PRODUCTION OF CANINE PANCREATIC ISLETS FROM AN IMMATURE PANCREAS

Abstract

The present invention relates to an in vitro method for preparing and producing canine pancreatic islets from immature pancreatic tissue. Such islets express, produce and secrete insulin upon glucose stimulation. The invention further encompasses canine pancreatic islets obtainable according to the present method, islet population of said islets and compositions comprising said islets. It also relates to transduced canine pancreatic islets, or tumours or cells derived thereof. The present invention also concerns the use of said canine pancreatic islets or cells derived thereof for treating a canine pancreatic disorder, such as canine diabetes, or for diagnosing canine diabetes.

Claims

1. A method for producing canine pancreatic islets, comprising the steps of: a) obtaining canine pancreatic endocrine cells from an immature canine pancreas or a portion thereof; and b) incubating the endocrine cells of step a) in an appropriate culture medium comprising glucose at a concentration comprised between 4 mM to 30 mM, allowing pancreatic islets to develop.

2. The method according to claim 1, wherein step a) comprises mincing said immature canine pancreas or portion thereof and/or digesting said immature canine pancreas or portion thereof with an appropriate enzyme.

3. The method according to claim 2, wherein the appropriate enzyme is collagenase.

4. The method according to claim 1, further comprising the step c) of encapsulating the pancreatic islets of step b) in a device.

5. The method according to claim 4, wherein the device is a protective device comprising: (i) a semi-permeable membrane of high polymer; (ii) a mesh reinforcement; and (iii) a microcapsule, a microparticle or a mixture thereof; or comprising: (i) a semi-permeable membrane of high polymer; and (ii) a microcapsule, a microparticle or a mixture thereof.

6. The method according to claim 1, wherein the pancreatic endocrine cells of step a) comprise beta cells, or wherein the pancreatic endocrine cells of step a) comprise beta cells and alpha cells.

7. The method according to claim 1, wherein the immature canine pancreas is an immature dog pancreas.

8. The method according to claim 1, wherein the immature canine pancreas is a foetal canine pancreas, or a neonatal canine pancreas, or is obtained from a non-adult canine.

9. The method according to claim 8, wherein the fetal canine pancreas is in the last third of gestation.

10. The method according to claim 8, wherein the fetal canine pancreas is at days 40 to 60 post conception.

11. Canine pancreatic islets obtainable by the method according to claim 1.

12. The canine pancreatic islets according to claim 11, wherein said islets possess at least one feature selected from of: presence of canine alpha cells; presence of canine beta cells; expression of canine-specific insulin; expression of canine-specific glucagon; and any combination thereof.

13. (canceled)

14. The canine pancreatic islets according to claim 11, wherein said pancreatic islets are positive to reaction with canine-specific anti-insulin, canine-specific anti-glucagon, anti-GAD and/or anti-IA2 antibodies.

15. The canine pancreatic islets according to claim 11, wherein said pancreatic islets are capable of secreting canine specific insulin and/or canine specific glucagon in response to glucose stimulation.

16. (canceled)

17. A culture comprising canine pancreatic islets according to claim 11 in an appropriate culture medium.

18. A veterinary composition comprising a pharmaceutically acceptable carrier and an effective amount of the canine pancreatic islets according to claim 11.

19. Method for reducing the risk of developing or treating a canine pancreatic disorder in an animal, comprising the administration of the canine pancreatic islets according to claim 11, or a veterinary composition comprising a pharmaceutically acceptable carrier and an effective amount of the canine pancreatic islets according to claim 11.

20. The method according to claim 19, wherein said canine pancreatic disorder is canine diabetes.

21. The method according to claim 19, wherein said canine pancreatic islets or said veterinary composition are (is) transplanted in the pancreas, the liver, a muscle, a subcutaneous tissue, the renal subcapsule, or the peritoneal cavity of said animal.

22. The method according to claim 19, wherein said canine pancreatic islets or said veterinary composition are (is) administered by injection in said animal.

23-32. (canceled)

33. A method for preparing transduced canine pancreatic islets, transduced canine pancreatic beta cells or canine beta cell tumours comprising the step of: a) transducing or co-transducing the canine pancreatic islets of claim 11 with i) a lentiviral vector expressing SV40 Large T antigen under the control of the insulin promoter, or ii) with a lentiviral vector expressing SV40 Large T antigen under the control of the insulin promoter and a lentiviral vector expressing hTert under the control of the insulin promoter, or iii) a lentiviral vector expressing both SV40 Large T antigen and hTert under the control of the insulin promoter.

34. The method according to claim 33, further comprising the step of: b) collecting the canine pancreatic islets obtained at step a) to form a homogenous transduced canine islet population.

35. The method according to claim 33, further comprising the step of: b) dissociating the transduced pancreatic beta cells from the transduced canine pancreatic islets of step a); and c) harvesting the pancreatic beta cells contained in the dissociated islets of step b), to form a homogenous transduced canine pancreatic beta cell population.

36. The method according to claim 33, further comprising the steps of: b) introducing the transduced pancreatic islets obtained in a) into the kidney capsule of a first severe combined immunodeficiency (scid) non-human animal; c) allowing the transduced pancreatic islets to develop insulinoma-like structures, wherein the canine pancreas cells in insulinoma-like structures have differentiated to insulin-producing pancreatic islets and/or beta cells; d) micro-dissecting the insulinoma-like structures obtained in step c), and dissociating the islets and/or cells thereof; e) sub-transplanting the islets and/or cells obtained in step d) into the kidney capsule of a second scid non-human animal; f) allowing the sub-transplanted islets and/or cells in step e) to develop and regenerate newly developed insulinoma-like structures, wherein said newly developed insulinoma-like structures are enriched in insulin-producing pancreatic islets and/or beta cells; and g) micro-dissecting the insulinoma-like structures obtained in step f), and dissociating and collecting the islets and/or cells thereof.

37-49. (canceled)

50. Canine pancreatic islets, canine beta cell tumours or canine pancreatic beta cells obtainable by the method according to claim 33.

51. The canine pancreatic islets, canine beta cell tumours or canine pancreatic beta cells according to claim 50, wherein said tumours or cells have at least one feature selected from: Carboxypeptidase-A negative transcriptional factor Pdx1 positive transcription factor MafA positive proconvertase Pcsk1 positive expression of Glucose transporter Glut2 expression of Kenj11 and Abcc8 coding for subunits of the potassium channel expression of zinc transporter Znt8 (Slc30a8) expression of canine-specific insulin positive to reaction with canine-specific anti-insulin, anti-GAD and/or anti-IA2 antibodies and any combination thereof.

52-73. (canceled)

Description

FIGURE LEGENDS

[0211] FIG. 1: Pancreatic neo-islets after three days of culture of immature pancreatic islets obtained from of a dog foetus at 53pc (E-53). X20

[0212] FIG. 2: Pancreatic neo-islets after 7 days of culture of immature pancreatic islets obtained from of a dog foetus at 53pc (E-53). X10

[0213] FIG. 3: The dog pancreatic neo-islets are functional and produce dog insulin and glucagon

[0214] A) Immunostaining of endocrine markers (insulin (light grey) and glucagon (white)) of pseudo-pancreatic islets after six days of culture of immature pancreatic islets obtained from of a dog foetus at 53pc (E-53). X20.

[0215] B) Immunostaining of endocrine markers (insulin (light grey) and glucagon (white)) of pseudo-pancreatic islets after eight days of culture of immature pancreatic islets obtained from of a dog foetus at 53pc (E-53). X10.

[0216] FIG. 4: Endocrine pancreas-like tissue obtained after grafting of non-transduced canine pancreatic neo-islets in a scid mice: the pancreatic neo-islets are functional and produce dog insulin and glucagon

[0217] Immunostaining of endocrine markers (insulin (light grey) and glucagon (white)) of Endocrine pancreas-like tissue obtained after grafting non-transduced pancreatic islets in scid mice, 2 months post-graft. X20

[0218] FIG. 5: Large T positive neo-islets obtained after grafting of Large T transduced canine pancreatic neo-islets in a scid mice: the transduced pancreatic neo-islets are functional and produce dog insulin and glucagon

[0219] Immunostaining of endocrine markers (insulin (light grey) and glucagon (white)), of Large T positive neo-islets obtained after grafting Large T transduced-pancreatic neo-islets in scid mice, 2 months post-graft. X10.

[0220] FIG. 6: Large T positive neo-islets obtained after grafting of Large T transduced canine pancreatic neo-islets in a scid mice: the transduced pancreatic neo-islets and produce dog insulin and regulate blood glucose concentration

[0221] Immunostaining of insulin (light grey) of Large T positive neo-islets, obtained after grafting Large T transduced-pancreatic neo-islets in scid mice.

[0222] FIG. 7: Beta cell lines obtanined from Large T positive neo-islets produce dog insulin

[0223] Immunostaining of endocrine marker insulin (light grey) of beta cells obtained from a Large T positive neo-islets obtained after grafting Large T transduced-pancreatic neo-islets in scid mice.

EXAMPLES

A) Material and Methods

[0224] A.1. Materials

[0225] HBSS (Hanks' Balanced Salt Solution) is supplemented with 5.6 mM glucose; 0.2 mg/mL BSA fat acid free and 1% penicillin-streptomycin.

[0226] The culture medium is made with a base of RPMI 1640 medium already containing 11 mM glucose and 25 mM Hepes and supplemented with 10% FCS and 1% penicillin-streptomycin.

[0227] A.2. Source of canine pancreatic tissue and collection procedure

[0228] Pancreases were obtained from Beagle dogs, a strain raised in the housing facilities of Maison-Alfort Veterinary School, at foetal stage 53 days pc (post conception, E-53). All foetal samples were obtained by elective caesarean section. The foetal age was determined according to the ovulation identified by the plasma progesterone surge

[0229] All the procedures involving animals were approved by the Ethic Committee of Maison-Alfort Veterinary School.

[0230] A.3. Generation of the Canine Pancreatic Islets

[0231] Immediately after surgery, all pancreases were dissected and minced into 1 mm square pieces in supplemented HBSS. The pancreas pieces were digested with collagenase A at 6 mg/mL at 37° C. for 4-6 min. The digestion was stopped by dilution with cold supplemented HBSS. The digested pieces were washed twice.

[0232] The digested pancreas pieces were collected and incubated in the culture medium defined in A.1.

[0233] A.4. Immunohistochemistry

[0234] The pseudo-islets were fixed in 4% PFA (paraformaldehyde) and embedded in gelatine-sucrose. Sections were cut them with cryostat.

[0235] Sections were stained with a guinea pig anti-insulin antibody (1/500; A0564, Dako-Cytomation) and rabbit anti-glucagon (1/1000; 20076-Immuno, Euromedex). The secondary antibodies were fluorescein Texas red anti-guinea pig antibody (1/2000; 706-076-148, Jackson and anti-rabbit antibody (1/200; 711-096-152, Jackson Immunodetect Laboratories, Beckman Coulter). Cell nuclei were stained with Hoechst or DAPI. Digital images were captured using an Axio Scan Z1 (Zeiss).

[0236] A.5. DNA Constructs and Recombinant Lentiviral Productions

[0237] The lentiviral vectors, pTRIP ΔU3.RIP405-SV40LT loxP and pTRIP ΔU3.RIP405-hTERT loxP, have been constructed by adding a loxP site in the 3′LTR region of the pTrip ΔU3.RIP405-SV40LT/hTERT previously described (Ravassard et al, 2009). Both pTRIP ΔU3 vectors were digested by KpnI and PacI to remove the 3′LTR region. The 3′LTRloxP region of the SIN-RP-LTcDNA-WHV-U3loxP (provided by Bernard Thorens) was amplify by PCR and next digested by KpnI and PacI and then ligated into the two linearized pTrip vectors. The Lentiviral vector stocks were produced by transient transfection of 293T cells by encapsidation of the p8.9 plasmid (ΔVprΔVifΔVpuΔNef), pHCMV-G that encoded the VSV glycoprotein-G and the pTRIP ΔU3 recombinant vector, as previously described (Zufferey et al., 1997). The supernatants were treated with DNAse I (Roche Diagnostic) prior to their ultracentrifugation, and the resultant pellets were re-suspended in PBS, aliquoted, and then frozen at −80° C. until use. The amount of p24 capsid protein was quantified by the HIV-1 p24 antigen ELISA (Beckman Coulter). All transductions were normalized relative to p24 capsid protein quantification.

[0238] A.6. Gene Transfer

[0239] The pseudo-islets to be transduced were incubated with a total amount of lentiviral vectors (pTRIP ΔU3.RIP405-SV40LT loxP) corresponding to 2 μg of p24 capside protein for 1 hour at 37° C. in of DMEM that contained 5.6 mM glucose, 2% bovine serum albumin fraction V (BSA, Roche diagnostics), 50 μM 2-mercaptoethanol, 10 mM nicotinamide (Calbiochem), 5.5 μg/ml transferrin (Sigma-Aldrich), 6.7 ng/ml selenite (Sigma-Aldrich), 100 U/ml penicillin, and 100 μg/ml streptomycin and 10 μg/ml DEAE-dextran (DEAE for Diethylaminoethyl). The transduction reaction was diluted in the culture medium and the transduced islets were kept on culture overnight until transplantation into scid mice.

[0240] A.7. Animals and Transplantation into Scid Mice

[0241] Male scid mice (Harlan) were maintained in isolators. Diabetes was induced in the scid mice by treating said mice with streptozotocine as described previously (Ravassard et al., 2009; Ravassard et al., 2011). Using a dissecting microscope, islets were implanted under the kidney capsule, as previously described (Ravassard et al., 2011). At different time points after transplantation, the mice were sacrificed, the kidney removed, and the graft dissected. All animal studies and protocols were approved by the Veterinary Inspection Office in compliance with the French legislation under agreement number B75-13-03.

[0242] A.8. Assay of Dog-Specific Insulin Levels

[0243] The levels of dog-specific insulin were assayed using an ELISA kit commercialized by MERCODIA, following the instructions of the manufacturer.

B) Production of Functional Canine Pancreatic Neo-Islets

[0244] Dog islets were prepared and grown as described in section A.3 above. The evolution of the cultures was monitored by microscopy. A network of fibroblastic type cells begin to form after two days of culture (D+3; FIG. 1). At D+4 pancreatic islet-like structures (pseudo-islets) begin to form. At D+7, spherical pancreatic neo-islet structures are formed (FIG. 2). These results show that the method developed by the inventors allows rapid, efficient and easy de novo generation of dog pancreatic neo-islets.

[0245] The dog islets were studied by immunohistochemistry. Cells were stained with an anti-insulin antibody (light grey), an anti-glucagon antibody (white) and the nuclei were stained with Hoechst (dark grey; FIG. 3). FIGS. 3 A and B shows that the size of the neo-islets increases with the time of culture and that high levels of insulin and glucagon are detected in all the pseudo-islets. Both insulin and glucagon expressions are detected after more than 21 days of culture (data not shown). Dog-specific insulin secretion in the culture medium was assayed as described in section A.8 above. Insulin was detected in all batches.

[0246] These results show that the dog neo-islets are homogeneous and are stably producing insulin and glucagon and are capable of secreting insulin.

[0247] Insulin secretion was further assayed upon glucose stimulation. Increasing the glucose concentration in the medium to 15 mM resulted in a 1.5 to 4-fold increase in insulin secretion. Therefore, the dog neo-islets are capable of responding to glucose stimulation.

[0248] These data show that the dog islets obtained de novo using the method developed by the inventors are fully functional and stable.

C) Grafting of Canine Pancreatic Islets

[0249] C1. Grafting of Non-Transduced Canine Pancreatic Islets

[0250] Dog islets were prepared as described in section A.3 above. The islets were implanted under the kidney capsule of scid mice. The development of endocrine pancreas-like tissue was confirmed by assaying dog-specific insulin in the transplanted mice (as described in EP2017/061401). The grafts were harvested two months after transplantation. The grafts were dissected and fixed in 3.7% formaldehyde prior to their embedding in paraffin.

[0251] Paraffin-embedded sections were cut and stained with an anti-insulin antibody (light grey), an anti-glucagon antibody (white) and the nuclei were stained with Hoechst (dark grey; FIG. 4), as described in section A.6 above.

[0252] FIG. 4 shows that endocrine pancreas-like structures have developed under the kidney capsule of the grafted scid mice. High levels of insulin and glucagon are homogeneously detected in the neo-islets in the structures. These data show that the neo-islets obtained after transplant of non-transduced dog islets are fully functional. These results show that the canine pancreatic islets produced by the method described above can be successfully grafted in animals and develop fully functional endocrine pancreas-like structures. This opens considerable perspective towards veterinary use of such pancreatic islets in the treatment of canine pancreatic disorders, such as diabetes.

[0253] Moreover, these result show that pancreatic islets obtained by the method of the invention may be further expanded and maintained in vivo by sub-grafting said islets in scid mice.

[0254] C2. Grafting of Large T-Transduced Canine Pancreatic Islets

[0255] C2.1. Dog islets were prepared and transduced with Large T expressing vectors as described in sections A.3 and A.6 above. Large T-transduced islets were implanted under the kidney capsule of scid mice as described in section C.1 above.

[0256] FIG. 5 shows that Large T-positive neo-islets have developed under the kidney capsule of the grafted scid mice. High levels of insulin and glucagon are homogeneously detected in the islets. These data show that the dog pancreatic islets produced by the method described above can be successfully grafted in animals and developed fully functional Large T-positive neo-islets.

[0257] C2.2. Dog neo-islets were prepared as described in section A.3 and transduced with lentiviral vectors, pTRIP ΔU3.RIP405-SV40LT loxP described in section A.S. For the transduction, a total amount of lentiviral vector corresponding to 1 μg of p24 capsid protein was used to transduce 10.sup.6 dog neo-islets in culture. 2 hours after transduction the culture medium is changed. 24 h later the transduced neo-islets were transplanted under the kidney capsule of a scid mice as described in section A.7. 10.sup.6 to 2×10.sup.6 transduced pseudo islets were transplanted per mouse. The glucose concentration in the blood of the SCID mice host was assayed.

[0258] The data show that a decline in blood glucose concentration from mild to severe hypoglycemia can be observed in the host SCID mice.

[0259] In addition, a significant amount of dog insulin was measured in the blood of the host SCID mice.

[0260] An insulinoma was obtained (FIG. 6). FIG. 6 shows that the cells of the insulinoma were stained for insulin (light grey) and are Large T-positive (white). The Large T oncogene has been transduced in the nucleus of the cells.

[0261] The insulinomas were dissociated and cells (beta cells) were collected and expended to obtained a master cell bank of immortalized cells. As shown in FIG. 7, the expended beta cells were positive for insulin, as shown by immune cyto-chemistry (insulin in stained in light grey and nuclei are stained in dark grey). In addition, cells were cultivated and insulin was measured in the milieu. The data show a high concentration of dog insulin secreted by those cells.

[0262] This opens considerable perspective towards veterinary use of such pancreatic islets in the treatment of canine pancreatic disorders, such as diabetes.

REFERENCES

[0263] Ahlgren K M, Fall T, Landegren N, Grimelius L, von Euler H, Sundberg K, “Lack of evidence for a role of islet autoimmunity in the aetiology of canine diabetes mellitus”. PLoS One. (2014); 9(8):e105473. [0264] Ausubel F. M. et al, eds., 1987 Current Protocols in Molecular Biology, and periodic updates. [0265] Barbas et al., 2001, Phage Display: A Laboratory Manual. [0266] Bonnet B N. a Egenvall A. “Age patterns of disease and death in insured Swedish dogs, cats and horses”, Department of Population Medicine, University of Guelph, Ontario, Canada. J Comp Pathol. 2010 January; 142 Suppl 1:S33-8. [0267] Bricout-Neveu E, Pechberty S, Reynaud K, Maenhoudt C, Lecomte M J, Ravassard P, and Czernichow P. “Development of the Endocrine Pancreas in the Beagle Dog: From Fetal to Adult Life”. Anat Rec. 2017 Mar. 14. doi: 10.1002/ar.23595. [0268] Castaing, M., Duvillie, B., Quemeneur, E., Basmaciogullari, A., and Scharfmann, R. (2005). “Ex vivo analysis of acinar and endocrine cell development in the human embryonic pancreas”. Dev Dyn. 234, 339-345. [0269] Catchpole B., Ristic J M., Fleeman L M. a Davison L I “Canine diabetes mellitus: can old dogs teach us new tricks?” Diabetologia, 2005; 48:1948-56. [0270] Davison L J, Weenink S M, Christie M R, Herrtage M E, Catchpole B. “Autoantibodies to GAD65 and IA-2 in canine diabetes mellitus”. Veterinary immunology and immunopathology. (2008); 126:83-90. [0271] Davison L I, Herrtage M E. a Catchpole B. “Study of 253 dogs in the United Kingdom with diabetes mellitus” inVet Rec. 2005; 156(15):467-71. [0272] Freshney R. I., ed., 1987. Animal Cell Culture. [0273] Gait M. J., ed., 1984, Oligonucleotide Synthesis. [0274] Gale E. A. M. «Do dogs develop autoimmune diabetes?» Diabetologia (2005) 48: 1945-1947. [0275] Hawkins, K. L., Summers, A., P. Kuhajda, P., Smith, C. A. “Immunocytochemistry of Normal Pancreatic Islets and Spontaneous Islet Cell Tumors in Dogs”. Vet. Pathol. 24: 170-179 (1987). [0276] Justice, D., Cruccioli, N., Roque, C., Galls, J, F., Remaudet, B., Cahard, D. «Etude morphometrique des ilots de Langerhans du pancreas du chien Beagle» Rev Fr Histotechnology 1997, 10:45-49. [0277] Kennedy L J, Davison L J, Barnes A, Short A D, Fretwell N, Jones C A, et al. “Tissue Antigens. Identification of susceptibility and protective major histocompatibility complex haplotypes in canine diabetes mellitus.” (2006); 68(6):467-76. [0278] Khalfallah, O., Ravassard, P., Serguera-Lagache C., Fligny, C., Serre, A., Bayard, E., Faucon-Biguet, N., Mallet, J., Meloni, R., and Nardelli, J. (2009). “Zinc finger protein 191 (ZNF191/Zfp191) is necessary to maintain neural cells as cycling progenitors”. Stem Cells 27:1643-1653. [0279] Kim A, Miller K, Jo J, Kilimnik G, Wojcik P, and Hara M. “Islet architecture: A comparative study”. Islets. 2009; 1(2): 129-136. [0280] Mullis et al, ed., 1994. PCR: The Polymerase Chain Reaction. [0281] Nelson R W and Reusch C E, (2014), “Animal models of disease: classification and etiology of diabetes in dogs and cats”. J Endocrinol. September; 222(3):T1-9. doi: 10.1530/JOE-14-0202. [0282] Niessen S J., Powney S. Guitian J., Niessen A P., Pion P D., Shaw J A. a Church D B. “Evaluation of a quality-of-life tool for dogs with diabetes mellitus” J Vet Med. 2012; 26(4):953-61. [0283] O'Kell A L, Wasserfall C, Catchpole B, Davison L J, Hess R S, Kushner J A, Atkinson M A. “Comparative Pathogenesis of Autoimmune Diabetes in Humans, NOD Mice, and Canines: Has a Valuable Animal Model of Type 1 Diabetes Been Overlooked?” Diabetes. 2017 June; 66(6):1443-1452. [0284] Perbal Bernard V., 1988, A Practical Guide to Molecular Cloning. [0285] Pictet, R. L., Clark, W. R., Williams, R. H., and Rutter, W. J. “An ultrastructural analysis of the developing embryonic pancreas”. Dev. Biol. 1972, 29, 436-467. [0286] Rand J S, Fleeman L M, Farrow H A, Appleton D J, Lederer R. (2004) “Canine and feline diabetes mellitus: nature or nurture?” J Nutr. 2004 August; 134(8 Suppl):20725-20805. [0287] Ravassard P, Emilie Bricout-Neveu, Hazhouz Y, Pechberty S, Mallet J, Czernichow P, Scharfmann R. (2009) “A new strategy to generate functional insulin-producing cell lines by somatic gene transfer into pancreatic progenitors”. PLoS One. 4(3): e4731. [0288] Ravassard P, Hazhouz Y, Pechberty S, Bricout-Neveu E, Armanet M, Czernichow P, Scharfmann R.; (2011) “A genetically engineered human pancreatic B cell line exhibiting glucose-inducible insulin secretion”. J Clin Invest. 2011 September; 121(9):3589-97. [0289] Remington's Pharmaceutical Science, 17.sup.th ed., Mack Publishing Company, Easton, Pa. (1985). [0290] Russ, H. A., Bar, Y., Ravassard, P., and Efrat, S. (2008). In vitro proliferation of cells derived from adult human beta-cells revealed by cell-lineage tracing. Diabetes 57:1575-1583. [0291] Sambrook et al, 1989, Molecular Cloning: A Laboratory Manual, second edition. [0292] Scharfmann R, Duvillie B, Stetsyuk V, Attali M, Filhoulaud G, Guillemain G. Beta-cell development: the role of intercellular signals. Diabetes Obes Metab. 2008 November; 10 Suppl 4:195-200. [0293] Shield E J et al, “Extreme Beta-cell deficiency in Pancreata of Dogs with canine Diabetes”, PloS one (2015); 10, 1719. [0294] Steiner D J, Kim A, Miller K, Hara M., “Pancreatic islet plasticity: interspecies comparison of islet architecture and composition”. Islets. 2010; 2(3):135-45. [0295] Tuch B. E. and Madrid J. C., “Development of fetal sheep pancreas after transplantation into athymic mice”. Cell Transplant. 1996 July-August; 5(4):483-9. [0296] Tuch B E, Madrid J C, Summers E, Smith M S. “Production and characterization of fetal sheep pancreatic islet-like cell clusters Cell Transplant”. 1996 July-August; 5(4):491-8. [0297] Woolcott O. O., Bergman R N., Richey J M., Kirkman E L., Harrison L N., lonut V., Lottati M., Zheng D., Hsu I R., Stefanovski D., Kabir M., Kim S P., Catalano K J., Chiu J D., Chow R H., “Simplified Method to Isolate Highly Pure Canine Pancreatic Islets”. Pancreas 2012; 41(1):31-38. [0298] Zufferey, R., Nagy, D., Mandel, R. J., Naldini, L., and Trono, D. (1997). “Multiply attenuated lentiviral vector achieves efficient gene delivery in vivo”. Nat Biotechnol 15, 871-875.