Human fetal pancreas transplants

Human Fetal Pancreas Transplants

Charles M. Peterson, MD,

Lois Jovanovic-Peterson, MD,

Bent Formby, PhD, D&i,

Bernard Gondos, MD,

Lauren M. Monda, MD,*

Liberty Walker, BS,

William Rashbaum, MD,?

Kristen Williams, MD

Sansum Medical Research Foundation,

Santa Barbara, California

l Department of Pathology, Cottage Hospital, Santa Barbara, California

TDepartment of Obstetrics and Gynecology,

Beth Israel Medical Center, New York, New York

Presented in part at the Renal-Retinal

Symposium, Nov 1987. Reprint requests: Charles M. Peterson, MD, Sansum Medical Research Founda- tion, 2219 Bath Street, Santa Barbara, CA 93105.

ABSTRACT

Human fetal pancreatic islet tissue has several advantages as a transplant source for the amelioration of insulin deflclency in patients with Type I diabetes mellltus. It Is now possible to obtain viable tissue, store and ship the tissue without adverse effects on the lnsulln secretory capacity, and transplant either minced tissue or Isolated lslet- like cell clusters following dlgestlon and culture into animal models or man. A number of centers have undertaken studies of human fetal pancreatic allografts in man. Optlmal results have occurred when pooled tissue from six to 20 donors has been implanted and a number of sites have been studied. The authors’ own experience in four recipients who did not receive lmmunosuppresslon has documented insulin secretion for up to 1 year in the absence of an antlcytoplasmlc islet cell antibody response on the part of the recipients. Nevertheless, the procedure has not resulted in insulin independence for the reclpl- ents and the implanted tissue has not secreted insulin In response to a glucose-amino acid challenge in a normal physlologlc pattern. Thus, human fetal pancreatic transplantation for the treatment of Type I diabetes remains an experimental approach. (The Journal of Diabetic Complications 3;1:27-34, 1989.)

INTRODUCTION

The idea of using fetal pancreata as a source of insulin secreting tissue is not new. A number of investigators including Banting and Best have favored the use of fetal tissue at one time or another because of the relative lack of development of exocrine tissue in the fetal environment and hence a relative enrichment of endocrine cells with lessened possibility of enzymatic digestion of insulin or insulin containing cells.1 In 1928, the human fetal pancreas was first used for transplantation purposes.2 In that year Fichera placed pancreatic tissue from three fetuses into various sites in an 18 year old man with diabetes mellitus. The experiment failed, as the recipient died in diabetic coma 3 days later.

Despite these inauspicious beginnings, a resurgence in interest in the procedure of fetal islet or whole pancreas transplantation began in about 1977. The impetus for this renewed consideration of an aban- doned procedure lay in the high morbidity and mortality associated with vascularized transplants that were performed in man, and the experiments of Brown and co-workers that documented that implantation of fetal rat pancreas could reverse experimental diabetes in adult

animals.3-5 At the present time, perhaps as many as 900 transplants of fetal pancreatic tissue into man have taken place, mostly in the Peo- ple’s Republic of China and the Soviet Union.6~9

ISOLATION, STORAGE, SHIPMENT, AN0 FUNCTION IN VITRO

Brown’s group also was the first to demonstrate that the histologically complex rodent fetal pancreas could be cryopreserved, stored indefi- nitely, and used successfully to reverse diabetes after transplantation and that such an approach might be feasible in humans.lO.ll Since that time, a great deal of effort in a number of laboratories has been de- voted to the study of the optimal source and handling of potentially

27

28 PETERSON ET AL.

transplantable human fetal tissue. Such studies are par- ticularly important because the p-cell content of a single human fetal pancreas is not sufficient to normalize the hyperglycemia of an adult diabetic recipient immediately. Therefore, successful transplantation must either await significant expansion and differentiation of the implanted fetal P-cell mass or material collected from more than one fetus needs to be implanted during a single procedure.

Quantitative immunofluorescent studies of the endocrine cell content in the developing human pancreas’* have also shown that 8-10 week fetuses show a sizeable population of endocrine cells, of which almost 50% react solely to glicentin antibody (a proglucagon peptide). It is not known whether such cells represent transitional cells or precursors to the A-cell.13 Somatostatin-containing cells are the second most abundant endocrine cells from the 17th week of gestation up to the 5th postnatal month but become the least numerous in the adult.14 As gestation advances, p-cell mass increases and the insulin to glucagon ratio rises from 1.5 at 20-24 weeks to 5 in newborn infants.15 At all fetal ages, the number of islets isolated from the splenic half of the pancreas is greater than that of the duodenal half.‘6

Prior to this decade, research on the fetal pancreas was limited to a large extent by the lack of usable tissue available to investigators. Due to the founding of the Na- tional Disease Research Interchange (NDRI) in Philadel- phia, Pennsylvania, the availability of tissue for study has become less of a problem. Any investigator may apply for tissue through the NDRI.

We have found that tissue obtained immediately after dilation and extraction procedures and placed in ice- cold RPM1 1640 culture media (Gibco, Grand Island, NY) containing 20 mM Hepes and glutamine at pH 7.4 gives the best results. Optimal conditions of media to assure growth and development of this tissue is an area of intense study. Viability can be assessed in two ways: by trypan blue exclusion and by analysis of the insulin secretory capacity. 17-*1

Prior to study, pancreata attached to the surrounding tissue are aseptically removed and stored in ice-cold sterile RPM1 1640 culture medium containing 20 mM Hepes and 10% fetal calf serum, pH 7.4. Pancreata are then removed from the surrounding tissue, chopped into fine fragments (l-2 mm), and digested in warm (38OC) magnesium-free Hanks’ buffered salt solution (HBSS) containing 7 mg/ml collagenase (TypeV, Sigma Chemical Co, St Louis, MO), 5 mM glucose, and lo/b human albumin for 75 min in an agitator water bath (shaking at a rate of 120/min). The digest is then washed three times with ice- cold HBSS, and samples of islets are selected under a microscope in sizes from 100 to 300 pm, transferred to clean petri dishes, and incubated for 48 hr at 37°C in 95O/o air and 5% CO2 with RPM1 1640 supplemented with 10% human adult serum. Each petri dish contains islets isolated from a single donor pancreas. The number of islets isolated from each pancreas usually is about 800-l 500 (Figure 1).

The adult pancreas is comprised of about lo/6 islets by weight or about l-2 million islets of 20-300 Ccm.22 It is estimated that diabetes occurs when the islet mass

decreases to lo-20% of normal. Therefore, it could be estimated that without replication of fetal 6-cells, the pooled islets from approximately 30 or more fetal donors would be required to eliminate glucose intolerance given the above yields. These calculations emphasize the need for optimizing isolation and culture procedures of this tissue.

To analyze staining with trypan blue, fetal islets are incubated in a solution of the dye containing 0.04% of the stain in isotonic Krebs-Ringer buffer (KRB) pH 7.40 for 15 min and carefully washed several times in KRB, after which the islets are counted under a microscope. The percentage of unstained islets can then be calculated and/or viable islets selected by hand.

Glucose-stimulated responses for insulin release are generally performed in our laboratories in two succes- sive 1 hr static incubations. After culture and staining, islets isolated from each fetal pancreas are transferred to plastic tubes and incubated in KRB, pH 7.4, containing 25 mM Hepes, 2 mM glucose, and 0.3% bovine serum albumin (BSA) at 37OC in an atmosphere of 95% O2 and 5% C02. After an initial period of 1 hour to stabilize basal secretion, buffer is removed; a fresh KRB solution, such as above but containing sufficient glucose to bring the total concentration to 25 mM with or without addition of 1 mM 3-isobutyl-1-methyl-xanthine (IBMX) as a positive control/potentiator is prepared, and the islets are rein- cubated for an additional hour. After each incubation, samples of the buffer are removed for radioimmunoas- say of insulin released; human insulin is used as a ref- erence standard. Insulin remaining within the islets at the end of the second static incubation was extracted with acidic ethanol and assayed. The fractional stimulated insulin secretion rate during the first hour (Fl) of incubation is the amount of insulin secreted as a percent of total insulin (sum of the insulin secreted during the first and second hour plus the nonsecreted insulin). The fractional secretion rate during the second hour of incubation (F2) is the amount of insulin secreted as a percent of total insulin (sum of insulin secreted during the second hour plus the nonsecreted insulin). The fractional stimulatory ratio FSR is defined as F2/Fl. Thus, a FSR value of one theoretically represents no stimulation.

Using these approaches we have determined that fetal pancreatic tissue remains viable under conditions of cold storage at 0-2OC for up to 144 hours, although warm- ing adversely affects survival. These observations allow the shipping of tissue on ice for transplantation and markedly enhance the potential use of this tissue. Up to 144 hr of culture of islets after 18 hr of cold storage also did not adversely affect islet viability. Finally, the insulin secretory response to glucose, while sluggish when compared to the adult, was found to be a function of gestational age (Figure 2) with relative insulin secretory capacity decreasing to its lowest point at 20-23 weeks and gradually increasing again thereafter. These latter findings may have implications for the development of macrosomia in the infant of the diabetic woman because glucose intolerance before 18 weeks may initiate excess insulin secretion in the fetus despite normal maternal glucose levels during the second trimester and beyond.

As documented recently by Sandler et al.,23 the pro-

HUMAN FETALPANCREASTRANSPLANTS

FIG. 1 (A) The isletlike cell clusters after 48 hours of culture that were isolated according to the procedures described in the text are shown at a magnification of X46. (6) An electron micrograph of a single isletlike cell cluster magnified 4,320-fold. Note the different cell types surrounding a central core.

30

.

.

i

.

.

. .* :

. . .

. . . 0: . :. .

. . . :

: . . : :: l

FIG. 2 This graph shows insulin secretory capacity as a func-

tion of gestational age. The fractional stimulated insulin secre-

tion rate during the first hour (Fl) of incubation is the amount of

insulin secreted as a percent of total insulin (sum of the insulin

secreted during the first and second hour plus the non-secreted

insulin). The fractional secretion rate during the second hour

of incubation (F2) is the amount of in&in secreted as a per-

cent of total insulin (sum of insulin secreted during the second

hour plus the non-secreted insulin). The fractional stimulatory

ratio (FM) is defined as F2lFl. Thus, a FSR value of one the-

oretically represents no stimulation.

liferative capacity of human fetal pancreatic cells in culture depends greatly on the condition and handling of the donor tissue. Thus, there is a wide variability in the viability of donor tissue as assessed both histologically and by growth in culture, depending on how the tissue was obtained and on the subsequent storage and culture conditions. The optimal handling of fetal islet tissue and the optimal environment for storage and induction of proliferation remain to be determined.

Our technique described is modified slightly from that of Agren et al.24 These investigators used pancreatic fragments, which were transferred to nonattachment sterile culture dishes containing either 5 ml medium TCM 199 or RPM1 1640 supplemented with lo-20% fetal calf serum and antibiotics. The cultures were maintained at 37% in an atmosphere of 5% COP in humidified air. By this procedure the fragments remain unattached by resting on the bottom of the dishes, thereby creat- ing some gas diffusion problems in the fairly large cel- lular fragments. It was found that after 6-14 days in culture, the fragments were composed of fibrous tissue with well-preserved ducts and islet cells, of which some were organized into islets, whereas acinar cells seemed to have disappeared. Insulin release experiments demonstrated that a high glucose concentration alone failed to stimulate insulin secretion; however, the addition of 5 mM theophylline to 15 mM glucose did elicit a slight to marked insulin response from the cultured pancreatic

PETERSON ET AL.

fragments. No difference in the secretory response was observed between medium TCM 199 and RPM1 1640. The investigators could not document a correlation of stimulated insulin release with fetal age.

Another method of culture uses tissue fragments cultured on a raft to maintain the tissue at the gas-medium interface.25F26 The respiring tissue is provided with optimal gas exchange and access to the nutrients of the culture medium. Andersson et al.26 used cultured minced fragments on Millipore filters (Bedford, MA) supported by a surgical gel foam in the presence of either medium RPM1 1640 or TCM 199 and found a considerable decrease in the insulin accumulation in the culture medium when explants were maintained in RPM1 1640 but not TCM 199. Nevertheless, a comparison between the air-liquid interface technique and free-floating sus- pension culture failed to demonstrate any clearcut differ- ences between the two methods with regards to the insulin output from the fragments to the culture media.

Using a similar culture technique, Maitland and co- workers reported that their fetal explants responded with an increased insulin release to either 1.5 PM glucagon, 5 mM leucine, 10 mM arginine, or 10 mM theophylline but only slightly to 19.3 mM glucose.27 Elevation of the glucose concentration in the TCM 199 medium seemed to maintain a higher insulin secretion in the cultures. Addition of an amino acid mixture also enhances the insulin secretion of human fetal pancreatic explants in culture.28 In addition, Maitland et a/. found that a high oxygen concentration (95% O2 and 5% COP) was toxic to the explants and suggested a gas phase of air plus 5% C02.zg A toxic effect of high O2 in the gas phase was also reported by Mandel and Koulmanda30 in experiments designed to decrease the immunogenicity of fetal mouse pancreas before allogeneic transplantation. Our own unpublished observations would concur with these observations on the effects of high oxygen concentra- tions.

Sandler et a/.22 recently described a method for tissue culture of human fetal pancreas in which isletlike cell clusters (ILCC), partly composed of endocrine cells, gradually develop in vitro. RPM1 1640 has been used as the culture medium and different biological sup- plements, i.e., fetal calf serum, human amniotic fluid, and human serum have been tested for their effects on growth and function of the endocrine cells.22J1J2 In the presence of either human amniotic fluid or human serum, the growth characteristics of the ILCC were found to change, as they became more abundant but smaller in size and free-floating when compared to those cultured in the presence of fetal calf serum. In the latter medium, ILCC were attached to a confluent monolayer of fibroblasts growing on the bottom of the culture dishes. A similar observation was made by Gold- man and Colle.33 These observations are confirmed by our own studies. We have found that the addition of human recombinant growth hormone both increases insulin output in vitro as well as produces an increase of intracellular messenger RNA for the insulin molecule.34 It previously has been documented in the rat that growth hormone stimulates islet p-cell replication in monolayer cultures.35 It would appear that the human fetal islet is

HUMAN FETALPANCREASTRANSPLANTS 31

highly sensitive to a number of growth factors in vitro and that the ontogeny of this tissue is rapidly being defined.

Cryopreservation of fetal islet tissue for potential transplantation is also a possibility, as noted previously.10 Sandler and co-workers have documented that these techniques can be used with human fetal pancreatic tissue as well,36 thus confirming the work of Kemp et ~1l.3~

STUDIES IN VW0

The availability of the immunosuppressed nude (Nu/Nu) mouse has provided investigators with the opportunity to study xenografted tissue without the administration of toxic immunosuppressive drugs. Transplantation of ILCC beneath the kidney capsule of nude mice after culture in the presence of fetal calf serum has been described.** Eight weeks later the recipient mice were in- jected with 3H-thymidine 1 hour before being killed. In seven of nine animals receiving transplants, histologic sections from the graft stained for insulin or glucagon showed groups of cells composed of p- or A-cells ar- ranged in an isletlike fashion and surrounded by non- stained epithelioid cells. There were also ductlike struc- tures composed of occasional hormone positive cells. The number and proportion of p-cells increased in the grafted ILCC as compared to the nongrafted ones ex- amined 5 days after culture. The labeling index for the entire cell population in the grafts was found to be about 5 times higher than the corresponding values for islets prepared from adult mice.

These findings agree with those of Tuch and co- workers who demonstrated a histologic differentiation of endocrine cells occurring in viva after implantation.36 One of the problems encountered by this group was in making nude mice of different strains diabetic, in contrast to other groups who have had little difficulty in this regard.3g140 Initially, this group placed both fresh and cultured human fetal pancreatic slices into the subcutaneous space of diabetic nude mice. The explants coalesced soon after transplantation, becoming visible within two weeks as the scar tissue from the wound re- gressed. Thereafter the implants grew macroscopically for at least 37 weeks; growth was inversely related to the time the tissue was cultured prior to transplantation, with most growth occurring in uncultured tissue. In all cases, when explants were cultured for up to 3 weeks prior to transplantation, tissue was macroscopically visible. In contrast, tissue cultured for periods longer than this was rarely visible. Experiments in mice that were not diabetic demonstrated that uncultured subcutaneous implants would continue to grow for up to 54 weeks,41 indicating a possible adverse affect of the diabetic environment per se. In these latter experiments a linear relationship was demonstrated between absolute age of the implant (absolute age = gestational age + duration in the nude mouse) and its wet weight. The maximum weight reached by an implant was 730 mg. The investigators concluded that it was likely that the limiting factor for growth of human fetal tissue in nude mice is more the life span of the mouse than an intrinsic factor in the

tissue. Insulin content of the tissue correlated with mor- phometric quantification of islet cell content in the transplanted tissue.

Thus, human fetal pancreatic tissue implanted into nude mice appears to differentiate selectively into endocrine tissue, ducts, and fibrous tissue; exocrine tissue has not been identified in the implants. These findings have been observed in both diabetic and nondia- betic mice whether the tissue is implanted peripherally or centrally, as reported by Tuch and co-workers as well as others.d*-44

Tuch and co-workers,45 Noonan and co-workers46 and Hullett et a/.47 have been able to demonstrate normaliza- tion of blood glucose after implantation of human fetal pancreatic tissue into diabetic nude mice. However, re- moval of the implanted tissue has not always resulted in a return of diabetes thus indicating, perhaps, a re- growth of mouse pancreas, something known to occur after treatment with streptozotocin.46 Figure 3 documents biopsy specimens taken from the

kidney capsule of NOD mice 7 days after implantation of human fetal ILCC. The top panel shows a specimen taken from a mouse that received no immunosuppression, while the bottom panel shows tissue taken from a mouse that received immunosuppression with 50 mg/kg cyclosporine A for 7 days prior to and including the day of implantation and 50 mg/kg for 6 days posttransplan- tation. While the doses of cyclosporine used were high, these studies do document the feasibility of transplantation of human fetal islet tissue, even as a xenograft, in an animal with an autoimmune form of diabetes mellitus.

These studies have led to renewed interest in the use of human fetal islet tissue as a potential treatment for Type I diabetes in man. The group in Uppsala, Sweden have transplanted six individuals with human pancreatic tissue since 1979. All were insulin-dependent diabetic patients who had received a kidney transplant and were thus already on immunosuppression. Three patients received pancreatic fragments; one who was treated with an intraportal injection showed C-peptide in the urine at a level 5% of normal. After four months, the C-peptide production subsequently ceased. Interestingly, the patient exhibited antibodies against an islet cell surface antigen at the same time as the urinary C-peptide disappeared. In the other two patients there was no evidence of graft function. In 1982, one patient was transplanted intraportally with cryopreserved human fetal pancreatic fragments from 24 donors. Two additional patients received injections of ILCC, which had developed in culture in the presence of fetal calf serum. In one of the patients, the material was given intraportally as a single injection, and in the other patient, an indwelling catheter was used for repeated intraportal injections over a period of 6 weeks. In none of these patients was there any sign of graft function.@Jl

Clinical transplants of human fetal pancreas were be- gun in Sydney, Australia in 1983 and, to date, five patients without detectable serum C-peptide have received human fetal tissue. Four of these patients received immunosuppression in the form of cyclosporine A, pred- nisone, azathioprin, and/or anti-lymphocyte globulin and have received tissue from l-6 fetal pancreata be-

32 PETERSON ETAL

FIG. 3 Xenografted fetal isletlike cell clusters were implanted under the kidney capsule of Sansum Strain NOD Mice and biopsy specimens were obtained 7 days after implantation. (A) shows a specimen obtained from a mouse that had received

no immunosuppression, while (B) shows a specimen obtained from a mouse which had received cyclosporine A. See text for details.

HUMAN FETAL PANCREAS TRANSPLANTS

tween 14 and 20 weeks gestational age. Tissue was transplanted into either the omentum or muscle tissue and was typed for histocompatibility antigens A, B, and DR.50 In only two patients who received tissue from six donors was C-peptide detected after a glucagon challenge and accompanied in one patient by a 50% drop in insulin requirement. It was notable that this latter patient had a normal glucosylated hemoglobin value before transplantation. Because the patient received implants at multiple sites, it was impossible to determine which implant was responsible for the C-peptide secretion at 3 months. From these experiments the investigators concluded that multiple donors will be needed for effective transplantation.51n52

To date, we have implanted four patients without detectable serum C-peptide and who were not on immunosuppressive therapy with fetal pancreatic fragments obtained from 6-12 donors. In two cases the tissue was implanted into the brachioradialis of the nondominant forearm. However, to find an easily accessible site with more room, the second two recipients received tissue transplanted into a pocket above the abdominus rectus in the left lower quadrant. In each case, blood glucose values were normalized using continuous subcutaneous insulin infusion prior to implantation, as documented by home blood glucose monitoring values and glucosylated hemoglobin levels. lmmunosuppression was not used. In each case C-peptide levels after a “Sustacal” (Mead Johnson, Evansville, IN) challenge were detected within 3 weeks of implantation and have remained detectable for up to 1 year postimplant. However, in each case, insulin was required to maintain normoglycemia,‘g with the exception of two patients who had 24-46 hours during which their blood glucose values continually dropped into the range of 50 mg/dl. This latter observation may reflect that donor pancreatic tissue in rodents has been shown to regulate recipient blood glucose to those levels generally seen in the donor rather than the recipient.53

DISCUSSION

These studies document that fetal islet tissue can be obtained, stored, cultured, and transplanted into rodents and man. Following transplantation into man, the tissue has been shown in certain cases to be viable and se- crete insulin for varying periods of time. The reasons for the erratic clinical results and the development of optimal techniques that will result in insulin independence for the transplant recipient require further study. Rea- sons for our own relative success may be due, in part, to the patients being healthy and maintained relatively euglycemic before implantation by continuous subcutaneous insulin infusions4 and that the tissue was obtained from dilation and extraction procedures. Nevertheless, the insulin secretory patterns of the transplanted tissue were never normal and the total output of insulin following three months was only 20% of the maximal secretion seen. Thus, considerable insulin secreting tissue was lost either through classic immunologic mech- anisms, failure of vascularization, or mechanical problems. It is noteworthy that our procedure has not been

33

found to stimulate the production of islet cell cytoplas- mic antibodies in the recipients.

While the use of fetal islets for transplantation purposes remains a tantalizing but unfulfilled possibility, much has been learned from the intense study of this tissue. The sensitivity of fetal tissue to glucose and a number of growth factors as early as 13 weeks indicates a vulnerability of this tissue to perturbations in glucose homeostasis at a much earlier stage in gestation than was previously thought. The rapid response of this tissue (hours to days) to changes in culture conditions also has implications in terms of response to reestablishment of the normal maternal/fetal metabolic milieu. While the results to date have not led to a normal internal milieu in patients receiving allografted human fetal tissue, the rapid progress made in this field provide hope that transplantation of viable fetal pancreatic tissue will provide an option for the amelioration of Type I diabetes in the not too distant future.

ACKNOWLEOGMENTS

This work was supported in part by The American Di- abetes Association, The Cottage Hospital Foundation, The Diabetes Research and Education Foundation, The Diabetes Treatment Centers of America Foundation, and The Juvenile Diabetes Federation International.

REFERENCES

1.

2.

3.

-4.

5.

6.

7.

8.

9.

10.

11.

12.

Bliss M: The Discovery of Insulin. Chicago, The University of Chicago Press, 1982, pp. 28-29. Downing R: Historical review of pancreatic islet transplantation. World J Surg 8:137-142, 1984. Sutherland DER: Pancreas and islet transplantation: II Clin- ical trials. Diabetolooia 20:435-450. 1981. Brown J, Clark WR,-Molnar IG, Mullen YS: Fetal pancreas transplantation for reversal of streptozotocin-induced diabetes in rats. Diabetes 2556-64, 1976. Brown J, Molnar IG, Clark W, Mullen Y: Control of experimental diabetes mellitus in rats by transplantation of fetal pancreases. Science 184:1377-1379, 1974. Sutherland DER, Goetz FC, Najarian JS: Review of world’s experience with pancreas and islet transplantation and results of intraperitoneal segmental pancreas transplantation from related and cadaver donors at Minnesota. Transplant Proc 13:291-297, 1981. Hu Y-F: Clinical studies on islet transplantation in 39 patients with insulin-dependent (type I) diabetes mellitus. Wuhan int. Symp. Organ Transplant 1985, pp. 39-40. Shumakov VI, Bljumkin VN, lgnatenko SN, et al: The prin- cipal results of pancreatic islet cell culture transplantation in diabetes mellitus patients. Int. Congress Transplant Sot. 11:40. 1986. Benikova EA, Turchin IS, Beliakova LS, Bolshova EV, Ly- senko AG: Experience with the treatment of children with diabetes mellitus using allo- and xenografts of cultures of pancreatic islet cells. Probl Endocrinol (MO&) 33:19-22, 1987. Kemp JA. Mullen Y, Weisman H, et al: Reversal of diabetes in rats using fetal pancreases stored at -196C. Transplan- tation 26:260-264, 1978. Brown J, Kemp JA, Hurt S, et al: Cryopreservation of human fetal pancreas. Diabetes 29(Suppl 1):170-173, 1980. Stefan Y, Grass0 S, Perrelet A, et al: A quantitative immunofluorescent study of the endocrine cell populations in the developing human pancreas. Diabetes 32:293-301,1983.

PETERSON ET/IL. 34

13.

14.

15.

16.

17.

18.

19.

20.

21.

22.

23.

24.

2;.

26.

27.

28.

29.

30.

31.

32.

33

Stefan Y, Ravazzola M, Grass0 S, et al: Glicentin precedes glucagon in the developing human pancreas. Endocrinol- ogy liO:2189-2191, 1982. - Orci L. Stefan Y. Malaisse-Laaae F. et al: lnstabilitv of oan- creatic endocrine cell populations’ throughout life. Lancet 1:615-616, 1979. Wirdnam PK, Milner RDG: Quantitation of the p and A cell fractions in human pancreas from early fetal life to puberty. Early Hum Dev 5:299-309, 1981. Goldman H, Wong I, Pate1 YC: A study of the structural and biochemical development of human fetal islets of Langer- hans. Diabetes 31:897-902, 1982. Formby B, Walker L, Peterson CM: Improved isolation yield of murine islets of Langerhans from a single donor can reverse experimental diabetes after isotransplantation. Di- abetes Research 2:217-219, 1985. Formby B, Walker L, Peterson CM: Rapid selection of viable transplantable human fetal pancreatic islets by trypan blue exclusion. Proc Sot Exp Biol Med 182:245-247, 1986. Peterson CM, Miller N, Walker L, Formby B: Effect of glip- izide on insulin secretion from cultured human fetal pancreatic islets. Diabetes Care 9:556-557, 1986. Peterson CM, Jovanovic L, Formby B, et al: Studies of human fetal pancreas. Proceedings of the Second Interna- tional Conference on the Use of Human Tissues and Or- gans for Research and Transplant. Philadelphia, National Diabetes Research Interchange, 1986, pp. .220-222. Formby B, Walker L, Peterson CM: Effects of duration of cold storage and gestational age on the insulin secretory capacity of human fetal pancreatic islets. Diabetes Res 4:113-116, 1987. Williams RH, Porte D: The Pancreas, in Williams RH (ed), Textbook of Endocrinology, Philadelphia, WB Saunders, 1974, p. 509. Sandler S, Andersson A, Schnell A, et al: Tissue culture of human fetal pancreas. Development and function of p- cells in vitro and transplantation of explants to nude mice. Diabetes 34:1113-l 119, 1985. Agren A, Andersson A, Bjorken C, et al: Human fetal pancreas. culture and function in vitro. Diabetes 29(Suppl 1):64-69, 1980. Auerbach R: Morphogenetic interactions in the development of the mouse thymus gland. Dev Bio/2:271-284, 1960. Andersson A, Christensen N, Groth C-G, et al: Survival of human fetal pancreatic explants in organ culture as re- flected in insulin secretion and oxygen consumption. Trans- plantation 37:499-503, 1984. Maitland JE, Parry DG, Turtle JR: Perifusion and culture of human fetal pancreas. Diabetes 29(Suppl 1):57-63, 1980. Hoffman L, Mandel TE, Carter WM, et al: Insulin secretion by fetal human pancreas in organ culture. Diabefologia 23:426- 430, 1982. Maitland JE, Caterson ID, Gauci RE, et al: Organ culture of human foetal pancreas: conditions which affect basal and stimulated insulin release. Acta Endocrinol 108:377- 385, 1985. Mandel TE, Koulmanda M: Effect of culture conditions on fetal mouse pancreas in vitro and after transplantation in syngeneic and allogeneic recipients. Diabetes 34:1082-1087, 1984. Sandler S, Andersson A, Swenne I, et al: Tissue culture and cryopreservation of fetal mammalian endocrine pancreas intended for transplantation, in Peterson CM, Jovanovic L, Formby B (eds), Human Fetal /s/et Transplantation, New York, Springer ieriag, 1988, pp. 9-42. Andersson A. Sandler S. Hellerstrom C. et al: Effects of amniotic fluid’on the deve’lopment of human fetal pancreatic p-cells in tissue culture. Transplant Proc 18:57-59, 1986. Goldman H, Colle E: Human pancreatic islets in culture: effects of supplementing the medium with homologous and heterologous serum. Science 192:1014-1016, 1976.

34.

35.

36.

37.

38.

39.

40.

41.

42.

43.

44.

45.

46.

47.

48.

49.

50.

51.

52.

53.

54.

Formby B, Ullrich A, Coussens L, et al: Growth hormone stimulates insulin gene expression in cultured human fetal pancreatic islets. J C/in Endicrinol Microb 66:1075-1079, 1988. Rabinovitch A, Quigley C, Rechler MM: Growth hormone stimulates islet P-cell replication in neonatal rat pancreatic monolayer cultures. Diabetes 32:307-312, 1983.

Sandler S, Andersson A, Hellerstrom C, et al: Preservation of morphology, insulin biosynthesis and insulin release of cryopreserved human fetal pancreas. Diabetes 31:238-241, 1982. Kemp JA, Hurt SN, Brown J, et al: Recovery and function of human fetal pancreas frozen to -196%. Transplantation 32:10-15, 1981. Tuch BE, Ng ABP, Jones A, et al: Histologic differentiation of human fetal pancreatic explants transplanted into nude mice. Diabetes 33:1180-l 187, 1984.

Paik S-G, Fleischer N, Shin S-l: Insulin-dependent diabetes mellitus induced by subdiabetogenic doses of streptozotocin: Obligatory role of cell-mediated autoimmune pro- cesses. Proc Nat/ Acad Sci USA 77:6129-6133, 1980.

Buschard K, Rygaard J: Restitution of streptozotocin induced diabetes mellitus in nude mice with pancreatic grafts from the rat. Acta Pafhol Microbial Stand (C) 84:221-226, 1976. Tuch BE, Grigoriou S, Turtle JR: Growth and hormonal content of human fetal pancreas passaged in athymic mice. Diabetes 351464-469, 1986.

Povlson CO, Skakkeback NE, Rygaard J, et al: Heterotrans- plantation of human foetal organs to the mouse mutant nude. Nature 2481247-249, 1974.

Usadel KH, Schwedes U, Bastert G, et al: Transplantation of human fetal pancreas: experience in thymus aplastic mice and rats and in a diabetic patient. Diabetes 29(Suppl 1):74- 79, 1980.

Mandel TE, Georgiou HM: Insulin secretion by fetal human pancreatic islets of Langerhans in prolonged organ culture. Diabetes 32:915-920, 1983. Tuch BE, Jones A, Turtle JR: Maturation of the response of human fetal pancreatic explants to glucose. Diaberologia 28:28-31, 1985. Noonan RA, Walthall BJ, Godfrey WL, et al: Reversal of diabetes in nude mice by xenograft transplant of cultured fetal pancreas. Diabetes 35(Supbl 1):138A, 1988. Hullett DA. Falanv JL. Love RB. Burlinaham WJ. Pan M. Sollinger H’W: Huhan ietal pancreas-a potential source fo; transplantation. Transplantation 43:18-22, 1987. Junod A, Lambert AE, Stauffacher W, et al: Diabetogenic action of streptozotocin: relationship of dose to metabolic response. J C/in invest 48:2129-2139, 1969. Groth C-G, Andersson A, Bjorken C, et al: Transplantation of fetal pancreatic microfragments via the portal vein to a diabetic patient. Diabetes 29(Suppl 1):80-83, 1980. Tuch BE, Doran TJ, Messel N, et al: Typing of human fetal organs for the histocompatibility antigens A, B and DR. Pathology 17:57-61, 1985. Tuch BE, Sheil ARG, Ng ABP, et al: Long term survival of human fetal pancreatic tissue transplanted into an insulin dependent diabetic patient. Diabetes Medicine 3:24-28, 1986. Tuch BE: From nude mouse to man, in Peterson CM, Jo- vanovic L, Formby B (eds), Human Fetal /s/et Transplanta- tion. New York, Springer tierlag, 1987 (in press). Georaious HM. Mandel TE: Transplanted fetal pancreas al- lograits regulaie blood glucose to bonor strain l&els. Trans- plant Proc 19:2922-2925, 1987. Mandel TE, Cuthbertson RA, Koulmanda M: The effect of chronic diabetes and continuous pretransplantation insulin infusion on the function of organ-cultured fetal islet iso- grafts. Transplant Proc 19:2916-2918. 1987.

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Human fetal pancreas transplants