Fetal pancreatic transplantation

Fetal Pancreatic Transplantation Ame Andersson andStellan Sandler

ince the first pancreas transplantation was performed in 19661 it has been repeatedly demonstrated that by transplanting most or all of the pancreas as a vascularized organ to patients with insulin-dependent diabetes mellitus (IDD~l), norrnoglycemia is achieved without the need for insulin injections. The procedure has become more frequent in recent years and success rates have been markedly improved," However, there arc three major problems that remain: (1) results with a pancreas transplantation that has not been combined with previous or simultaneous kidney transplantation have, in most studies, been discouraging and therefore the treatment can not be offered until late complications (ie, renal failure) have already occurred; (2) it is difficult to foresee that recipients ofvascularized organ grafts will not have to commit to a life-long regimen of immunosupprcssives; (3) because the primary objective of transplanting endocrine pancreatic tissue is to achieve insulin independency sufficiently carl}' in the course of the disease for prevention oflate complications, most patients with IDD~1 should be candidates for this kind of therapy and, taking this into account, there \\;11 be a considerable donor organ shortage. It should also be kept in mind that, because the whole-organ procedure per se is not lifesaving, it has become a much debated procedure and is questioned by many diabctologists.' Concurrent with the development of the wholeorgan transplantation technique as an alternative approach, islet cell transplantation has been pursued. The idea of replacing only the desired endo-

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From the Department '!f Medical Cell Biology, Uppsala Uniursity, Uppsala.Sucden. Supported l!>' grantsfrom the Suedisli Medical Research Council(,\'os. 19P-8982, 12X-I09, 12X-8237, and 12X-9237-' lI:n. Saunders Company 0955-170:\/92/0601-000355.00/0

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crine component and thereby circumventing most of the surgical problems became a practical reality when Hcllcrstrorn,' Moskalcwski,' and Lacy and Kostianovsky" described techniques for the preparation of pure mammalian islet tissue. It is now almost 20 years since Ballinger and Lacy first reported on the amelioration of streptozotocin-induced diabetes by intraperitoneal syngeneic rat islet transplantation.' Since then, considerable effort has been put into this field of research, but the promising results in experimental animals have been difficult to realize in clinical practice. However, during the last 2 years, there have been reports indicating that several diabetic patients who have undergone islet transplantation have achieved at least a temporary insulin independence,"?" No doubt, these arc extremely encouraging results because it has now been proven that it is possible to achieve success with this simple surgical procedure. l\iore experimental work can now be performed in the area of immunoalteration and immunoisol~tion of the grafted tissue. Once these attempts have been shown to be clinically feasible, the problems with immunosuppression should disappear. Such progress might imply that xenografting also could become a possible thcrapcutie choice. The challenge that has now been successfully met is that of developing methods for islet isolation capable of producing pure, viable human islets for clinical trials.":" However, there arc many remaining problems to be solved, such as isolating sufficient quantities of endocrine material. Pancreases from as many as five different donors were used for each recipient in the study performed by \Varnock et al." If not solved, this \\;11 constitute an even greater demand on the donor organ supply than is the case with whole-organ transplants. Therefore, other potential sources of endocrine pancreatic tissue must be considered. One of these sources is fetal pancreatic tissue. Actually, the usc of this latter type of islet cell preparation as an insulin-producing graft was suggested by Ssobolcw in 1902. 13 Other early investigators proposed and demonstrated that the endocrine component of grafted fetal pancreatic tissue survivcd, grew, and matured." The advantages of this procedure included the fact that the transplanted

Transplantation Recieus, 1'016, No 1 (jalluaT)), 1992:P/J 20-38

FetalPancreatic Transplantation

exocrine cells did not survive the transplantation and that the ratio of endocrine to exocrine cells is much higher in the fetal tissue. This article reviews the effect of fetal pancreatic transplantation on the amelioration of chemically induced diabetes in experimental animals and describes different techniques for the preparation and storage of the fetal pancreatic material. Special attention is given to the ontogeny of the 13-cell, the regulation of the growth of the graft, the impact of implantation site, and the mechanisms for the functional maturation of the insulin-producing cells. Finally, some clinical trials offetal pancreas transplantation using either human or porcine material arc reviewed.

Effect of Fetal Pancreas Transplantation in Diabetic Animals Control of experimental diabetes mellitus in rats by transplantation of fetal pancreases was first demonstrated by Brown et al in 1974.13 They used three fetal pancreases (15 to 18.5 days gestation) per recipient and, without further treatment, placed them beneath the kidney capsule of syngeneic diabetic rats. About 3 weeks later the recipients were norrnoglyccmic and, on removal of the implant, they returned to the serum glucose levels of nontreated diabetic controls. Complete reversal of diabetes in streptozotocin-injected rats by transplantation of a single pancreas, provided the grafted organ was first grO\m in a normal syngeneic carrier before transfer to the diabetic recipient, was later achieved, by the same investigators." A series of articles by l\lcEyoy et al confirmed that transplanted fetal pancreatic slices were effective in restoration of normoglyccmia in diabetic rats. 17• 19 They gave special attention to the effect of insulin treatment on the growth and differentiation of the fetal pancreatic implant. Although these invcstigators used as many as eight fetal pancreases for implantation, more than II weeks passed before reversion of diabetes occurred. To some extent, this seemingly lower efficacy could be explained by the long (8 weeks) period of alloxan-diabetes prior to transplantation. There arc also some reports to indicate that much shorter time periods (ie, a couple weeks) arc needed before full reversal of diabetes is achicvcd.Y" Mandel et al were successful in using just one syngeneic fetal pancreas to cure streptozoto-

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cin-diabetic mice by either intrasplcnic" or renal subcapsular" implantation. However, they cultured their fetal grafts for about 3 weeks prior to transplantation because there was a lO-fold increase in the insulin content of the organ during the culture period. The feasibility of fetal pancreas transplants for reversing diabetes in a large animal model was tested by Sasaki et al and Mullen in the genetically defined miniature pig. 21.2'> They found that porcine pancreas slices taken from 48-day-old fetuses grew considerably by at least a 20-fold increase in the insulin content of the grafts 2 months after implantation under the kidney capsule ofathymic rats." There was also evidence to suggest that pig fetal pancreas can reverse streptozotocin-induced diabetes in pigs. 25.26 Human fetal pancreas (HFP) obtained from therapeutically terminated pregnancies also has been used for implantation experiments. Overnight-cultured HFP (13 to 17 weeks gestational age) transplanted beneath the kidney capsule of diabetic nude mice cured the recipients within 6 to 8 weeks." Likewise, Tuch et al carried out a number of studies in which streptozotocin-diabetic nude mice served as recipients. 2H.2'l However, these investigators used a somewhat unconventional procedure by inducing the diabetic state of the recipient after the transplantation had been performed. In all the studies previously referenced, the implanted fetal tissue had been sliced or left intact before transplantation. By collagenase incubation of minced fetal rat pancreas and subsequent culture of the crude digest in attachment culture dishes, Hcllcrstrom et al introduced a new technique for production of fetal islet tissue." After culture for 5 to 7 days a substantial number of islets, each consisting of more than 80% 13-cells, can be harvested (subsequently discussed). Such islets have been found to be useful for transplantation purposes both in rats31,32 and in mice." However, in these rodent models a greater number of fetal as compared with adult islets had to be used in order to cure the recipients, and it usually took a longer period of time before norrnoglyccmia was achieved, A collagenase culture isolation method also has been used on fetal pancreases from large animals including humans. In two recent studies it was shown that islet tissue from porcine fetuses prepared in this manner cured diabetic nude mice (Fig I ).31,35 Human fetal pancreatic tissue treated with a mild collagenase digestion was also capable of curing nude mice with streptozotocin-induced diabetes."

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Andersson andSandler

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20

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o

U

:J Cl

10

E

:J .... Q) en

o -t-.--.----.---,--.--....---......-,.-,--r---r---.--...-...., o 2 3 4 5 6 7 Months posttransplantation

Figure 1. Serum glucose concentrations at different times after transplantation of porcine ICC beneath the kidney capsule of normal (closed symbols) and alloxandiabetic (open symbols) nude mice."

Ontogeny of the Fetal Endocrine Pancreas Several articles and reviews on the evolution of the endocrine pancreas of rodents have previously been published.V'" However, from a clinical perspective these rodent studies may be of little relevance because the period of fetal development in rodents is short and the neonate is very immature. Therefore, in this section attention will focus mainly on the ontogeny of the 13-cell in the human and porcine fetal pancreas. The latter because it is thought to be similar to human fetal development, and because there is good access to fetuses of all developmental stages for experimental purposes. With the increased interest in xenotransplantation, the porcine fetal pancreas has also become a source of xenogeneic insulin-secreting islet tissue that is being intensely investigated at present.2' .33,' 2-43 To date, the most detailed study that has been published on the ontogeny of porcine pancreatic endocrine cells is that ofAlumets et al.-l6 By immunocytochemical means, they demonstrated that at 4 weeks gestation, which was the earliest stage studied, the 13-cells, the glucagon-containing a-cells, and the somotostatin-containing o-cells were present in the dorsal pancreatic primordium. However, in the vcntral primordium there were only PP-cells to be found. At 10 weeks, 13-cells were found in this region also, but were few and weakly immunoreactive, ie, contained only minute amounts of insulin. Three weeks later, the number of 13-cells had increased markedly and were intensely immunoreactive. However, they

were still randomly distributed in the exocrine parenchyma, and not until a week or 2 after birth did the 13-cells cluster together in small islets (pigs are born during the 17th week of gestation). Taking these facts into account, it is quite obvious that the use of a traditional collagenase isolation method for the preparation of free fetal islets is not worthwhile because there are actually no pancreatic islets to isolate in the fetal porcine pancreas. In two quantitative morphological studies ofendocrine cells in the HFP it was demonstrated that 13-eells and also the other endocrine cells are present in 8-week-old 17 and 9-week-old ' 3 fetuses. In the study by Stefan et al,.J7 no fetuses younger than 8 weeks were investigated; in the study by Clark and Grant," only one 7-week-old fetus was examined. Thus, it is unclear whether 13-cells appear earlier in fetal development than has been reported in these two investigations. Whereas in the youngest fetuses (7 to 9 weeks gestation) most endocrine cells were found in the walls of the ducts or adjacent to them, a number ofwcll-defined" or primitive" islets were found in the pancreases of 12-week-old fetuses. In older fetuses (20 weeks," and 28 weeks to 5 months after birth") most endocrine cells were grouped into individual islets. Quantitative morphometrical estimations showed that the relative and total volumes of 13-cells increased in parallel with the increase of the total glandular tissue ..J7 Thus, there was a 3D-fold increase of the total Bvccll volume between the 12th week of gestation and the 5th postnatal month. However, the corresponding figure for the a-cells was only half that of the 13-cell figure, whereas the o-cells increased on a proportional basis as much as the 13-cells. It is noteworthy that in neonates the o-cells make up no less than 38% of the endocrine pancreas, which decreases to about 5% in the adult pancreas. Altogether, these figures show that there is a preferential increase in the number of13-cells with time. This may be caused by a replicatory activity that is higher than in the other endocrine cells or because progenitor cells in later stages ofdevelopment become 13-cells. To what extent this latter differentiation process can be enhanced is of great interest to investigators in the field of transplantation. It was found that when preparations of fetal porcine islet-like cell clusters (ICC),3w which contain only a minority ( < 10%) of 13-cells, were transplanted beneath the kidney capsule of nude mice there was a considerable and preferential growth of these cells." By applying both autoradiographic and immunohistochemical techniques it was concluded that this growth was not

FetalPancreatic Transplantation

caused by an increased rate of DNA synthesis of the l3-cells as compared with the other endocrine cells, but because of an influence on the undifferentiated epitheloid cells constituting the predominating cells of the porcine ICC so that they become l3-cells. In the secretory granules of the fetal porcine endocrine pancreas there is colocalization of two or more islet hormones that was recently shO\\11 by means of a double immuno-gold-Iabelling technique at the ultrastructural level." These findings confirm results obtained by Alpert et al from experiments with transgenic mice." Thus, they demonstrated that at an early developmental stage in the fetal mouse pancreas there is coexpression of genes whose later activity is restricted to distinct endocrine cell types. The results were interpreted as suggesting a common precursor for the endocrine cells of the pancreas. For the purpose of transplantation, knowledge of the ontogeny of the fetal endocrine pancreas may form the basis for selecting a donor gestational age that is optimal with regard to the frequency of differentiated l3-cells. However, as suggested by the findings from transplantation of porcine ICC, it seems that a significant number of the ICC cells that do not stain immunohistochemically positive for any of the islet hormones do differentiate into l3-cells posttransplantation." More detailed information on the molecular mechanisms underlying this phenomenon is urgently needed. Thus, the possibility of using younger fetuses and less differentiated tissue may have substantial practical implications.

Preparation, Culture, and Cryopreservation of Fetal Pancreas In most cases, transplantation of fetal pancreatic tissue has been found to require that the tissue is explanted in culture for various periods of time prior to implantation. Ideally, this culture period should not decrease the viability of the explantcd material, but instead promote differentiation and growth of the Ictal Bvcclls. Furthermore, specimens for bacteriologic and viral screening can be secured, and functional tests of the intended graft performed. To achieve this objective, a number of methods for culture of fetal pancreatic tissue have been presented and will subsequently be described in more detail. However, a crucial step before culture is the procurement and preparation of the fetal pancreas, especially when using HFP. Ifmore than I 1/2 hours elapse between completion of a prostaglandin-

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induced abortion and dissection of the pancreas, the viability of the HFP in culture is much reduced.51 The method ofabortion also affects the subsequent viability of an HFP explant in culture; mechanical procedures and hysterotomies yield the best results. 52-51 Once the HFP has been removed from the fetus, it can usually be stored for up to 10 hours at 4°C in a tissue culture medium supplemented with antibiotics before further handling," When using experimental animals (eg, mouse, rat, pig), the dissection of the fetal pancreas can be commenced immediately after killing the pregnant animal and there will be almost no risk for warm ischemic injuries.

Suspension or Air-liquid Interface Culture of Fetal Pancreatic Fragments Suspension culture of fetal pancreatic fragments has been used in several studies.56,57 For this purpose, the dissected gland is minced into fragments about 2 to 3 !-LL in size and transferred to nonattachment sterile culture dishes in which the fragments will remain unattached. However, with this method there arc some gaseous diffusion problems and, during prolonged culture periods, a gradual loss of tissue will occur. A method that was originally developed for thymic explants," is that of maintaining fetal pancreatic fragments at the air-liquid interface. 59-6 1 This provides the tissue with both a sufficient oxygen supply and nutrients from the culture medium. The tissue fragments arc pipetted on to Milliporcs' filters (Millipore Corporation, Bedford, ~IA) (pore size < 8 urn), which arc supported by a block of surgical gel foam. Provided the culture medium is exchanged e\'ery 2nd to 3rd day, viable explants ofHFP can be maintained in vitro for at least 2 months." However, after 100 days none of 122 cultured HFP remained functional in terms of insulin secretion." Lafferty and Trujillo designed another type of air-liquid interface culture by adding only a small volume of medium to fragments of HFP. 53 This was combined with an oxygenrich gas atmosphere that may also reduce the immunogcnicity of the preparation.

Collagenase Digestion and Preparation of Fetal Rat Islets. Large-scale isolation of pancreatic islets from the fetal rat pancreas was first described by Hcllerstrom et al." For this technique, rat fetal pancreatic glands are removed and harvested I to 2 days before the expected delivery, The glands arc finely minced and subsequently digested with a high collagenase concen-

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Andersson andSandler

tration in Hanks' balanced salt solution. The resulting pancreatic digest is washed and seeded out into culture dishes that allow cellular attachment. This results in selective degeneration of exocrine pancreatic cells but survival of the endocrine cells. During the next few days endocrine and fibroblastoid cells form a monolayer attached to the bottom of the culture dishes, and within I week, attached or freefloating islets appear. These islets can easily be harvested, and immunocytochemical examinations have demonstrated a pure endocrine structure composed of more than 80% 13-cells (Fig 2).3o,67_'A difference in the cytoskeleton and the adhesive properties between fibroblasts and the endocrine cells 'may explain why the latter cells become organized into islets in vitro," Moreover, fibroblast-derived factorsj" in particular collagcn" may promote both 13-cell differentiation and islet formation. A procedure similar to that developed for the fetal rat pancreas has been described for the fetal mouse pancrcas.i':" However, these investigators have chosen the designation fetal mouse proislets for their preparations.

Isolation of Human Fetal Islets Because of the structural organisation of the endocrine cells within the HFP at the gestational stage at which they usually become available (previously discussed), it can be anticipated that the yield and purity of these islets will be low. Nevertheless, it was reported that it is possible to identify and collect isolated islets after a long collagenase incubation.f" Unfortunately, no immunocytochemical data on these

Figure 2. Light micrograph of a section, immunocytochemically stained for insulin, of a fetal rat pancreatic islet fixed on the 5th day of culture. A large majority of dark l3-cells can be seen (magnification X900).

islets were provided. More recently, it was reported that "true" islets were obtained from the HFP after they had been minced and had undergone a 14 minute collagenase digestion period before explantation of the tissue into nonadhesive culture dishes.;; During the subsequent 6 to 7 weeks in culture the islets were identified and picked out based on their size and appearance.

Preparation of ICC Based on experience gained from the preparation of fetal rat pancreatic islets (previously discussed), a culture method has been developed in which socalled ICC are formed in vitro on explantat ion of collagenase-digested fetal human pancreas. This method was first applied on HFP obtained after legal abortions performed at 12 to 20 weeks gestation.":" Later, the same technique was also found suitable for the porcine fetal pancreas obtained at 60 to 75 days gestation." When compared with the preparation offetal rat islets, the procedure for HFP involves a milder collagenase digestion procedure in combination with the use of a Ca2+- andMg'"-free buffer, which does not lead to complete disintegration of the gland. After cxplantation in vitro of the collagenasedigested pancreatic tissue, an initial growth of fibroblastoid cells occurs and is followed by the appearance of spherical cell aggregates, ie, ICC, which are loosely attached to the bottom cell layer. An analysis of the cultures has shown that the great majority of the total insulin content is found within ICC and not in the bottom cell layer; however, the collagenase treatment may cause an initial loss in 13-cells of the cxplants." If the culture period is extended for more than I week, the ICC may become overgrown by the fibroblasts and disappear. By harvesting human ICC on day 3 of culture and then subculturing ICC in petri dishes coated with bovine corneal matrix, it has been possible to establish a fibroblast-free monolayer with a high proportion of13-celIs.77 In contrast to the fetal rat pancreas, the HFP glands used for experimental studies represent a much less mature tissue because they are usually obtained in the early 2nd trimester. When considering that the spheroid cell aggregates formed in vitro contain only a minority of insulin-positive cells,51.55 they were designated ICC rather than fetal islets (Fig 3). It is the opinion of the authors that a structure should contain a majority of endocrine cells in order to be denominated as a pancreatic islet. This criterion is not fullfilled by most preparations ob-

FetalPancreatic Transplantation

.'

Figure 3. Light micrograph of a sectioned human ICC fixed on the 5th day of culture and immunocytochemically stained for insulin.The crown-heellength of the fetus was 18em. A group, and also some scattered 13-cells, as well as some duct-like struct ures can be seen. Kate the difference in the frequencyof13-cells compared with the fetal rat islet depicted in Fig 2 (magnification x990). tained after collagenase digestion and primary culture of both the HFP and porcine fetal pancreas. A methodology almost identical to that used for the production orICC was recently applied to 75-day-old porcine fetal pancreas, but in this case the preparations were designated as fetal porcine proislcts."

Cryopreservation of Fetal Pancreas The ultimate method for in vitro preservation of fetal pancreas prior to transplantation is cryoprcscrvation because it would allow indefinite periods of storage of viable p-cclls in a tissue bank. When designing protocols for cryoprcscrvation, it is important to determine the specifie optimal cooling and thawing rates and modes of addition and removal of the cryoprotcctant for each individual tissue in order to avoid freezing injuries." With regard to the fetal P-cclls, this raises some additional problems, because the most abundant cellular constituents of the fetal pancreas are non-Bscclls and, thus, subsequent viability tests reflect the predominant action of these other cells. Simple tests for screening of p-cell function, such as glucose-stimulation of insulin secretion, are essentially precluded because of their immature function (subsequently discussed). In 1977, Mazur et al were the first investigators to publish a report on cryoprcscrvation of whole fetal rat pancreas." Based on measurements of'incorporation rates oflabelled amino acids into proteins, it was concluded that the best cryoprescrvation results required a slow cooling rate (O.3°C/min) and a high

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concentration of the cryoprotcctant dimethylsulfoxide (D~ISO). It was subsequently demonstrated that syngeneic transplantation beneath the renal capsule of such frozen-thawed preparations of rat pancreas reversed experimental diabetcs.f'" Another study examining several cryoprescrvation variables indicated that the permeation of the cryoprotcctant into the tissues was the most decisive factor for cl)'opreservation offetal rat pancreas." When cryoprcscrvation techniques were applied to fragments ofHFP, a 50% deterioration in function as compared with nonfrozen material was observed. This was mainly attributed to D~lS0 toxicity." The viability was improved when the HFP was cultured for 18 to 24 hours after thawing that resulted in an insulin secretory capacity in vitro similar to that of nonfrozen HFP.8t Two studies examined cl)'Opreservation of HFP glands obtained after prostaglandininduced abortions.f''" Parts of the minced glands were cryoprcservcd at a slow cooling rate (O.3°C/ min) after a I-day period of suspension culture, whereas the remaining fragments were morphologically and functionally studied in the nonfrozen state. After storage at -196°C forup to 5 months, the frozen material was rapidly thawed at 37°C and cultured for an additional day. Cryoprcservation caused some morphological damages in comparison with the non frozen control HFP fragments. Ncverthcless, in three of four cases the frozen-thawed HFP fragments showed evidence of an active (pro)insulin biosynthesis. Furthermore, a significant insulin response to stimulation with glucose plus theophylline was observed to the same extent as before cryoprcservation. Shiogama et al reported an improved viability and p-cell function ofcryoprcservcd HFP fragments when a high concentration of D~ISO was added by a two-step method." Using a slow cooling rate and a step-wise addition of D~ISO, Rajotte et al llll demonstrated that cryoprcscrvcd HFP tissue could normalize the hyperglycemia ofstrcptozotocin-diabctic nude mice 2 to 3 months after implantation under the kidney capsule." Dawidson et al attempted intraportal transplantation of similarly cryoprcservcd HFP fragments in six IDD~l patients, but there was no evidence of in \;\'0 insulin or C-peptide production during a l-ycar follow-up period." Transplantation of slowly frozen syngeneic fetal mouse proislets suggested a morphological differentiation ofP-cclls, although only normoglycernic recipients were studied." 'After culturing by the air-liquid interface technique and freezing at a rate of lOCI

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Andersson andSandler

min, Mandel and Carter grafted either fetal mouse pancreas syngcncically or HFP to nude mice." This seemed to be a suboptimal procedure because a loss of function ensued following cryoprescrvation and transplantation. Current observations indicate that fetal endocrine pancreas is suitable for cryoprcservation. However, thc methods for cryoprcservation offetal f3-cells require further evaluation, particularly in the area of the more recently described preparations of fetal f3-cclls such as ICC or monolaycrs,

destruction with insulin leakage. If estimations ofcell rcplicatory rates with 3H -thym id inc- Iabelled autoradiographs or bromodcoxyuridinc labelling arc not combined with immunocytochemical identification of thc f3-cells, the results essentially will reflect only the replication of non-f3-cells. Therefore, cxplorations of f3-cell differentiation in thc fetal pancreas must be performed with these difficulties and possiblc pitfalls in mind.

Growth and Differentiation of Fetal J3-Cells

The most commonly used culture medium seems to bc RPMI 1640,93 which was originally found to be suitable for maintenance of f3-ccll function in adult mouse islets." Similar comparative studies arc difficult to perform on fetal preparations because fetal f3-cells may exhibit different in vitro characteristics depending on the preparation technique, fetal age, and species used. Although the f3-cell content in cultures of fetal rat islets can be preserved without serum supplcmcntation,9:;.'ll; prolonged culture of fetal pancreas preparations requires' a serum supplementation of 10% to 20%. Experiments with HFP show that human serum promotes the development of islet s" or ICC;; better than fetal calf serum. The same was found with regard to the formation of porcine ICC.~2 If the serum supplement is added 12 to 18 hours after the cxplantation of the tissue, there is a restriction in the fibroblast outgrowth." Supplementation with human amniotic fluid also has been found to promote formation of human ICC.911

Experiments on animals have demonstrated a latent period of'sevcral weeks between fetal pancreas transplantation and reversal of hypcrglyccmia.P'" This delay presumably reflects thc differentiation and growth of the grafted tissue after implantation and before diabetes can be corrected. To enhance this process, much attention has been focused on factors that might regulate thc differentiation and replication of fetal f3-cells in vitro and in vivo, Particular attention has been givcn to thc possibility of influcncing these variables in vitro before implantation in order to promote an adequate graft function in \;\"0. This sccms particularly important for HFP allografting because thc availability of human fetal material is greatly restricted in most countries. Because of thc strong growth capacity of the fetal pancreas, it is vital that the proliferation of th e tissue after transplantation be controlled. Thus, thc transformation of thc graft into neoplastic cells could create a major hazard to the recipient. This aspect has not been the object of many studies, although data on graftcd porcine ICC indicate that the growth ratc slowed considerably with time."

Methodological Considerations TIlC assessment of thc growth and diffcrentiation requirements for fetal islet cells is rather cumbersome because fetal f3-cclls lack a normal insulin response as is subsequently discussed. Moreover, the majority of the cells within the preparations arc usually non-f3-cells. Experiments with HFP and also, to a lesser extent, porcine fctal pancreas arc associated with a marked experimental variation, which is imposed by the noninbred origin of thc donor material. Increased insulin concentrations in the culture medium of cultured fctal pancreatic preparations may indicate both development of f3-cells and f3-cell

Culture Medium and Serum Supplements for Fetal f3·Cells

Effects of Glucose on the Differentiation and Replication of Fetal f3-Cells A key factor for thc physiological regulation of f3-cell differentiation is glucose. In fetal rat islets cultured at a high glucose concentration, the fraction of f3-cells capable of replication increased from 3% to 7% by recruiting more cells from thc resting Gophase of the cell cycle into the G 1 phase." The effect of glucose seems most prominent late in gestation, whereas at earlier stages amino acids arc more effective in promoting f3-cell replication.'?' Glucose also stimulates the proinsulin gene expression of fetal rat f3-cells,IOI.IrrZ which is accompanied by a hypertrophy of the f3-cells and an expansion of differcnt organelles invoked in insulin production.!" However, these findings do not necessarily imply that fetal pancreatic grafts should be cultured at a very high glucose concentration (> 20 mrnol/L). Thus,

FetalPancreatic Transplantation

experiments with cultured fetal mouse isografts indicated that tissue grm\TI under "normoglycemic" conditions functioned better than tissue grm\TI in "hyperglycemic" media.22.lol

Other Compounds Stimulating Formation and Replication of Fetal p-Cells. Numerous factors have been reported to affect both adult and fetal l3-cell replication and differentiation and have been reviewed in dctail. llo , ' 06 Subsequently, those compounds that might be the most relevant for transplantation purposes will be discussed in detail. Growth hormone (GH) has been found to increase DNA replication of both fetal 1OO, I07 and neonatal 108,109 rat islets. Addition of human GH to HFP cultures stimulated the formation and production of insulin by ICC"O,I" and enhanced the expression of the insulin gene in human fetal l3-cells.1I2 Also porcine ICC were formed in larger numbers when exposed to GH, but the insulin content was not incrcascd.l" Other experiments have suggested that part of the GH-induced islet cell DNA replication is mediated by a local production from islet cells of insulin-like growth factor-I (IGF-l ).111,115 Recently, it was reported that IGF-l supplementation of cultures of HFP proislets enhanced l3-cell activity in vitro." When such proislets were transplanted into diabetic nude mice, the time required to restore norrnoglycemia was reduced from 12 to 6 weeks when compared with a control group. Another recent study showed that a composite graft of fetal liver cells and fetal pancreas caused a 50% reduction in the time required to cure streptozotocin diabetes in rats.!" This may reflect the release in vivo by the liver cells of l3-cell-promoting growth factors such as IGF-I. Otonkoski et al compared the addition ofGH or IGF-I on the functional maturation of human ICC and concluded that IGF-I could not reproduce the effects of GH." 1 Instead they suggested that GH had a direct effect on the ICC that was not mediated by IGF-I. Experiments with prolactin (PRL) on neonatal rat islets have indicated that this pituitary hormone also may enhance insulin secretion and cell proliferation in \itro. 108,1I7 Furthermore, when homologous preparations of GH and PRL were compared, the effects ofPRL on insulin production and cell replication in rat islets were much more pronounccd.l'V" However, when human GH was added, an effect of similar potency as that exerted by rat PRL was observed. This may imply that heterologous human GH acts as a lactogen rather than a somatotropin on rat islcts.!" It is of interest that placental lactogen,

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when present with an elevated glucose concentration, also enhanced the insulin content and insulin release by fragments ofHFP in culture'" and stimulated the replication of neonatal rat islet cells. lOll Another mode of affecting cellular differentiation and replication is by influencing adenosinediphosphate-(ADP-)ribosylation reactions."! This can be performed by adding an inhibitor of the enZ}1TIe poly(ADP-ribose) synthetase, eg, nicotinamide or 3aminobenzamide. It has been demonstrated that treatment of partially depancreatized rats with nicotinamide-induced l3-cell regeneration in the pancreatic rcmnant.F" Moreover, newly diagnosed IDD~I patients treated with nicotinamide showed an extended remission pcriod.i" Increased rates of DNA replication were found in adult mouse islets cultured in media supplemented with nicotinamide.!" Cell replication in syngcneically grafted adult mouse islets was increased in animals given nicotinamidc.l" Addition of nicotinamide to cultures of HFP increased both the yield and insulin content ofICC. 126 Similarly, nicotinamide increased the insulin content of porcine ICC I 13 and doubled the number of insulinpositive cells." Transplantation of nicotinamidecultured ICC significantly decreased the time needed to cure alloxan-diabetic nude mice as compared with implantation of identical volumes of control ICC.97 \Vhen examining the subcapsular grafts 8 weeks after transplantation, the insulin content of the graft consisting of nicotinamide-cultured ICC was more than double that of the control group."

Stimulation of Insulin Secretion by the Fetal p-Cell The hallmark of the adult l3-cell is the ability to secrete insulin promptly when exposed to an increased ambient glucose concentration. Even I day after birth, this capacity had not been attained by the neonatal rat pancreas.!" Six days postnatally.!" .or after I week of culture of fetal rat islets in ll.l mmol/L glucose.!" a nearly normal function had developed. Extensive biochemical investigations have aimed at characterizing the mechanisms for this defective insulin secretion. Theoretically, this could be localized to one or several steps in the chain of events connecting the l3-cellmetabolism ofglucose in the cytoplasm and mitochondria with the ionic fluxes over the l3-cell membrane preceding insulin secretion. Based on experiments with fetal rat islets, it was suggested that the attenuated glucose-induced insulin release in fetall3-cells is caused by an immature glucose metabolism. This leads to a too low produc-

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Andersson andSandler

tion of cellular adenosine triphosphate (ATP), which in turn is insufficient to block the ATP-sensitive membrane-bound potassium channel.!" The ensuing deficient depolarization ofthe j3-eell membrane would not allow an opening of the voltage-dependent Ca2+ channels, which is a prerequisite for glucose-stimulated insulin secretion. Alternatively, it has been suggested that the metabolism of glucose fails to couple its signal(s) with the potassium channel in fetalj3-cells. l3O The available data on the insulin secretory capacity of the human fetalj3-cell relate essentially to HFP at approximately 12 to 20 weeks gestation, ie, during the 2nd trimester. At this stage, the j3-cell response to glucose in vitro is either lacking or only minute.51.57•00.62.63.l3 1. 133 However, addition of theophylline, a phosphodiesterase inhibitor, to a high glucose concentration elicits a noteworthy HFP insulin rcsponse.51.56.57.00.62.65.132. 131 Arginineoo. 132 as well as elevated concentrations of amino acid mixturcs'" potentiate insulin secretion from the human fetal j3-cell. A short-term stimulatory, but chronic inhibitory, effect on the insulin secretion of both HFP and fetal rat islets by activation of protein kinase-C, a signal transducing enzyme, has been reported. 135• l3S Tuch et al have tried to assess the time point when glucose-sensitive insulin secretion develops by following 14- to 20-week-old HFP preparations either in culture, or after a passage in vivo consisting of transplantation to diabetic nude mice. 63. l3 1 An initial weak insulin response was observed at 25 weeks of age, but it was not until after 55 weeks of absolute (fetal plus postnatal) age that a pronounced insulin secretion in response to glucose was evident. \Vhen similar experiments were performed with norrnoglycemic nude mice as HFP recipients, there was no maturation of the insulin secretory capacity to glucosc.!" In line with this, 6 months after transplantation, the insulin release from perfused porcine ICC was higher when the graft had been implanted into diabetic nude mice than into normoglycemic rccipients." Thus, it seems that either hyperglycemia per se or a factor in the diabetic environment promotes maturation of the glucose-induced insulin secretion. Otonkoski and colleagues reported a glucose-stimulated insulin release during the first half of the gestational period in perifusion experiments with human ICC, whereas a normal biphasic insulin release pattern did not begin until after birth.!" Much less is known about the secretory behaviour of the porcine fetalj3-cell at different developmental stages. However, it seems to be quite similar to the

human fetalj3-cell in terms of insulin release, despite the fact that various culture techniques have been used. 3t ,H.n .n Furthermore, using a perfusion technique of the graft-bearing kidney," a mature pattern of glucose-stimulated insulin secretion by transplanted porcine ICC was found to evolve with time."

Effects of Hyperglycemica on the Transplanted Fetal j3-Cells One much debated question concerning transplantation of both adult islet and fetal pancreas is to what extent hyperglycemia in the graft recipient is deleterious to the transplanted j3-cells in the immediate posttransplantation period. As previously discussed, glucose seems to promote growth and function of fetal j3-cells, but some data indicate that fetal pancreas maintained in vitro at a high glucose concentration functioned less well after transplantation.F'?' Experiments with fetal mouse pancreas suggested that chronic hyperglycemia impaired isograft engraftmcnt,'!" whereas allografts were not similarly afIcctcd.!" ~lcEvoy and Hegre reported that some diabetic rats treated with fetal syngeneic grafts and insulin administration for 7 days had a greater implanted j3-cell mass and insulin content than similarly treated rats not given insulin." It was reported that insulin was advantageous to the growth of HFP after implantation to diabetic nude micc.!" This could be ascribed either to the insulin-induced norrnoglyccmia, or to a growth promoting effect of insulin. In the latter case it could not be excluded that high concentrations of insulin affected IGF-I receptors. Other important processes such as the neovascularization and blood flow of an islet graft may also be impaired by a diabetic state as was suggested by experiments with autotransplanted adult rat islcts.!" Prolonged hyperglycemia may also affect the survival of j3-cells, and an actual loss of grafted j3-cells can occur. IH-1I6 This response to hyperglycemia by the j3-cells seems to be at least partially regulated by genetic factors.l'"!" Although so far not supported by actual experimental data, it can be anticipated that the fetal graft recipient would benefit from a strict normoglyccrnia during the first 10 days after transplantation, but subsequently a moderate hyperglycemia might promote the growth and maturation offetalj3-cells.

Significance of Implantation Site Although still a controversial issue, it seems as if none of the three most commonly used sites for

29

FetalPancreatic Transplantation

experimental islet implantation-intraportal, intrasplenic, renal subcapsular space-is superior to the others with regard to the acute normalizing effect on the hyperglycemia. There arc some reports to suggest that islet grafts with portal venous drainage arc more efficient than systemically draining grafts.HH.119 Other studies have shown that the renal subcapsular space represents a relatively immunoprivilegcd site. I30•131 Perhaps most important in this context is the recent demonstration that there is a progressive deterioration of endocrine function after intraportal, but not kidney subcapsular, rat islet transplantation. 132 Because grafted fetal islet tissue has to grow and to mature after engraftment, special attention has to be focused on the possibilities for the fetal cells to achieve this at the different implantation sites. So far there arc no comparative studies carried out on this issue with fetal islet tissue, perhaps simply because it is difficult to implant fetal pancreatic slices intraportally and intrasplcnically, However, with more recently introduced ICC/proislet preparations, such studies should be possible. Using adult mouse islets and autoradiographic techniques, the importance of the transplantation site for the replication of grafted islet cells has been investigatcd.P' Fourteen days after islet implantation the alloxan-diabetic mice were killed subsequent to an injection of tritiated thymidine. Each animal had islets implanted into two graft-bearing organs (liver, spleen, kidney) that were processed for autoradiography followed by determination of the labelling index of the islet cells. When intrasplenic islets were compared with both intraportal and subcapsular islets, they were less active in their rate of DNA synthesis. In the final comparison, the subcapsular islets had a labelling index twice as high as the intraportal islets. The reason for the superiority of the subcapsular space is not easy to ascribe, but it could be because of a better supply of well-oxygenized blood. It is worthy of note that local growth factors do influence the growth of grafted islets. Islets located either in the liver that is partially resected, or in the remaining kidney when unincphrcctomy is performed, grew concurrently with the surrounding parenchyme cells (Fig 4).';t In support of such a transfer of growth stimulation, Tafra et al showed a beneficial effect of fetal liver cells on the engraftment and function of fetal pancreatic grafts when implanted as a composite graft. II 6 Little is known about the rate of differentiation of fetal islet grafts implanted at different sites. 110st probably, this process is very much influenced by the

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Figure 4. Labelling indices of different tissues in shamoperated control (C) mice and in mice subjected to a partial hepatectomy (I'll). Values are means + SE~l for 7 animals in each group. Asterisks denoteP < O.Oj andP < 0.0 I, respectively, versus corresponding control tissue. m efficiency by which engraftment takes place. Revascularization and reinnervation of the graft arc two crucial mechanisms and both have been given considerable attention during recent years. Based on a microsphere technique introduced by Jansson and Hcllerstrorn for studies of islet blood flow regulation.!" it was found that persistent hyperglycemia after transplantation reduced the blood flow through the grafted islcts.l" Moreover, intraportally implanted islets revascularizcd from the arterial side, most probably by ingrowth of capillary sprouts emanating from the hepatic artery.l" It should be mentioned for the sake of clarity that all these studies have been performed with adult rodent islets. Knowledge of the ingrowth of new nerves into the implanted islets has so far been scarce, but it was recently shown that this process takes place at a slower rate than that of the rcvascularization.l" There were no nerve fibers to be found in adult mouse islets transplanted beneath the kidney capsule until the 6th to 10th week after implantation. Some of the observed nerve fibers contained tyrosine hydroxylase (TH) or neuropcptidc-Y (NPY) (markers for sympathetic nerves) and extended from the kidney parenchyma under the graft. Other fibers positive for vasoactive intestinal peptide (VIP) and acetyl cholinesterase (AchE) (presumably paras)mpathetic nerves) were derived from the capsule. Ongoing studies have shown that the islet reinnervation process is markedly influenced by the organ into which they had been implanted. Thus, intraportal and intrasplenic islets, in contrast to the subcapsular

30

Jindemon andSandler

ones, did not contain ill- and Nl'Ypositivc fibers, whereas VIP- and AchE-containing fibers were idcntified. ' 511 In contrast to what has been found in studies with adult islets, fetal pancreatic tissue seems already to contain certain neural elements at the time of implantation. These components do survive, as demonstrated by the presence of dopamine-beta-hydroxylase-positive nerves in fetal rat pancreatic tissue 32 days after transplantation into the anterior eyechambcr.!" In support of this idea, it was found that undifferentiated cells within porcine ICC grafted into nude mice developed into VIP-containing neurons.l'" By using species-specific antibodies, 'it was possible to demonstrate that these nerve fibers grew from the grafted ICC into the kidney parenchyma underneath. The functional significance ofthis apparent difference between adult and fetal tissue and for graft reinnervation in general is difficult to assess. Because the phenomenon as such is a fairly slow process, it is conceivable that influences on the long-term function are the most important. Thus, it is tempting to speculate that the recent demonstration oflong-term functional failure of intraportal rat islets might be explained by an impaired reinncrvation. 152

Immunogenicity of Fetal Pancreatic Tissue The issue has been raised whether fetal cells arc less immunogenic than adult cells and therefore, less prone to rejection following allotransplantation. 20,' 61, ' 63 Studies on the vulnerability of grafted fetal pancreas and adult islets to adoptive immunity in tolerant animals have indirectly supported this theory. For this purpose, diabetic Fischer rats made tolerant to Lewis antigens were cured by either a fetal graft or adult islets. Subsequently, they were given normal Fischer rat lymph node and spleen cells in order to break the tolerance. The animals with the fetal graft did not become diabetic, whereas those grafted with adult islets did. ' 61 This suggests that fetal f3-cells are less vulnerable to the cellular and molecular processes causing cell damage during allograft rejection. On the other hand, both fresh" and culturcd'f fetal rat pancreases were promptly rejected on allotransplantation. In addition, after allograft transplantation, freshly isolated fetal mouse pancreases were more actively rejected than adult mouse islets. ' 63 This was attributed to contaminating lymphoreticular cells in the mesentery surrounding

the mouse fetal pancreas. Even when fetal mouse pancreases were cultured for 10 days in 95% 0z' the tissue was rapidly rejected after transplantation.l'" However, when fetal mouse proislets were used, a prolonged, but not indefinite, allograft survival was obsenTd. ' 6I,' 61 Because the cells expressing class II DR-antigens are believed to trigger the primary signals for allograft rejection, the occurrence of such cells in fetal pancreatic grafts has been explored. Examinations of imrnunopcroxidasc-staincd sections ofHFP at 12 to 2+weeks gestation showed numerous I-ILA-DR antigen-expressing cells.' 65 One such cell type was large with a dendritic appearance and was distributed throughout the whole gland. A second cell type was also dendrite-shaped and was localized in close association with insulin-producing cells. Moreover, clusters of Dk-positivc cells, probably within small lymph nodes in the pancreas, were found to be T lymphocytes. Most endothelial cells seemed devoid of DRantigens. A subsequent study of the HFP endothelium until 18 weeks of age showed that Dlt-positivc endothelial cells were located in the intralobular connective tissue, but not in the pancreatic parenchyma.l" By 24 weeks, endothelium of medium-sized blood vessels expressed HLA-DR, whereas the endothelium of islet blood vessels and duct cells were Dk-negativc. The density of DR-positive cells increased about fourfold between 12 to 14 weeks and 18 to 22 weeks of gestation. Another study of class II antigen expression in HFP at 14 to 19 weeks of gestation not only demonstrated the occurrence of Dk-positivc cells in the connective tissue and pancreatic parenchyma, but also showed a small proportion of DP-positive cells appearing at 17 weeks."? However, these investigators did not observe any c1ass-IIpositive cells in the endothelium of the pancreas. Examinations of HFP graftcd nude mice demonstrated that after an initial increase in the amount of DR-expressing passenger leukocytes, the graft content of DR cells became greatly reduced by 32 weeks posttransplantation." In view of this, it was suggested that nude mice could act as interim hosts of I-IFP grafts in order to reduce the number of passenger leukocytes of a graft. Another study demonstrated that it was possible virtually to abolish the content of DR-expressing cells in HFP fragments by culture at a high oxygen tension.'?" Using stimulator cells obtained from monolayer cultures prepared from human ICC, it was reported that the response by human peripheral blood cells in mixcd-lymphoC)1e cultures was markedly reduced." However, it is

FetalPancreatic Transplantation

yet not known if either of the latter two procedures enhances the allograft survival after transplantation. In conclusion, the data indicate that the fetal pancreas contains class II antigen-expressing cells and that their number seems to increase with gestational age. It is possible to deplete the number of class II cells within the preparations with different in vivo or in vitro techniques including tissue culture. However, this property of the fetal pancreas is not different to that which has also been observed for adult pancreatic islets. Thus, it is unlikely that usc of the fetal pancreas will be advantageous when com:, pared with adult preparations because ofa decreased immunogenicity. Therefore, in clinical trials conventional immunosuppression must be considered mandatory, prodded no specific methods arc adopted to reduce the immunogenicity of the graft.

Clinical Experiences of Fetal Pancreas Transplantation Despite the considerable amount ofdata showing the capacity of fetal pancreatic grafts to correct experimentally induced diabetes, clinical trials reporting successful cure oflDD~1 patients, including reversal of late complications, with either fetal pancreatic allografts or xenografts arc difficult to evaluate. In many studies essential information has been lacking, eg, monitoring of the pretransplant diabetic state and details of the immunosuppressive regimens used. In several of these cases no immunosuppressive therapy seems to have been given. Because clinical fetal pancreas transplantations have not been internationally registered to the same extent as arc whole pancreas transplantations, it is difficult to estimate the total number performed. In 1988, Hering et al reported approximately 400 fetal pancreas transplantations.l" but as of December 1990, a total of 1,770 cases from 64 institutions had been rcportcd.!" The largest numbers of these transplantations are reported from Moscow (350 cases),17I,172 Kiev (316 cases),173 Riga (187 cases), Lvov (127 cases), and Shanghai (75 cases)."! Another 376 cases arc reported from 29 hospitals in China. There arc two circumstances worth noting concerning the Chinese transplantations: (I) the water-bag abortion method used may be less harmful to the fetal pancreas than other conventional abortion methods; and (2) very old fetuses, up to 34 weeks gestation, were used. At the Huddinge Hospital in Stockholm six patients given imrnunosupprcssivcs because of previous

31

kidney grafts were grafted intraportally with various numbers ofHFP fragrnents.l" In one patient, C-peptide was noted in the urine and reached a level of about 5% of normal after 4 months, but then disappeared. Interestingly, at that time, increased titers of islet cell surface antibodies were found in the blood of the recipient. Two other immunosuppressed patients received intraportal injections of human ICC obtained from 3 and II HFP, respectively. However, over a fi-month period, no signs of graft function were observed." jovanO\ic-Peterson et al observed a significant C-peptide production starting after 5 days and having a duration of 12 months following transplantation of pooled HFP glands into skeletal muscles of IDD~1 patients who received no immunosuppression.i" However, Valente et al failed to observe function in HFP grafted to an intramuscular site in nonimmunosuppressed paticnts.!" Recently, Yanhu et al reported that 7 of 10 IDD~1 patients had stopped insulin therapy after intracerebral transplantation of 7 to 10 cultured HFP glands.!" Some of these patients received dexamethasone treatment for 6 to 10 days to avoid cerebral edema, but otherwise no immunosuppression was given. In a clinical trial with three out of four patients receiving immunosuppression and grafts of cultured HFP in their forearm muscle, Tuch et al observed evidence of C-peptide production in one of the patients at 3 months, but not thcrcaftcr.t" Biopsies performed on the graft of one patient at 9 to 13 months after transplantation showed various stages of rejection, some pancreatic duct tissue, and a few l3-cells. lllO Usadel et al failed to observe any evidence ofHFP graft function in a diabetic paticnt.!" Voss et al reported data on 25 patients who had been either concurrently grafted with a kidney, or grafted after a kidney implant with HFP cells produced in culture. IH2,183 Low C-peptide levels were observed in ll patients in at least one sample point. However, it seemed as if the grafts had no major impact on the glucose homeostasis. The cells and the culture technique used for these transplantation attempts were not clearly described. In an ongoing clinical study at the Huddinge Hospital, Sweden, attempts have been made to usc porcine ICC for treatment oflDD~1 in patients with established kidney grafts."! So far, five patients have each been intraportally injected with material from 30 to 50 fetuses. In order to maintain a rigid blood glucose control, insulin was admininstered by con tin-

32

Andersson andSandler

uous intravenous infusion for 2 weeks. Initially, the doses of Cyclosporin A, azathioprine, and prednisolone were increased, but were later tapered off AIl patients have tolerated the procedure well and no side effects have so far been recorded. There was evidence to indicate engraftment of the injected ICC, because IgG anti-pig antibodies were noted at abou t 10days after transplantation and later reached a plateau level in all patients. Moreover, in one patient, urinal)' excretion of porcine C-peptide was detected after 2 months, and a peak value of about 4,000 pmol/24 hr was observed after 2Y:! months. The excretion then varied, but C-peptide was still present in the urine 9 months posttransplant. Significant levels of porcine C-peptide in the serum were detected in this patient during the lst, 2nd, and 3rd posttransplantation months.

Fetal Versus Adult Islet Tissue At present, the choice between using fetal or adult islet tissue for clinical transplantation purposes seems quite an easy one to make. There are, as previously described, a few diabetic patients who achieved insulin independence after adult islet transplantation. Comparative results and experience concerning the usc of fetal pancreatic tissue arc essentially lacking. If the perspectives for wider usc of islet transplantation techniques arc considered, it is clear that there arc beneficial features of both sources of tissue. A comparison of the critical features of the two available types ofislet tissue is listed in Table I. It is immediately evident that fetal tissue, especially when processed into ICC/proislets, has a number of potential advantages versus adult islet tissue. The

Table 1. Comparison Between the Usefulness oflslet Grafts of Fetal or Adult Origin

Feature Growth capacity Preparation procedure Implantation volume Tolerance to ischemia Purity of graft Immunoalteration/ isolation Availability Time to cure Monitoring of rejection Ethical constraints

FetalIslets

Adult Islets

+++ +++ +++ ++ ++

+

-- (+++)

+

- (+++) ++

++ - - - (+)

- (+)

Note: Degrees of advantage are denoted by plus (+, + +, + + +) signs; degrees of disadvantage are denoted by minus (-, --, - - -) signs; parentheses denote use ofxenogeneic islet material.

preparation procedures arc simpler and result in smaller and purer tissue volumes for implantation. These two latter features arc most certainly of crucial importance for the safety of the recipient. It is also obvious that with the poor development of exocrine tissue in the fetal gland it tolerates ischemia better than the adult gland. The two major problems in the usc of fetal tissue arc: (I) it acts slowly to reverse diabetes; and (2) its availability, which is partly linked to ethical constraints. In resolving the first problem, it may be helpful to be aware of the fact that the difference between fetal and adult islets is possibly not that great. In their most successful case, \Varnock et al administered exogenous insulin to their patient for about 10 weeks to avoid hyperglycemia and thereby protect the islet graft." Under such a therapeutic regimen, it is not easier to detect rejection ofan adult islet graft than iffetal islets had been implanted, nor was it possible to follow the engraftment process by monitoring the acute normalizing effect on the diabetic state. Therefore, it seems the drawback of the functional immaturity of fetal tissue is not as detrimental when compared with adult tissue and, thus, the scoring in Table I may have been more equal. Without doubt, the many ethical and practical problems associated with the usc of human fetuses for research have led to a shortage of fetal donor organs. Although less of an ethical dilemma, there is an insufficiency in the number of donated adult organs as well. In the United States there arc presently 3,500 human organ donors per year, but about 10,000 to 15,000 new cases of IDD~1.'1l3 The corresponding figures for Sweden arc presently about 100 suitable donors and 800 newly diagnosed IDD~l patients per year. For both types of procedures the successful usc of xenogeneic material has to be the ultimate solution of the problem. Thus, ethical constraints probably \\111 be reduced. Indeed, the experiences from our clinical trials with fetal porcine ICC transplantations in five patients arc positive in this respect.

Conclusion Experimental studies, mainly in rodents, have shown that islets arc effective in curing diabetes irrespective of donor age. Encouraging support has recently been gained for the continuation of clinical trials of adult islet transplantation when insulin independence was achieved in a few diabetic patients allotransplanted

FetalPancreatic Transplantation

with isolated islets. However, there are still two major problems to be solved; namely, the supply of donor tissue and the prevention of rejection, both of which could be successfully surmounted if efficient islet immunoisolation procedures became available and made possible the use ofxenogeneic material. In the future, it may be found that adult and fetal islet grafts do not differ much in effectiveness for successful immunoisolation, but it is noteworthy that the fetal islets must have the opportunity both to grow and to mature in vivo inside the insertion device. While waiting for the clinical introduction of such immunoprotection procedures, it might be worthwhile to pursue trials with xenogeneic islet grafts. Thus, the overwhelming problem with hyperacute rejection of xenografted vascularized organs should be csscntally obviated with islet grafts because animal studies have shown that they revascularizc with capillaries emanating from the arteries of the recipient. Indeed, experience from ongoing clinical trials (previously discussed) is in support of this theory, although a delayed anti-pig antibody response may constitute a major problem. The use of new immunosuppressive drugs such as 15-deoxyspergualin and RS-61443 that are more efficient in suppressing B lymphocytes "ill be of great interest in this area.

Acknowledgment \\'e are thankful to ~Is. Agneta Snellman for secretarial assistance, and to Drs. Claes Hellerstrom, C.G. Groth, and Leif Jansson for reviewing the manuscript.

References I. Lillehei RC, Idezuki Y, Feemster jA, et al: Transplantation of stomach, intestine and pancreas: Experimental and clinical observations. Sugery 1967,62:721 2. Goetz FC, Moudry-Munns K, Sutherland DER: Wholeorgan pancreas transplantation in the 19905.Clin Diabetes 1991,9:33 3. Pyke D: Pancreatic transplantation for type I diabetes. Lancet 1990, 335: 1538 4. Hellerstrom C: A method for the microdissection of intact pancreatic islets of mammals. Acta Endocrinol (Copenh) 1%1,45:122 5. Moskalewski S: Isolation and culture of the islets of Langerhans of the guinea-pig. Gen Comp Endocrinol1965, 5:342 6. Lacy PE, Kostianovsky ~I: Method for the isolation of intact islets of Langerhans from the rat pancreas. Diabetes 1967, 16:35 7. Ballinger \\1:, Lacy PE: Transplantation of intact pancreatic islets in rats. Surgery 1972,72:175 8. Scharp DW, Lacy PE, Santiago jV, et al: Insulin independence after islet transplantation into type I diabetic patient. Diabetes 1990,39:515

33

9. Scharp DW, 1-'11:}' PE, SantiagojV, ct al: Results of our first nine intraportal islet allografts of type I, insulin diabetic patients. Transplantation 1991,51:76 10. Warnock GL, Kneternan i'\~I, Ryan E, et al: Normoglyccmia after transplantation of freshly isolated and cryopreservcd pancreatic islets in type I (insulin-dependent) diabetes mellitus. Diabetologia 1991,34:34 I I. Gray DWR, ~lcShane 1', Grant A, et al: A method for isolation of islets of Langerhans from the human pancreas. Diabetes 1984,33: 1055 12. Ricordi C, Lacy PE, Finke EH, et al: Automated method for isolation of human pancreatic islets. Diabetes 1988,37:413 13. Ssobolcw LW: Zur norrnalen und patologischen Morphologic dcr inneren Secretion der Bauchspcicheldruse. Virchows Arch [A] 1902, 168:91 H. Hegre OD, Lazarow A: Islet transplantation, in Volk B\\', Wellman KF (eds): The Diabetic Pancreas. Plenum, i'\ew York,1'."Y 1977,pp517-550 15. Brown}, Molnar IG, Clark W, et al: Control of experimental diabetes mellitus in rats by transplantation of fetal pancreases. Science 1974, 184:1377 16. Mullen YS, Clark \\'R, Molnar IG, et al: Complete reversal of experimental diabetes mellitus in rats by a single fetal pancreas. Science 1977, 195:68 17. ~IcEyoy RC, Schmitt RV, Hegre OD, et al: Syngeneic transplantation of fetal rat pancreas. I. Effects of insulin treatment on the reversal of alloxan diabetes. Diabetes 1978, 27:982 18. ~lcEyoy RC, Hegre Ol): Syngeneic transplantation of fetal rat pancreas. II. Effect of insulin treatment on the growth and differentiation of pancreatic implants fifteen days after transplantation. Diabetes 1978,27:988 19. ~lcEyoy RC, Hegre OD: Syngeneic transplantation of fetal pancreas. III. Effect of insulin treatment on the growth and differentiation of the pancreatic implants after reversal of diabetes. Diabetes 1979,28: HI 20. Garvey jF\\', Morris PJ: Early rejection of allogeneic foetal rat pancreas. Transplantation 1979,25:342 21. ~Iillard PR, Gan'eyjFW,jeffel)' EL, et al: The grafted fetal rat pancreas.Amj Patho11980, 100:209 22. Mandel TE, Collier S, Carter W, et al: Effect of in vitro glucose concentration on fetal mouse pancreas cultures used as grafts in syngeneic diabetic mice. Transplantation 1980, 30:231 23. Hoffman L, Mandel TE, Carter W: Insulin content of fetal mouse pancreas in organ culture and after transplantation. Diabetes 1982,31 :826 24. Sasaki i'\, Yoncda K, BiggerC, et al: Fetal pancreas transplantation in miniature swine. Transplantation 1984,38:335 25. Mullen Y: Fetal pancreas transplantation for treatment of type I diabetes: Xliniaturc swine model, in Peterson C~I, jo\
34

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p an creas after inducti on of d iabet es. Transplantation 1988, -1 6:608 29. Tuch BE: From nude m ouse to m an , in Peterson C~I , jovanO\ic-Peterson L, Formby B (eds ): Fet al Islet Tran spl antation . Impl ication s for Diabet es. Sp ring er -Ve rlag, New York, ;\Y , 1988, p p 127-16-1 30. H ellcrstrom C , Lewis ;g, Bor g II , et a l: M ethod for large sca le iso la tio n of pancre at ic isle ts by tissu e culture offet al rat pancreas. D iabetes 1979, 28:76 31. :-IcEvoy RC, Leung I' E: T ransplanta tio n of fetal rat islets in to th e ce re bra l ve nt ric les ofa lloxan-diabe tic rats. Amelioration of diabetes by syn gen eic but not allogeneic islet s. D iabetes 1983,32:852 32. Kruszynska YT, Home I'D, Alberti KG~l\l: Comparison of portal and peripheral insulin deliv ery on carbohydrate metabolism in streptozotocin-diab eti c rat s. Diabctologia 1985, 28 :167 33. S im eonovic Cj, Lafferty Kj : The isola t ion and transplantation of fetal m ouse proislets . Australas ] Exp Bioi Med Sci 1982, 60:383 3-l. Korsgr cn 0, J ansson L, Eizi rik D, ct a l: Fu ncti onal a nd m orphologi cal d ifferen tia tion of fer al porcine islet-like ce ll clu st e rs after transplantation in to n ud e m ice. D iabetologia 1991 ,34:279 35. Liu X , Federlin KF , Bretzel RG , ct a l: Persistent reversal of di a bet es by t ra nspl ant ati on of fet al p ig proislet s into nude mi ce. D iab et es 1991,-10:858 36. Eckhoff DE, Sollinge r HW, Hu llet D:\: Sel ect ive enhancem e nt of 13 cell a ct ivity by p rep a rati on of fetal pan cre atic p roi slets and culture wi th insulin growt h fa ctor I. Tran splantation 1991 ,51 :1161 37. Pict et R , Ruit er ,\]: D evelopm ent of the emb ryo nic endocr ine pa ncreas in St ein e r D F, Frcinkcl N (ed s) H andboo k of Physi ology, Am Phys iol Soc, Washingl on , DC, 1972, pp 25- 66 38. Fuji S: D evelopment of pa ncreat ic en docrine cells in the rat fetus. Arch Ilistol Cy1011979, -1 2:,167 . 39. Yoshinari ~I, Daik oku S: Ontogen eti c a ppearance ofimmunore active endocrine cells in rat pan cr eatic islets. Anat Ernbryol (Berl) 1982, 165:63 -10. S andler S, Andersson A, Swenne I, et al: Culture and cryopreservation of fetal e ndoc r ine pancreas, in Peterson C~I, jovanO\ic-Peterson L, For m by B (eds): Fetal Islet Transplantation. Implicat ion s for D iabet es. Springer-Verlag, 1\"ewYork,;\Y,1988,pp 9-4 2 4l. Teite lma n G , LeejK.: C ell linea ge an al ysis of pancreatic islet ce ll devel opm ent : Glucago n and ins u lin ce lls arise from cat echolamlnergic pr ecu rsors p re sent in th e pan cr eatic du ct. D evBio11987, 121 :-I;}~ ·~2 . Korsgr en 0 , Sa ndle r S, Sc h ne ll La nd strom A, et al: La rgesca le product ion of fetal porcine pancre a tic islet-like cell clu sters: An experimental tool for studies o f isle t ce ll d iffer entiation a nd xenotrans plant a tion, T ransplan tati on 1988,45:509 ·~3 . Thom pson SC, ~ land el TE: Fet a l pig pa nc reas. Preparation an d a ssessment of t issue for transplantation , and its in vivo developm ent and fun ction of a thymic (n ud e) m ice. Transplantation 1990,49:571 -H. W ilsonjD, Simeonovi c Cj, T ing j H L, et a l: Rol e of T '-lymphocyt es in rej ecti on by o f feta l pig proislet x enogr a fts. Dia bet es 1989,38:217 (suppll) 45. S im pso n A.\I , Tuch BE , Vincent PC: Pig fet al pancreatic rnon olaye rs, Transpl a nt ation 1990, 49:1 133 46. Alum et s ], Hfikanson R, S undlcr F: O nt ogeny of endocrine

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cur

65.

cel ls in porcine gu t a nd pa ncrea s. An immun ocytochemical s tu dy. G astroenterology 1983,85:1359 Stefan Y, Gras so S, Perrelet A, e t al: Q uanritati vc immunoflu ore sce nt st udy of th e endocrine ce ll populations in the d eveloping human pancr ea s. D ia be tes 1983,32:293 Clark A, G rant A.\I : Quan titative m orphology o f endocrin e ce lls in human fet al pan cr ea s. Diabctologia 1983, 25 :31 Korsgr en 0, Lukiniu s A, Sc h ne ll R, ct al: T he major islet horm ones a re co- loca lized within th e secre tory g ra nu lae in the fet al po rci ne pancreas . Diabet ologia 1990, 33:A388 (abstr) Al pert S, H anahan D, Te itel rnan G : H ybr id ins u lin gen es r eveal a d evel opment lin ea ge for pancreatic e ndoc ri n e cells and imply a relationship with n eu ron s. C ell 1988,53:295 Sandler S, Andersson A, Schnell A, ct a l: Ti ssue culture of human fetal pancreas: Development and fun ction ofB-cells in vitro and transplantation of explants to nude mice. Diab etes 1985, 3-1:1113 Mand el TE, G eorgiou H~I: In suli n secret ion by fetal human pa nc reatic islets of Langcrhan s in p ro lon ged or gan culture. D iab et es 1983, 32:915 Lafferty Kj, Trujillo S: In vitro a nd in vivo behaviour of human fet al pa ncreas (Hl'P), in Bri dges 1..\1, C ad es j S, Lu skin HA (ed s):llle Use o f Hu ma n Tissues a nd Organ s for Research and Transpl ant. Nat ional Diabe tes Research Interch an ge, University City Science C e nt e r, Ph iladelphia, PA, 1986, pp52-iO Oton koski T , Knip ~I , Panula P, e t a l: ~Iorphology, yield and fu nct ion al integrity of islet -like ce ll cluste rs in tissue cu lt ure of human fe tal pancreat a ob tained a fte r differ ent m eans of abortion. Act a Endocri nol (C op en h) 1981l, 118:68 Sand ler S, Ander sson A, Schnell La ndstrom A, et al: Tissue culture of human pancreas : Effects of human se ru m on t he d evel opment of isle t-like cell clu st ers. Di abetes 1987, 36: HOI Le ach F1\", Ash worth :-IS, Barson ,\} , e t a l; Insulin rel eas e from h uman foet al pan cre as in tissue cu ll ure. j Endocrinol 1973, 59:65 Agr en A, Anders son A, Bjork cn C, e t a l: Human fetal pa ncr eas. Culture and function in vivo . D iab etes 1980,29:61 (suppll) A uerbach R: Morphogenetic int er ac tions in the d evelopment of th e mouse thymus gland. De\'!lioI1 960, 2:27 1 Fuj im oto WY, Williams RH : In sulin rel eas e from cultured human fetal pancreas. Endocrinology 1972,91: 1133 ~la itlandjE, Parry DG, Turll ejR: Perifu sion a nd cult ur e of human fet al p an cre as . Di abet es 1980,29:57 (suppl I) C oll ier S, Mand el TE, H offm an 1., e t al: O rgan cu lture of fct nl m ou se pancr eas . The effec t of cu lt ure. co ndit ions on insulin a nd glucagon secretion . Diabet es 1981, 30:804 Ho ffm an 1.., Mandel TE, Carter W~I, et a l: Insul in secretion by fe tal human pancrea s in orga n cu lture. D ia bctologia 1982, 23 :426 Tuch BE,jones A, TurtlejR: Maturat ion of the response of human fetal p ancreatic explan ts to glucose. Diab etol ogia 1985, 28:28 Ande rsson A, Christ en sen 1\", Grot h C oG, c t a l: Survival of h u man fet al p an creat ic explants in organ cul ture a s refl ected in ins u lin secre tion and oxygen co nsu mption . Transplantation 1 98~ , 37 :-199 ~l a it landjE, C a ter son !D, Ga uci RE, e t a l: Orga n cult ur e of hu m an foetal pancreas: Conditions whic h a ffect basal a nd st imulated in sul in rel ea se. Acta E ndoc ri no l (Copenh) 1985, 108:377

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66. Tuch BE,jones A, J\g ABT, et al: Characteristics of human fetal pancreas cultured in vitro, Transplant Proc 1986, 18:322 67. Dudek RW, FreinkelJ\, Lewis i'\J, et al: Morphologic studyof cultured pancreatic fetal islets during maturation of the insulin stimulus-secretion mechanism. Diabetes 1980,29:15 68. Masquelier D, Amory B, Mourrneaux jl., et al: Cell interactions during the in vitro neoformation offetal rat pancreatic islets. Cell Differentiation 1986, 18:199 69. Rabinovitch A, Russel T, Mintz DH: Factors from fibroblasts promote pancreatic islet B cell survival in tissue culture. Diabetes 1979,28: 1108 70. Montesano R, Mouron P, Amherdt ~I, et al: Collagen matrix promotes reorganization of pancreatic endocrine cell monolayers in islet-like organoids.j Cell Bioi 1983, 97:935 71. Hegre Ol), Simeonovic C], Lafferty Kj: Syngeneic transplantation of cryoprcserved fetal mouse proislets. Diabetes 19(H, 33:975 72. Espinosa de los Monteros ~IA, Driscoll SG, Steinkej: Insulin release from isolated human fetal pancreatic islets. Science 1970,168:111 73. Goldman H, Colle E: Human pancreatic islets in culture: Effects of supplementing the medium with homologous and heterologous serum. Science 1976, 192:10H 74. Goldman HY, Wong I, Patel YC: A study of the structural and biochemical development of human fetal islets of Langerhans. Diabetes 1982,31 :897 75. Kover K, Moore W: Development of a method for isolation of islets from human fetal pancreas. Diabetes 1989,38:917 76. ~lcEvoy RC, Thomas J\~I, Bowers C, et al: Maintenance of fetal human pancreatic beta cells in tissue culture. Med Bioi 1986, 64:271 77. Simpson A.\I, Tuch BE, Vincent PC: Characterization of endocrine-rich rnonolayers of human fetal pancreas that display reduced immunogenicity. Diabetes 1991,40:800 78. Mazur P: Freezing of living cells: Mechanisms and implications. Amj Physiol1984, 247:C 125 79. Mazur 1', KempjA, Miller RH: Survival of fetal rat pancreas frozen to -79° and -196°C. Proc J\atl Acad Sci USA 1976, 73:4105 80. KempjA, Mazur 1',Mullen Y, et al: Reversal of experimental diabetes by fetal rat pancreas. I. Survival and function offetal rat pancreas frozen to -196"C. Transplant Proc 1977,9:325 81. KempjA, Mullen Y, Weissman II, et al: Reversal ofdiabetes in rats using fetal pancreas stored at -196°C. Transplant Proc 1978, 26:260 82. Rajotte RV, Mazur P: Survival of frozen-thawed fetal rat pancreas as a function of the permeation of dimethylsulfoxide and glycerol, warming rate and fetal age. Cryobiology 1981,18:17 83. BrO\\11j, KempjA, Hurt S, et al: Cryopreservation of human fetal pancreas. Diabetes 1980,29:70 fl.!. KempjA, Hurt SX, Brown}, et al: Recovery and function of human fetal pancreas frozen to -196°C. Transplantation 1981,32:10 85. Sandler S, Andersson A, Hellerstrorn C, et al: Preservation of morphology, insulin biosynthesis and insulin release of cryopreserved human fetal pancreas. Diabetes 1982,31 :328 86. Sandler S, Andersson A, Swenne I, et al: Structure and function of human fetal pancreas before and after cryopreservation, Cryobiology 1983,20:230 87. Shiogama T, Mullen Y, Klandorf H, et al: An improved

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cryopreservation procedure for human fetal pancreas tissues. Transplantation 1987,44:602 88. Rajotte RV, Warnock GL, Bruch LC, et al: Transplantation of cryoprcservcd and fresh rat islets and canine pancreatic fragments: Comparison of cryopreservation protocols. Cryobiology 1983,20: 169 89. Huller DA, Bethke KP, Landry AS, et al: Successful longterm cryopreservation and transplantation of human fetal pancreas. Diabetes 1989,38:448 90. Dawidson I, Simonsen R, Aggarwal S, et al: Cryopreserved human fetal pancreas: A source of insulin producing tissue? Cryobiology 1988, 25:83 91. ~Iandel TE, Carter W~I: Cryopreservation and transplantation of organ-cultured fetal islets. Transplant I'roc 1984, 16:842 92. I1egre Ol), Leonard Rj, Schmitt RV, et al: Isotransplantation of organ-cultured neonatal pancreas: Reversal of alloxan diabetes in the rat. Diabetes 1976,3:180 93. Moore GE, Gerner RE, Franklin HA: Culture of normal human lcukocytcs.] Am ~Ied Assoc 1967, 199:8792 94. Andersson A: Isolated mouse pancreatic islets in culture: Effects of serum and different culture media on the insulin production of the islets. Diabetologia 1978, 14:397 95. ~IcEvoy RC, Leung I'E: Tissue culture of fetal rat islets. Comparison of serum-supplemented and serum-free, defined medium on the maintenance, growth and differentiation of A, Band D cells. Endocrinology 1982, III: 1568 96. Kinard F, deClerq L, Billen B, et al: Culture of endocrine pancreatic cells in protein-free, chemically defined media. In Vitro Cell Dev Bioi 1990, 26:100l 97. Korsgren 0, Andersson A, Sandler S: Pretreatment of fetal porcine pancreas in culture with nicotinamide accelerates reversal of diabetes after transplantation to nude mice. Surgery (in press) 98. Andersson A, Sandler S, Hellerstrom C, et al: Effects of amniotic fluid on the development of human fetal pancreatic B-eells in tissue culture. Transplant Proc 1986, 18:57 99. Swenne I: TIle role ofglucose in the in vitro regulation of cell cycle kinetics and proliferation of fetal pancreatic B-eells. Diabetes 1982,31:75l 100. Swenne I: Glucose-stimulated DXA replication of the islets during the development of the rat fetus. Effects of nutrients, growth hormone, and triiodothyronine, Diabetes 1985,34: 803 101. Nielsen DA, Welsh ~I, Casadaban ~U, et al: Control of insulin gene expression in pancreatic l3-eells and in an insulin-producing cell line, RI;'\-5F cells. I. Effects of glucose and cyclicA.\lP on the transcription of insulin mRt'\'A.j Bioi Chern 1985, 260: 13585 102. Welsh ~I, Nielsen DA, ~lacKrell Aj, et al: Control of insulin gene expression in pancreatic l3-eells and in an insulinproducing cell line, RI;,\-5F cells. II. Regulation of insulin mRt'\'A stability.} BioI Chern 1985,260: 13590 103. Dudek RW, Kawabc T, Brinn jE, et al: Glucose affects in vitro maturation of fetal rat islets. Endocrinology 1981, 114:582 HH. Collier S, Mandel TE, Hoffman I., et al: Organ culture of fetal mouse pancreas. The effect of culture conditions on insulin and glucagon secretion. Diabetes 1981, 30:81}l 105. Hellerstrom C, Swenne I: Growth pattern of pancreatic islets in animals, in Volk BW, Arquilla ER (eds): TIle Diabetic Pancreas. Plenum, ;'\ew York, xv 1985, PI' 53-79

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106. l\eilsenJH: Growth and function of the pancreatic 11 cell in vitro. Effects of glucose, hormones and serum factors on mouse, rat and human pancreatic islets in organ culture. Acta Endocrinol (Copenh) 1985, 108:I (suppI266) 107. Dudek RW, Kawabe T, Brinn JE, et al: Effects of gro\\th hormone on the in vitro maturation of fetal islets. Proc Soc F.xp BioI :\Ied 198\, 177:69 108. l\ielsenJH: Effects of growth hormone, prolactin and placcntal lactogen, on insulin content and release, and dcoxyribonucleic acid synthesis in cultured pancreatic islets. Endocrinology 1982, 110:600 109. Rabinmitch A, Quigley C, Rechler :\1:\1: Growth hormone stimulates islet B-cell replication in neonatal rat pancreatic monolayer cultures. Diabetes 1983,32:307 110. Sandler S, Andersson A, Korsgren 0, et al: Tissue culture of human fetal pancreas: growth hormone stimulates the formation and insulin production of islet-like cell clusters. J Clin Endocrinol :\Ietab 1987,65: 115-\ Ill. Otonkoski T, Knip :\1, Wong I, et al: Effects of growth hormone and insulin-like growth factor I on endocrine function of human fetal islet-like cell clusters during longterm tissue culture. Diabetes 1988,37: 1678 112. Formby B, Ullrich A, Cousscns L, et al: Growth hormone stimulates insulin gene expression in cultured human fetal panereat ic islets.J Clin Endocrinol :\letab 1988,66: 1075 113. Korsgren 0, Sandler S,Jansson L, et .'II: Effects of culture conditions on formation and hormone content of fetal porcine isletlike cell clusters. Diabetes 1989,38:209 (suppll) 114. Swenne I, Hill DJ, Strain I\], et al: Growth hormone regulation of somatomedin C/insulin-like grm\th factor I production and D:'\A replication in fetal rat islets in tissue culture. Diabetes 1987,36:288 115. Hill DJ, Frazer A, Swenne I, et al: Somatomedin-C in the human fetal pancreas: Cellular localization and release during organ culture. Diabetes 1987,36:-165 116. Tafra L, Berezniak R, Dafoe DC: Beneficial effects of fetal liver tissue on fetal pancreas transplantation. Surgery 1990, 108:73-1 117. Sorenson RL, Brelje TC, Hegre OD, et al: Prolactin (in vitro) decreases the glucose stimulation threshold, enhances insulin secretion, and increases dye coupling among islet B cells. Endocrinology 1987, 121:2+17 118. BreljeTC,Allaire P, Hegre 0, et al: Effect of prolactin versus growth hormone on islet function and the importance of using homologous mammosornatotropic hormones. Endocrinology 1989,125:2392 119. Brelje TC, Sorenson RL: Role of prolactin versus growth hormone on islet B-cell proliferation in vitro: implications for pregnancy. Endocrinology 1991,128:-15 120. Swennc I, Hill DJ, Strain I\], et al: Effects of human placental lactogen and growth hormone on the production of insulin and somatorncdin C/insulin-like growth factor I by human fetal pancreas in tissue culture.J Endocrinol1987, 113:297 121. Ueda K, Hayashi 0: ADP-ribosylation. Annu Rev Biochern 1985,51:73 122. Yonemura Y, Yakashima T, :\Iiwa K, et al: Amelioration of diabetes mellitus in partially depancreatized rats by poly(ADP-ribose)sYllthetase inhibitors. Evidence of islet B-cell regeneration. Diabetes 198-\,33:-101 123. Vague I'll, Vialettes B, Lassmann-Vague V, et .'II: l\icotinamide may extend remission phase in insulin-dependent diabetes. Lancet 1987,1:619

12-1. Sandler S, Andersson A: Long-term effects of exposure of pancreatic islets to nicotinamide in vitro on D:'\A synthesis, metabolism and B-cell function. Diabetologia 1986,29:199 125. Sandler S, Andersson A: Nicotinamide treatment stimulates cell replication in transplanted pancreatic islets. Transplantation 1988,-16:30 126: Sandler S, Andersson A, Korsgren 0, et al: Tissue culture of human fetal pancreas. Effects of nicotinamide on insulin production and formation of islet like cell clusters. Diabetes 1989,38: 168 (suppl I) 127. Asplund K: Dynamics of insulin release from the foetal and neonatal rat pancreas. EurJ Clin Invest 1973,3:338 128. Freinkel N, Lewis !\J,Johnson R, et al: Differentia! effects of age versus glycemic stimulation on the maturation of insulin stimulus-secretion coupling during culture of fetal rat islets. Diabetes 198-1,33:1028 129. Rorsman P, Arkharnrnar 1', Bokvist K, et al: Failure of glucose to elicit a normal insulin secretory response in fetal pancreatic beta cells results from glucose insensitivity of the ATI'-regulated K+ channels. I'roc Natl Acad Sci USA 1989, 86:-1505 130. Hole RL, Pian-Smith :\IC:\I, Sharp GWG: Development of the biphasic response to glucose in fetal and neonatal rat pancreas. AmJ Physiol1988, 251:E167 131. Tuch BE, Jones fl., Turtle JR: Human fetal pancreatic explants: Maturation ofresponse to glucose. Transplant Proc 1985,17:-\02 132. Otonkoski T: Insulin and glucagon secretory responses to arginine, glucagon, and 'theophylline during peri fusion of human fetal islet-like cell clusters.J Clin Endocrinol Metab 1988,67:731 133. Otonkoski T, Andersson S, Knip :\1, et al: Maturation of insulin response to glucose during human fetal and neonatal development. Studies with perifusion of pancreatic iseletlike cell clusters. Diabetes 1988,37:286 \3-1. Milner RDG, Barson 1\], Ashworth :\L\: Human foetal pancreatic insulin secretion in response to ionic and other stimuli.J EndocrinoI1971,51:323 135. Tuch BE, Williams PF, Handelsman D, et al: Effect of phorbol and glucose on insulin secretion from the human fetal pancreas. Life Sci 1987,-10:H05 136. Tuch BE, Palavidis Z, TurtleJR: Activators of proteine kinase C stimulate insulin secretion from the human fetal pancreas. Pancreas 1988,3:675 137. Tuch BE, Osgerby KJ, Turtle JR: Chronic stimulation of human fetal pancreas with phorbol inhibits insulin secretion. Biochem Biophys Res Commun 1988, 15:1269 138. :\lounneauxJL, Remacle C, HenquinJC: Effects ofstimulation of adcnylatc cyclase and protein kinase-C on cultured fetal rat B-cells. Endocrinology 1989, 125:2636 139. Tuch BE, Grigoriou S, TurtleJR: Effect of normoglycemia on the functional maturation of the human fetal beta cell. Pancreas 1989, -1:587 1-10. Cuthbertson RA, Koulmanda :\1, Mandel TE: Evidence that chronic diabetes is detrimental to growth and function of fetal islet isografts in mice. Transplantation 1988, -16:650 HI. Gillies :\IC, Mandel TE: Evidence that chronic hyperglycemia in mice does not adversely affect fetal islet cell allograft function. Transplantation 1989, -18:523 1-12. Tuch BE, Lcnord KA: Insulin is advantageous to the growth of human fetal pancreas after its implantation. Transplant Proc 1989, 21:3803

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1-13. Sandler S,Jansson L: Blood flow measurements in autotranspla nted pan crea tic isle ts of t he ra t: Im pairm ent of the blood perfusion of the g ra ft during prolonged hype rglycem ia. ] Clinlnvesl1987,BO:17 1+1. Gray D\\'R, Cranston D, ~lcShan e 1', et al: The effect of hyperglycaernia on pancreat ic islets tr an splanted into rat s ben eath the kidn eycapsule. D iabetologia 1989,32:663 H 5. Leiter Ell: Analy sis ofd ifferential survi va l of syn geneic islets transplanted into hyperglycemi c C57BL/6] versus C57BLI KsJ mice. Transplantation 19R7,44:-102 H 6. Sandler S, Jansson L, Korsgren 0, et al : Engraftrnent of isolated islets after syn geneic transplantation into normal and diabetic mice. Transplant Proc (in pr ess) 1-17. Korsgren 0, Jansson L, Sandler S, ct al: Hyperglycemiainduced B cell toxicity, The fate of pancreatic islets transplanted into diabetic mice is dep endent on their genetic background.J Clin Invest 1990, 86:2161 H 8. Brown ], ~Iullen Y, Clark \\'R, ct al: Importance of hepatic portal circulation for insulin action in streptozotocin-diabctic rat s transplanted with fetal pan cr eascs v] Clin Invest 1979, 6 kl688 H9. Reece-Smith H, ~[cShan e 1', ~[orris [:1: Glucose and insul in changes following a rcnoportal shunt in s trcptozotocin diabetie rats with pancreatic isogr afrs under th e kidney' capsule. Diabetologia 1982, 23:3n ISO. R ecce-Smith H, DuToit DF, ~l cShane P, c t al ; Prolonged survival and pancreatic islet allografts transplanted ben eath the renal cap sul e. Transplantation 19BI, 32:305 151. L, cr PE, Finke Ell, J anney CG, ct al: Prol on gation of islet xen ogr aft survival by in vit ro culture of rat rncgaislets in 95% 0 /. Transplantation 1982 ,33:588 152. Hiller WFA, Klernpnaucr ], Luck R, et al; Progressive d eterioration of endocrine function after iniraportal but not kidn ey subcapsular rat islet transplantation- Diabetes 1991, 40: 13 1 153. ~Icllgren A, Schnell Landstrom A, Petersson B, et al: The renal subcapsular sil l' offers better growth conditions for transplanted mouse pancreatic islet cells than the liver or spleen. Diabetologia 1986, 29:6iO 15t Dungcr A, Korsgren 0, Andersson A: D:"A replication in mouse pancreatic islets tran splanted subcapsularly into the kidney and intraportally int o th e liver. Transplantation 1990, 49:686 155. Jansson L, Hellerstrom C : Stimulation by glucose of th e blood now to the pancreat ic islets of the rat. Diabetologia 1983,25:45 156. And ersson A, Korsgrcn 0 , J an swn L: Intraportally transplanted pa ncreatic islets rcva scular izcd fro m hepatic arteria l system.Diabet es 19l19,38:192 (suppl J) 157. Korsgren 0 , And ersson A,J ans son 1.,et al: Reinnervation of syngene ic mouse pancreatic islet s tr ansplant ed into the renal subcapsular space. Diabetes (in pr ess) 158. Korsgren O,Jansson 1.,Ande rsson A, et a]: Reinnervation of transplanted pancreatic islets : A compari son bel\,'een islet s impl ant ed into the kidn er, spleen o r liver . T rallSplant Proc, (in press) 159. Ade gh ate E, Donath T: Dopamine-bet a-hydroxybse-positive nen'es in normal and transplant ed pancreatic tissue in the anterior eye
\i'~11

37

of porcine nerves within the graft s. Fir st International Cong ress on Xenot ra nspla nta tion, ~I i nneapolis, ~Ii':, A ugus t 25-28,1991 :70 (abstr) 161. Ba rker CF, i\"aji A, Silvers W5 : Immunologic problems in islet transplantat ion. Diabetes 1980,29:86 (suppll) 162. Garvey JFW, Klein C, ~liIlard PR , et a l: Rejection of organ-cultured all ogeneic fetal rat pan crea s. Surgery 1980, 87: 157 163. Prowse S], Lafferty KJ, Sim eon ovic CJ, et al : The reversal of dia be tes by pancreatic islet transplantation . Diabet es 1982, 31:30 (suppl-l) 16L Sim conovic CJ, Lafferty KJ: Immu nogcnic ity of mouse fetal pancreas and proislcts. A comparison. Transplantation 19R8, -15:82-1 165. Danilovs j A, Hofmann nl, Taylor CR, ct a l: Expression of lILA-Dr antigens in human fetal pancreas tissue, Diabetes 1982,31:23 (suppl-l) 166. M otojirna K, Matsuo S, Mullen Y: Dr a ntige n expression on vascular endothelium and duct epithelium in fresh or cultured human fetal pancreata in the presen ce of gammaint erferon. Transplantat ion 1989, -18:1022 167. Ol iver A.\I, 111OII1son 1\W, Sew ell lIF, et a l: ~13jor histocompatibility complex (:\IHC) clas s II a nt igen (lILA-Dr, Dq , a nd 01') expression in human fetal endocrine o rgans and gut. Sc.l ndJ Imll1ulloI 1988,27:73 1 16R. Thomson i\"~I, Hancock \\~I , Lafferty K] , et a): Organ culture reduced la-positive cells pre sent wirh in the human fetal pan creas. Transplant Pr oc 1983, 15:1373 169. Hering BJ,Brelzcl RG, I'-ededin K; Current s tatus ofclinical islet transplantation. Harm Metab Res 1988, 20:537 I iO. Int ernational Islet Transplant at ion Regi stry'. Hering BJ (ed), Justus-Liebig Unh'ersityofG iessen ,ITR"e\\slell cr 1991,1:9 171. Shumakov VI, Bliumkin Vi\", Ignar enk o S:\, c t al: Results of tr an splantation of pan creatic islet cell cultures to patients with diabet es mellitus, Probl End okrinol (~I osk) 1986,31 :67 J72. Shurnakov VI, Bliurnkin Vi\", Ign at cnk o S:\, e t al: The principa l results of pa ncrea ric islet cell culture tran splantation in diab et es mellitus patients. Tran splant Proc 1937, 19:2372 173. Benikova EA, Turchin IS, Belyakova LS, et al: Experience of treatment of diabetes mellitus pediatric patients with the help of alia- and xcnotransplantation of pancreatic islet cell cultu re. Probl Endokrinol (~Iosk) 1987,33: 19 17-1. Hu Y, Zhang II, Hong-de Z, et al: Culture of human fetal pancreas and islet transplantat ion in 2·1 pati ents with type I d iab etes mellitus. Chin Med] [En gl] 1986, 93:236 175. G roth CG,AnderssonA, I3j(jrke~ C, ct a k Tran splantation of fetal pancreatic m icrofragrnerus via th e portal vein lO a d iabetic patient . D ia bet es 1980,29:80 (suppll) 176. ] O\'anO\ie-l' eterson L, Will ia ms K, Brennan :'\1, et a l: Studies of transplantation of human Ietal rissue in m an, in Peterson C~I, JO'-:lnO\ic-l'clerson L, Formby B (cd s): Fetal Islet Tran splantation. Implications for Diabetes, Springer-Verlag, i\"ewYork,1I.Y,1988,pp 185-195 177. Val ente U, Ferro ~I, Barroci S, et al : Report of clinical cases of human felal panercas transplantation. Transplant Proc 1930, /2 :213 (suppI2) 178. Ya nhu D,Jie Q, Sh anpn W: Treatment of lype I d iabetes m ellitus \\ith intracerebral pancreas islet transplant. Curr TIl er Rcs 1991,-19:7-1 li9. Tuch B, Sheil AGR, i\"g Am', et a l; Transplantation of cuhured human fetal pancrea s into insulin-
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IRO. Tuch B, Sheil AGR, i:\g ABP, et al: Recovery of human fetal pancreas after one year of implantation in the diabeti c patient. Transplantation 1988, -16:865 181. Usadel KH, Schwedcs D, Bastert G, et al: Transplantation of human fetal pancreas. Experience in thymus-aplastic mice and rats and in a diabetic patient. Diabetes 1980, 29:74 (suppll) 182. Voss F, Brewin A, Dawidson I, ct al: Transplantation of proliferated human pre-islets into diabetic patients with renal transplants . Transplant Proc 1989,21:2751

183. Walthall B], Elias KA,Godfrey WI., et al: Rodent xenografts of human and porcine fetal tissue, in Peterson C~I,Jo\"anO\ie­ Peterson 1., Formby B (cds): Fetal Transplantation. Implications for Diabetes. Springer-Verlag, NewYork, "t' 1988, pp 93-110 18-1. Groth CoG, Korsgrcn 0, Andersson A, et al: Evidence of xenograft function in a diabetic pat ient grafted with porcine fetal pancreas. Transplant Proc (in press) 185. Scharp D\\', Lacy PI-:: The clinical feasibility of human islet transplan tation . Clin Diabetes 1991,9:-12