Successful Intracerebral Allotransplantation of Purified Pancreatic Endocrine Cells in Diabetic Rat WAH JUN TZE and JOSEPH TAI

Successful Intracerebral Allotransplantation of Purified Pancreatic Endocrine Cells in Diabetic Rat WAH JUN TZE AND JOSEPH TAI

viated by employing a natural semipermeable membrane, SUMMARY the blood-brain barrier, which permits ready transmembrane Single pancreatic endocrine cells (PEC) have been demonstrated to restore normoglycemia when trans- passage of O2 and CO2 and diffusion of glucose and insulin planted intracerebrally in diabetic rat recipients across across the membrane barrier while it limits the passage of 56 a major histocompatibility barrier without immuno- antibody and restricts lymphocytic infiltration. Reports of suppression. Twelve out of twelve transplants of puri- functional allograft and xenograft of neural and endocrine fied PEC have achieved long-term survival of over 176 tissues in the brain also suggest that brain tissue can provide days. This approach would provide a model to identify a suitable environment for the survival of transplanted PEC.78 the specific cell(s) as inducer of graft rejection and an In this article we report the successful allotransplantation of opportunity for transplantation study in spontaneous purified PEC in the brain of diabetic rat recipients across a diabetic BB rats and larger diabetic animal models. major histocompatibility barrier without immunosuppression. DIABETES 32:1185-1187, December 1983.

MATERIALS AND METHODS ransplanted pancreatic islet tissue has been ob- Male rats of the inbred Lewis (Le; AgB 1/1) strain weighing 350-500 g, were selected as donors of pancreatic islets and served to restore normoglycemia and prevent sec- of the ACI (AgB 4/4) strain as streptozotocin-induced (55 ondary complications in the recipient diabetic an- mg/kg i.v.) diabetic recipients. Pancreatic tissue was di- imals. Allotransplantation of pancreatic islets for the T gested with collagenase and the islets were handpicked treatment of diabetes mellitus is limited by immune rejection. under a dissection microscope. Contaminating acinar tis- The present hypothesis considers that certain cellular ele- sues and blood vessels were removed from the islets by the ments within the donor islets other than the endocrine cells, single-layer Hypaque-Ficoll (H-F) separation technique.9 e.g., passenger leukocytes, are responsible for the induction Clean islets collected at the interface were either cultured of graft rejection. Various manipulations of pancreatic islets overnight at 26°C before transplantation or immediately dis- before transplantation have only been able to achieve vary- sociated into single cell suspension. For dissociation into ing degrees of success in the prolongation of allograft sur- 1 single cells, islets were suspended and resuspended every vival, one approach being the modification of islet immu- 2+ 2+ 23 minute in 1 ml 0.04% EDTA in Ca -Mg -free Hanks' bal- nogenicity by in vitro culture before transplantation. anced salt solution (HBSS), pH 7.2, at 25°C for 5 min. One However, the duration of prolonged graft survival was vari- milliliter of 0.02% VMF trypsin (Worthington, Freehold, New able and a better result was achieved in those recipients Jersey) solution in HBSS at 37°C was added to the islets.10 that received short-term immunosuppression. A second ap- After 2 min of incubation, the islets were dispersed gently proach was immunoisolation by means of a membrane bar- with a 19-gauge needle until the completion of their disso- rier, which can isolate the tissue graft from the host immune 4 ciation as observed under an inverted microscope. The cells system. Though theoretically sound, the use of an artificial were filtered through a sterile 66-mesh nylon screen in a 13- semipermeable membrane has encountered many practical mm Millipore holder to remove cell clumps. Single cell sus- 1 problems. However, these problems probably can be ob- pension in 1 ml HBSS was subsequently layered on top of 4 ml single layer H-F solution with a specific gravity of 1.080. Centrifugation was carried out at 22°C for 8 min at 800 x g From the Departments of Paediatrics and Pathology, University of British Co- lumbia, Vancouver, British Columbia, Canada. after which cells at the interface were collected. Cells from Address reprint requests to Dr. W. J. Tze, Children's Hospital, 4480 Oak Street, a quantity of 1500 islets were suspended in 15 ml of culture Vancouver, B.C. V6H 3V4, Canada. medium (M199 containing 15% fetal calf serum) and dis- Received for publication 24 August 1983.

DIABETES, VOL. 32, DECEMBER 1983 1185 INTRACEREBRAL ALLOTRANSPLANTATION OF PURIFIED PANCREATIC ENDOCRINE CELLS

TABLE 1 Effect of donor tissue and transplantation site on pancreatic islet allograft survival

Transplantation Individual graft Donor tissue site survival days Mean ± SD

Whole islets Liver 5,5,5,5,5,5,6,6 5.3 ± 0.5 Whole islets Brain > 90.8 ± 87.8 Pancreatic Brain >176,>177,>178,>179, endocrine >183,>183,>184,>185, >184.9 ± 8.4 cells > 186, > 190, > 191, >207

> indicates that the graft is still functional.

pensed into a 100 x 20-mm plastic dish and cultured in a pentia, California) and prepared for microscopic examina- humidified 5% CO2 incubator at 26°C overnight. Viable PEC tion. collected at the interface of H-F gradient after centrifugation for 8 min at 800 x g were subsequently collected for trans- RESULTS plantation study. Table 1 shows the allograft survival time after intracerebrai ACI diabetic rats were divided into four groups: (1) sham- or intraportal transplantation. All the intraportal transplants operated controls (N = 6), (2) intraportal transplant of whole were rejected within 6 days. Although both types of intra- islets (N = 8), (3) intracerebrai transplant of whole islets cerebrai allografts have prolonged survival, all the PEC trans- (N = 6), and (4) intracerebrai transplant of PEC (N = 12). plants remained functional, whereas three of six whole islet Fifteen hundred whole islets of 2-3 x 106 viable PEC sus- transplants were rejected. Figure 1 shows that the trans- pended in 50 |xl vol were stereotaxically injected with a 26- planted PEC normalized the blood glucose levels in all the gauge needle into the brain of the diabetic recipient through aliogeneic diabetic recipients within 4 days after transplan- a burr hole 3.8 mm in depth, 0.5 mm posterior to bregma, tation and maintained the normoglycemic state throughout and 1.5-2.0 mm lateral to the rostrocaudal axis.11 Identical the period of observation. The sham-operated rats sustained surgical procedures were repeated with the sham-operated their hyperglycemia with gradual deterioration of the diabetic control animals, which received an injection of physiologic state. Figure 2 demonstrates the normal pattern of 24-h serial saline. The plasma glucose and 24-h urine volume for each blood glucose in all the aliogeneic recipients of PEC. recipient rat were assessed daily after transplantation. In Histologic examination revealed grafted cells with normal addition, 24-h serial blood glucoses were performed on appearance in the subarachnoid space as well as the site these rats at around 4 wk after successful transplantation. immediately adjacent to the lateral ventricle of a recipient Rejection was considered to have occurred when the blood rat with functional aliogeneic PEC for 2 wk. There is no evi- glucose level after normalization of the diabetic state ex- dence of lymphocytic infiltration or inflammatory reaction ceeded 200 mg/dl for 2 successive days. To determine the around the grafts. The presence of insulin granules in the location and morphology of the transplanted cells, one re- endocrine cells is confirmed with immunoperoxidase stain. cipient rat with functional aliogeneic PEC for 2 wk was killed and the brain tissue was fixed in situ with 10% phosphate- DISCUSSION buffered formalin, sectioned, and stained with hematoxylin- The present study demonstrates that the brain has provided eosin and immunoperoxidase stain (Immunlok, Inc., Car- a hospitable environment for the survival and function of the

Sham-operated diabetic 600 r-

500

400 o 5 30° o I 200 Transplanted a. 4—f 1 100 -1

I II -5 0 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 Time (days) /

FIGURE 1. Nonfasting plasma glucose levels (mean ± SD) of ACI diabetic rat recipients of intracerebrai transplantation of aliogeneic Le PEC (•—•; N = 12) and sham-operated diabetic controls (• •; N = 6). Shaded area represents the range of plasma glucose in normal control rats (N = 10).

1186 DIABETES, VOL. 32, DECEMBER 1983 WAH JUN TZE AND JOSEPH TAI

600 site was used. Further studies with intracerebral transplan- 1 1 1 tation of purified PEC suspensions plus various known cell 1 types within the islet would make it possible to identify the ^ 500 1 i 1 specific inducer of graft rejection. This approach can be extended to the study of allotransplantation in spontaneous — 400 diabetic BB rats and xenotransplantation in larger diabetic animal models. 300 O o ACKNOWLEDGMENTS E 200 We thank S. Ng, D. Mallory, C. Graves, M. Thakchuk, and J. E. van den Broek for their technical assistance, Dr. M. G. 100 Norman for her expertise in reviewing the histology, and R. Bogyo for secretarial help. This work was supported by grants from the Medical Re- 0800 1200 1600 2000 2400 0400 0800 search Council of Canada, Funds For Medical Research from Time (hrs) the Royal Canadian Legion (Branch no. 177, Vancouver, British Columbia), and C. K. Chan. FIGURE 2. Twenty-four-hour serial plasma glucose levels (mean ± SD) at 4 wk after intracerebral transplantation of PEC. The three groups of rats are represented by • normal control (N = 10); a PEC recipients REFERENCES (N = 12); m sham-operated diabetic (N = 6). 1 Tze, W. J., and Tai, J.: Manipulation of pancreatic islet cells in allotransplantation. Transplant. Proc. 1982; 14:714-23. 2 Lacy, P. E., Davie, J. M., and Finke, E. H.: Transplantation of insulin- producing tissue. Am. J. Med. 1981; 70:589-94. grafted PEC and that single endocrine cell suspension of 3 Prowse, S. J., Lafferty, K. J., Simeonovic, C. J., Agostino, M., Bowen, pancreatic islet is functional in vivo in maintaining blood KM., and Steele, E. J.: The reversal of diabetes by pancreatic islet trans- glucose homeostasis in the diabetic recipient rat. plantation. Diabetes 1982; 31 (Suppl. 4):30-37. 4 Tze, W. J., Wong, F. C, and Tai, J.: Immunological isolation of allo- In an earlier study we have observed that intraportal al- geneic or xenogeneic cells in an implantable artificial endocrine pancreas. lografts of pseudoislets (Le-» ACI) formed by aggregation Transplantation 1979; 28:67-70. 5 of purified PEC had only marginally longer graft survival time Fishman, R. A.: Cerebrospinal Fluid in Diseases of the Nervous Sys- 12 tem. Philadelphia, W. B. Saunders, 1980. than fresh and cultured (at 37°C) whole islets. Prolonged 6 Barker, C. F., and Billingham, R. E.: Immunologically privileged sites. survival of intracerebrally transplanted purified PEC or whole Adv. Immunol. 1977; 25:11-15. 7 islets observed in this study would confirm that the brain is Gash, D., and Slader, J. R., Jr.: Function development of grafted vasopressin neurons. Science 1980; 210:1367-69. an immunoprivileged site. Despite this, the blood-brain bar- 8 Kolata, G.: Grafts correct brain damage. Science 1982; 217:342-44. rier is not absolute and graft rejection can occur if antigenic 9 Tze, W. J., Wong, F. C, and Tingle, A. J.: The use of Hypaque-Ficoll in the isolation of pancreatic islets in rats. Transplantation 1976; 22:201-205. dosage is sufficiently high as seen in our whole islet trans- 10 Tze, W. J., and Tai, J.: Preparation of pseudoislets for morphological plants and also in the recent observation that allogeneic fetal and functional studies. Transplantation 1982; 34:228-31. rat islets, contaminated with lymph nodes, failed to correct 11 Hart, B. L: Experimental Psychobiology: A Laboratory Manual. San Francisco, W. F. Freeman, 1976. the diabetic state of recipients after intraventricular trans- 12 13 Tze, W. J., and Tai, J.: Pseudoislet allotransplantation study in rats. plantation. Consistent long-term survival of islet allograft Diabetes 1983; 32 (Suppl. 1):168A. can be achieved without immunosuppression when the com- 13 McEvoy, R. C, and Leung, P. E.: Transplantation of fetal rat islets into the cerebral ventricles of alloxan-diabetic rats. Amelioration of diabetes bination of purified PEC as graft and brain as transplantation by syngeneic but not allogeneic islets. Diabetes 1983; 32:852-57.

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