-glucose.
• Fig. 8 illustrates Islets embedded in collagen matrix and maintained in DMEM/F12-CT. Photos from under the inverted microscope (A, C, E) and corresponding histological sections stained for pancytokeratin AE1/AE3 by immunocytochemistry (B, D, F) . (A, C, E, xlOO; B, D, F, x200)
• Fig. 9 illustrates Islets at an intermediate stage of cystic transformation still contain cells that (A) express the pro-insulin mRNA and that (B) synthesize and store insulin protein. (x400)
• FIG. 10 A illustrates Intracellular level of cAMP during the time course of islet -cystic transformation. Note the relatively constant level of intracelluar cAMP in islets maintained in CMRL 1066 alone .
• Fig. 10 B illustrates the integrated amount of cAMP (area under the curve in A) measured at 120 hours. There were no differences observed between islets cultured in DMEM/F12-CT, CMRL-CT and CMRL-forskolin. Note, however, that islets maintained in CMRL alone had significantly less intracellular cAMP .
• Fig. 10 C illustrates the percentage of islets undergoing cystic transformation increased over the time course of the culture period in the DMEM/F12-CT, CMRL-CT and CMRL-forskolin groups. Islets maintained in CMRL 1066 had a very low level of cystic transformation that remained constant. * p ⁇ 0.05, ** p ⁇ 0.01, *** p ⁇ 0.001 Fig. 11 illustrates the progressive loss of tissue insulin content during the time course of cystic transformation. Note the steep decline in islets maintained in DMEM/F12-CT, CMRL-CT and CMRL-forskolin, which corresponds to the early onset of apoptosis by 16 hours. * p ⁇ 0.03
• Fig. 12 illustrates Apoptotic activity (A) and BrdU labeling index (B) of islets cultured in DMEM/F12- CT and CMRL 1066 over the time course of cystic transformation. Note the shift to the left in the onset of apoptosis in islets in DMEM/F12-CT. *p ⁇ 0.02; **p ⁇ 0.01; ***p ⁇ 0.001.
• Fig. 13 illustrates the effect of integrin-binding peptides GRGDSP and GRGESP (A) , extracellular matrix proteins laminin and fibronectin (B) and a combination of GRGDSP or GRGESP and laminin (C) on islet -cystic transformation. *p ⁇ 0.05, **p ⁇ 0.01. ***p ⁇ 0.001.
• Fig. 14 illustrates the effect of extracellular matrix on islet-cystic transformation in isolated canine islets.
• Transdifferentiation is a change from one differentiated phenotype to another, involving morphological and functional phenotypic markers (Okada TS., Develop . Growth and Differ. 1986;28:213-321).
• the best-studied example of this process is the change of amphibian iridial pigment cells to lens fibers, which proceeds through a sequence of cellular dedifferentiation, proliferation and finally redifferentiation (Okada TS, Cell Diff . 1983; 13: 177- 183; Okada TS, Kondoh H, Curr. Top Dev. Biol . , 1986;20:1-433; Yamada T, Monogr. Dev. Biol . , 1977; 13:1- 124) .
• transdifferentiation involves a sequence of steps. Early in the process, intermediate cells appear that express neither the phenotype of the original nor the subsequent differentiated cell types, and therefore they have been termed dedifferentiated. The whole process is accompanied by DNA replication and cell proliferation. Dedifferentiated cells are assumed a priori to be capable of forming either the original or a new cell type, and thus are multipotential (Itoh Y, Eguchi G, Cell Differ. , 1986;18:173-182; Itoh Y, Eguchi G, Develop . Biology, 1986;115:353-362; Okada TS , Develop . Growth and Differ, 1986;28:213-321).
• Stability of the cellular phenotype in adult organisms is probably related to the extracellular milieu, as well as cytoplasmic and nuclear components that interact to control gene expression.
• the conversion of cell phenotype is likely to be accomplished by selective enhancement of gene expression, which controls the terminal developmental commitment of cells.
• pancreas is composed of several types of endocrine and exocrine cells, each responding to a variety of trophic influences.
• the ability of these cells to undergo a change in phenotype has been extensively investigated because of the implications for the understanding of pancreatic diseases such as cancer and diabetes mellitus.
• Transdifferentiation of pancreatic cells was first noted nearly a decade ago. Hepatocyte-like cells, which are normally not present in the pancreas, were observed following the administration of carcinogen (Rao MS et al . , Am . J. Pathol . , 1983;110:89-94; Scarpelli DG, Rao MS, Proc . iVat. Acad . Sci .
• MF Mesenchymal Factor
• Mesenchymal Factor has been extracted from particulate fractions of homogenates of midgestational rat or chick embryos. MF affects cell development by interacting at the cell surface of precursor cells (Rutter WJ. The development of the endocrine and exocrine pancreas. In: The Pancreas. Fitzgerald PJ, Morson AB (eds) . Williams and Wilkins, London, 1980, pp.30) and thereby influences the kind of cells that appear during pancreatic development (Githens S. Differentiation and development of the exocrine pancreas in animals. In: Go VLW, et al . (eds) .
• MF is comprised of at least 2 fundamental components, a heat stable component whose action can be duplicated by cyclic AMP analogs, and another high molecular weight protein component (Rutter WJ, In: The Pancreas. Fitzgerald PJ, Morson AB (eds) . Williams and Wilkins, London, 1980, pp.30).
• Rutter WJ In: The Pancreas. Fitzgerald PJ, Morson AB (eds) . Williams and Wilkins, London, 1980, pp.30.
• cells divide actively and differentiate largely into non- endocrine cells.
• Soluble peptide growth factors have also been implicated in endocrine maturation. Soluble peptide growth factors
• GF GF are one group of trophic substances that regulate both cell proliferation and differentiation. These growth factors are multi-functional and may trigger a broad range of cellular responses (Sporn & Roberts,
• IGF-I Insulin-like growth factor-I
• fetal and neonatal rat islets Hill DJ, et al . , Diabetes, 36:465-471, 1987; Rabinovitch A, et al . , Diabetes, 31:160-164,1982; Romanus JA et al . , Diabetes 34:696-792, 1985.
• IGF-II has been identified in human fetal pancreas (Bryson JM et al . , J. Endocrinol . , 121:367-373,1989).
• IGF's may be important mediators of ⁇ - cell replication in fetal and neonatal rat islets but may not do so in post-natal development (Billestrup N, Martin JM, Endocrinol .
• Platelet-derived growth factor also stimulates fetal islet cell replication and its effect does not require increased production of IGF-I (Swenne I, Endocrinology, 122:214-218, 1988). Moreover, the effect of growth hormone on the replication of rat fetal B-cells appears to be largely independent of IGF- I (Romanus JA et al . , Diabetes 34:696-792, 1985; Swenne I, Hill DJ, Diabetologia 32:191-197, 1989). In the adult pancreas, IGF-I mRNA is localized to the D-cell .
• IGF-I is also found on cell membranes of ⁇ - and A- cells, and in scattered duct cells, but not in acinar or vascular endothelial cells (Hansson H-A et al . , Acta Physiol . Scand . 132:569-576, 1988; Hansson H-A et al . , Cell Tissue Res . , 255:467-474, 1989). This is in contradistinction to one report (Smith F et al , Diabetes, 39 (suppl 1):66A, 1990), wherein IGF-I expression was identified in ductular and vascular endothelial cells, and appeared in regenerating endocrine cells after partial pancreatectomy .
• IGF-I insulin growth factor
• Fibroblast growth factor has been found to initiate transdifferentiation of the retinal pigment epithelium to neural retinal tissues in chick embryo in vivo and in vi tro (Hyuga M et al . , Int . J. Dev. Biol . 1993;37:319-326; Park CM et al . , Dev. Biol . 1991;148:322-333; Pittack C et al . , Development 1991;113:577-588).
• Transforming growth factor-beta TGF- ⁇
• TGF- ⁇ Transforming growth factor-beta
• epithelial growth factor EGF
• cholera toxin cholera toxin
• the search for the factors mediating cell differentiation and survival must include both the cell and its microenvironment (Bissell MJ et al . , J. Theor . Biol . , 1982; 99:31), as a cell's behavior is controlled by other cells as well as by the extracellular matrix
• ECM ECM
• ECM is a dynamic complex of molecules serving as a scaffold for parenchymal and nonparenchymal cells. Its importance in pancreatic development is highlighted by the role of fetal mesenchyme in epithelial cell cytodifferentiation
• BM interstitial matrix and basement membrane
• BM is a macromolecular complex of different glycoproteins, collagens, and proteoglycans .
• the BM contains laminin, fibronectin, collagen types IV and V, as well as heparan sulphate proteoglycans (Ingber D. In: Go VLW, et al (eds) The Pancreas: Biology, Pathobiology and Disease (New York: Raven Press, 1993) p. 369) .
• the specific role of these molecules in the pancreas has yet to be determined.
• ECM has profound effects on differentiation. Mature epithelia that normally never express mesenchymal genes, can be induced to do so by suspension in collagen gels in vi tro (Hay ED. Curr. Opin . in Cell . Biol . 1993; 5:1029). For example, mammary epithelial cells flatten and lose their differentiated phenotype when attached to plastic dishes or adherent collagen gels (Emerman JT, Pitelka
• ECM has more recently been recognized as a regulator of cell survival (Bates RC, Lincz LF, Burns GF, Cancer and Metastasis Rev. , 1995 ; 14 : 191) .
• Disruption of the cell- matrix relationship leads to apoptosis (Frisch SM, Francis H. J " . Cell . Biol . , 1994 ; 124 : 619 ; Schwartz SM, Bennett MR, Am . J. Path . , 1995 ; 147 : 229) , a morphological series of events (Kerr JFK et al . , Br. J. Cancer, 1972 ; 26 : 239) , indicating a process of active cellular self destruction.
• the platform technology is based on a combination of the foregoing observations, incorporating in a basal feeding medium the following components that are necessary and sufficient for the preparation of dedifferentiated intermediate cells from adult pancreatic islets of Langerhans:
• a solid matrix permitting "three dimensional" culture; 2. the presence of matrix proteins including but not limited to collagen type I and laminin; and 3. the growth factor EGF and promoters of cAMP, including but not limited to cholera toxin and forskolin.
• the preferred feeding medium is DMEM/F12 with 10% fetal calf serum.
• the starting tissue must be freshly isolated and cultured without absolute purification.
• the use of a matrix protein-containing solid gel is an important part of the culture system, because extracellular matrix may promote the process of transdifferentiation.
• pancreatic acinar cells which transdifferentiate to duct-like structures when entrapped in Matrigel basement membrane (Arias AE, Bendayan M, Lab Invest . , 1993;69:518-530), or by retinal pigmented epithelial cells, which transdifferentiate into neurons when plated on laminin- containing substrates (Reh TA et al . , Nature 1987;330:68-71).
• Gittes et al demonstrated, using 11 -day embryonic mouse pancreas, that the default path for growth of embryonic pancreatic epithelium is to form islets (Gittes GK et al . , Development 1996; 122:439-447).
• the pancreatic strom epithelium appears to programmed to form ducts. This finding again emphasizes the interrelationship between ducts and islets and highlights the important role of the extracellular matrix.
• stage 1 the production of dedifferentiated intermediate cells of the process.
• islets undergo a cystic transformation associated with (Arias AE, Bendayan M, Lab . Invest . , 1993;69:518-530) a progressive loss of insulin gene expression, (2) a loss of immunoreactivity for insulin protein, and (3) the appearance of CKA 19, a marker for ductal cells.
• CKA 19 a marker for ductal cells.
• the cells have the ultrastructural appearance of primitive duct-like cells. Cyst enlargement after the initial 96h is associated, at least in part, with a tremendous increase in cell replication.
• the cells are moved from the digested matrix and resuspended in a basal liquid medium such as CMRL 1066 supplemented with 10% fetal calf serum, with the addition of soluble matrix proteins and growth factors that include, but are not limited to, fibronectin (10- 20 ng/ml) , IGF-1 (100 ng/ml) , IGF-2 (100 ng) , insulin (10-100 ⁇ g/ml) , NGF (10-100 ng/ml) . In addition, the glucose concentration must be increased to above 11 mM. Additional culture additives may include specific inhibitors of known intracellular signaling pathways of apoptosis, including, but not limited to a specific inhibitor of p38.
• Evidence for the return to an islet cell phenotype includes: (1) the re-appearance of solid spherical structures; (2) loss of CK-19 expression; (3) the demonstration of endosecretory granules on electron microscopy; (4) the re-appearance of pro- insulin mRNA on in si tu hybridization; (5) the return of a basal release of insulin into the culture medium.
• Canine islets were isolated using Canine
• Total intracellular cAMP at 120 hr coincided with the % of islets undergoing transdifferentiation (63 ⁇ 2, 48 ⁇ 2, 35 ⁇ 3, 8 ⁇ 1) , as determined by routine histology, immunocytochemistry for cytokeratin AE1/AE3, and by a loss of pro- insulin gene expression on in si tu hybridization.
• islets were embedded in collagen type I, MatrigelTM and agarose gel and cultured in DMEM/F12 plus CT. Islets in collagen type I and MatrigelTM demonstrated a high rate of cystic transformation
• pancreata from six mongrel dogs of both sexes were resected under general anesthesia in accordance with Canadian Council for Animal Care guidelines (Wang RN, Rosenberg L (1999) J Endocrology 163 181-190) .
• the pancreatic ducts Prior to removal, the pancreatic ducts were cannulated to permit intraductal infusion with Liberase CI ® (1.25mg/ml) (Boehringer Mannheim, Indianapolis, IN, USA) according to established protocols (Horaguchi A, Merrell RC (1981) Diabetes 30 455-461; Ricordi C (1992) Pancreatic islet cell transplantation . pp99-112. Ed Ricordi C. Austin: R. G. Landes Co.) .
• CMRL1066 GEBCO
• CMRL1066 GEBCO
• 10%FBS and cholera toxin LOOng/ml
• 16.5mM D- glucose CMRL1066
• CMRL1066 supplemented with 10%FBS and 2 ⁇ M forskolin (Sigma, St. Louis, MO, USA)
• CMRL1066 supplemented with 10% FBS.
• Approximately 3000 islets per group per time point were used. Islets were cultured in 95% air / 5% C0 2 at 37°C, and the medium was changed on alternate days. Representative islets from each group were examined after isolation (0 hour) , and then on hours of 1, 16, 36, 72 and 120 using the following investigations.
• islets were cultured in suspension in DMEM/F12 with 10% FBS plus CT and EGF. To determine whether a solid gel environment and extracellular matrix proteins were independent requirements, islets were embedded in 1.5% agarose gel and maintained in DMEM/F12 with 10% FBS plus CT and EGF. Alternatively, islets were cultured in suspension with in DMEM/F12 with 10% FBS plus CT and EGF in the presence of soluble Laminin (50 ⁇ g/ml) or Fibronectin
• islets were pre-incubated at 37°C for 60 min either in the presence of the RGD-motif containing
• Tissue was fixed in 4% paraformaldehyde (PFA) and embedded in 2% agarose following a standard protocol of dehydration and paraffin embedding Wang RN, Rosenberg L (1999) J Endocrology 163 181-190) .
• PFA paraformaldehyde
• a set of six serial sections was cut from each paraffin block. Consecutive sections were processed for routine histology and immunostained for pancreatic hormones
• AE1/AE3 (Dako, Carpinteria, CA. , USA) , using the AB complex method (streptavidin-biotin horseradish peroxidase; Dako), as described previously (Wang RN et al . (1994) Diabetologia 37 1088-1096).
• cytokeratin AE1/AE3 sections were pretreated with 0.1% trypsin. The sections were incubated overnight at 4°C with the appropriate primary antibodies. Negative controls involved the omission of the primary antibodies.
• In situ hybridization for human proinsulin mRNA was performed on consecutive sections of freshly isolated islets and epithelial cystic structures at 120 h. The sections were hybridized with a fluorescein labelled oligonucleotide cocktail solution for 2 h at 37°C. Slides were then incubated with rabbit Fab anti-FITC conjugated to alkaline phosphatase antibody (diluted 1:200) for 30 min at room temperature.
• the reaction product was visualised by an enzyme-catalysed colour reaction using a nitro blue tetrazolium and 5'-bromo-4- chloro-3-indolyl-phosphate kit (Wang RN, Rosenberg L (1999) J " Endocrology 163 181-190, Wang RN et al . (1994) Diabetologia 37 1088-1096) . Analysis of Intracellular cAMP Level
• Cellular insulin content was measured using a solid-phase radioimmunoassay (Immunocorp, Montreal, Quebec, Canada) (Wang RN, Rosenberg L (1999) J Endocrology 163 181-190) with a sensitivity of 26.7 pmol/1 (0.15 ng/ml), an inter-assay variability of ⁇ 5%, and an accuracy of 100%.
• the kit uses anti-human antibodies that cross-react with canine insulin. Obtained values were corrected for variations in cell number by measuring DNA content using a fluorometric DNA assay (Yuan S et al . (1996) Differentiation 61, 67- 75) . The data are expressed as ⁇ g per ⁇ g DNA.
• Cells cultured in DMEM/F12-CT and CMRL1066 were harvested from the gel using collagenase XI (0.25 mg/ml) (Sigma, Montreal, Que . ) and processed for a specific programmed cell death ELISA, that detects histone-associated DNA fragments in the cell cytoplasm- a hallmark of the apoptotic process (Roche Molecular, Montreal, Que.) (Paraskevas S et al . (2000) Ann . Surgery in press) . Cells were incubated in lysis buffer for 30 min, and the supernatant containing cytoplasmic oligonucleosomes was measured at an absorbance of 405nm. Variations in sample size were corrected by measuring total sample DNA content (Yuan S et al . (1996) Differentiation 61, 67-75) .
• Islets cultured in CMRL 1066 alone maintained a solid spheroid appearance for the duration of the study and did not undergo epithelial transformation. Immunocytochemical staining did not demonstrate co- localization of cytokeratin and islet cell hormones. This is in keeping with the observation in the intact pancreas, that cytokeratin staining was only seen on duct epithelial cells. Pro-insulin gene expression and insulin protein were progressively lost during the period of duct epithelial differentiation (Fig. 9) Intracellular cAMP
• intracellular levels of cAMP of islets maintained in DMEM/F12-CT, CMRL1066-CT and CMRL1066-forskolin were significantly elevated compared to freshly isolated islets or to islets maintained in CMRL 1066 alone (Fig. 10A) .
• the intracellular level of cAMP of islets cultured in CMRL 1066 alone did not increase at all during the time course of the study.
• the total intracellular cAMP measured over 120 hr was similar for islets cultured in DMEM/F12-CT, CMRL 1066 -CT and CMRL 1066-forskolin (15 ⁇ 3, 16 ⁇ 2, 17 ⁇ 3 respectively), although the most sustained elevation of cAMP was in the DMEM/F12-CT islets, which were exposed to both EGF and CT.
• islets cultured in CMRL 1066 alone had the lowest level of total intracellular cAMP (4 ⁇ 1, p ⁇ 0.001) (Fig. 10B) , and this translated into the lowest level of islet-duct transformation (Fig. 10C).
• Fig. 11 The cellular content of insulin (Fig. 11) was highest in freshly isolated islets (ll ⁇ 2 ⁇ g/ ⁇ g DNA) . After 16 hours in culture, the insulin content of cells cultured in DMEM/F12-CT, CMRL1066-CT and CMRL1066- forskolin declined dramatically, falling to 7% of the initial value by 120 hours. Islets cultured in CMRL1066 alone did not undergo epithelial transformation, and maintained a higher level of intracellular insulin compared to the other three groups (p ⁇ 0.03, Fig. 11) . Analysis Of Cell Death And Proliferation
• cytoplasmic oligonucleosome enrichment was significantly higher in islets cultured with DMEM/F12- CT compared to islets cultured in CMRL1066 alone (p ⁇ 0.02, Fig. 12A) . After 36 hours, there was no difference between the groups. Looking at the data as a whole (Fig. 12A) , it appears that a wave of apoptosis occurred in both groups of islets, but that the time course of cell death was shifted to the left for islets undergoing cystic transformation in DMEM/F12-CT.
• islets were embedded in agarose gel, type 1 collagen gel or Matrigel . Only islets embedded in the latter two gels underwent cystic transformation (Table 1) . Furthermore, islets maintained in suspension in DMEM/F12-CT supplemented with either soluble laminin or fibronectin, failed to undergo ductal transformation. These experiments indicated that the process of transformation required the presence of ECM proteins presented in a solid gel environment.
• islets were pre-incubated with the RGD motif -containing GRGDSP peptide prior to embedding in collagen. This reduced cystic transformation to 57% of the control DMEM/F12-CT group (p ⁇ 0.001) at 72 hours (Fig. 14A) .
• the control peptide, GRGESP had little influence on the transformation process.
• Pre-treatment islets with either soluble fibronectin or laminin prior to embedding decreased cystic transformation to 50% of control (p ⁇ 0.01) at 72 hours (Fig. 14B) . Cystic transformation was further reduced to 33% of control, when islets were pre-incubated with both GRGDSP and laminin (p ⁇ 0.001, Fig. 14C) .
• extracellular matrix may also promote the process of transdifferentiation.
• extracellular matrix may also promote the process of transdifferentiation. This point is emphasized by isolated pancreatic acinar cells that transdifferentiate to duct-like structures when entrapped in Matrigel (Arias AE, Bendayan M (1993) Lab Invest 69, 518-530) , and by retinal pigment epithelial cells, which transdifferentiate into neurons when plated onto laminin-containing substrates (Reh TA et al. (1987). Nature 330, 68-71).
• cavitation is the result of the interplay of two signals, one from an outer layer of endoderm cells that acts over a short distance to create a cavity by inducing apoptosis of the inner ectodermal cells, and the other a rescue signal mediated by contact with the basement membrane that is required for survival of the columnar cells (Coucouvanis E, Martin GR (1995) Cell 83, 279-287).
• a central feature of this model is the direct initiation of apoptosis by an external signal that causes cell death.
• the second key feature of the model is a signal that appears to be mediated by attachment to ECM and rescues cells from cell death.
• Transdifferentiation may involve cell proliferation and the appearance of a multipotential dedifferentiated intermediate cell (Yuan S et al . (1996) Differentiation 61, 67-75) which can express markers characteristic of several alternative phenotypes . It is possible that this is the case in our system (Yuan S et al . (1996) Differentiation 61, 67-75) .

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