Transplantation of human fetal pancreas: fresh vs. cultured fetal islets or ICCS

The paucity of human adult islets available for transplantation in IDDM makes the use of human fetal pancreas a potential alternative. Fetal pancreatic endocrine cells grow and differentiate over time when fresh explants or cultured islet-like cell clusters (ICCs) are transplanted under the kidney capsule in athymic nude mice. We have recently developed a procedure to isolate fetal islets, which differ from ICCs in their β-cell content. This study was undertaken to compare the maturation and growth of grafts from purified fetal islets, containing mostly β-cells, to grafts of mostly undifferentiated endocrine cell precursors, cultured as ICCs, and fresh, uncultured tissue. Total insulin content was highest in the fresh tissue pre-transplant while insulin levels fell precipitously during culture as either fetal islets or ICCs. Although 500 fetal islets contained more insulin than 500 ICCS before transplantation, the insulin content of the resulting grafts was the same 3 months post-transplantation. The degree of stimulation following glucose challenge was comparable, as was the histological appearance. However 70 mg of fresh tissue was needed to generate the fetal islets while only 30 mg was needed for the ICCs. Grafts of 30 mg fresh tissue also had similar total insulin contents and stimulation following glucose challenge, but, when normalized to DNA there was a significantly higher concentration of insulin in the grafts from ICCs or fetal islets. Moreover there were distinct morphological differences, with fibrous and ductal elements prominent in the grafts from fresh tissue, which were also much larger and more diffuse, with cystic elements evident macroscopically. Quantitative immunohistochemical analysis showed that grafts from cultured tissue were 48.3±5% positive for immunoreactive insulin compared with grafts from fresh tissue which were only 13.3±1.4% positive for insulin. In conclusion cultured ICCs, a heterogeneous mixture of hormone-containing and undifferentiated endocrine cells, are a preferable source for transplantation than either purified fetal islets or uncultured tissue.     

Transplantation of the fetal rat pancreas: Quantitative morphological analysis of islet tissue growth

When pancreases from fetal rats were transplanted beneath the kidney capsule of isologous normal adult recipients, continued growth and differentiation of the endocrine portion of the pancreas occurred. While limited amounts of acinar tissue were identifiable in the early transplant period (7 days), such cells were absent in long term transplants (14 and 21 days). In contrast, while few definitive islet beta cells were present at the time of transplantation, following 21 days at the kidney site large circumscribed islets comprised of heavily granulated beta cells in association with duct epithelial cells predominated. Mitotic figures were seen in both these cell populations. Total islet mass had increased over 20-fold during the transplantation period. Similar results were observed if fetal pancreases were grown in organ culture for ten days prior to transplantation. Continued islet and duct cell growth, as evidenced by mitotic figures and an increase in absolute islet cell mass was obtained in such cultured explants when transplanted to either isogenic or allogenic recipients. These observations support the hypothesis that fetal pancreas may be the best source of donor material for transplantation to diabetic recipients, in part, due to the continued growth and differentiation of the islet tissue during the transplantation period.     

Continuous-Perifusion tissue culture of fetal and adult pancreas of the lizard Anolis carolinensis

The differentiation of the fetal saurian pancreas in continuousperifusion tissue culture (CPTC) was examined. Splenic pancreases from 24-day postoviposition fetuses of the green anole, Anolis carolinensis, were grown for 8 to 31 days by CPTC following successful preliminary studies with adult pancreas. Adult anolian endocrine pancreas was maintained for up to 7 days by CPTC. The pancreatic explants were examined morphologically by light and electron micros-copy. The functional integrity of the endocrine cells was evaluated by measuring hormone levels of the explants and in the basal medium and by determining the kinetics of hormone release. The pancreatic endocrine cells from fetal and adult anoles were functionally and morphologically intact after CPTC. The exocrine pancreas was not maintained during culture. This study demonstrates for the first time the growth of the reptilian endocrine pancreas in culture.     

Expression during rat fetal development of GLUT12--a member of the class III hexose transporter family

Glucose is an essential molecule for most mammalian cells, and is particularly important during fetal development, when cells are rapidly dividing and differentiating. In rats, GLUT1 is present at high levels in most fetal tissues, with levels decreasing after birth. We used immunohistochemistry to localise GLUT12 protein, a recently identified member of the sugar transporter family, and GLUT1 during rat fetal development. GLUT12 staining was observed in heart muscle from gestational days 15 to 21. GLUT12 staining in skeletal muscle increased from gestational days 17 to 21, and GLUT12 was also detected in brown adipose tissue. The expression of GLUT12 in insulin-responsive tissues supports a potential role for GLUT12 in the provision of glucose to these tissues before the appearance of GLUT4. GLUT12 protein was also expressed in fetal chondrocytes from gestational day 15 onward, in kidney distal tubules and collecting ducts from day 19, and in lung bronchioles from day 19. The specific pattern of expression observed in the rat fetus suggests that GLUT12 may be important in hexose delivery to developing tissues.     

Effect of thyrotropin-releasing hormone on development of the pituitary–thyroid system in fetal rats in organ culture

Six groups of thyroid glands from 18-day fetal rats were explanted to organ culture for 2 days. In one group, thyroid was cultured alone and in the remaining five groups thyroid was cocultured with pituitaries from fetuses ranging in age from 17 to 21 days. In each of the groups, half of the cultures had thyrotropin-releasing hormone (TRH) added to the medium. Histometric parameters of the thyroid follicle such as diameter and cell height were used as indicators of development of the thyroid gland. When 18-day thyroid was cultured alone, addition of TRH did not accelerate development. When either one 18-day or two 17-day pituitaries were cocultured with thyroid, a significant increase in diameter and cell height was seen. Addition of TRH to the medium induced little or no further change. When the thyroid was cultured with 19- to 21-day pituitaries, a marked increase in thyroid development was observed; and the addition of TRH caused further acceleration in thyroid development. These results suggest that, in organ culture, 17- to 18-day pituitary glands can release some thyrotropin (TSH) with or without additional TRH. Older pituitaries (19- to 21-day) apparently can release an amount of TSH in the presence of TRH that is greater than their own spontaneous TSH secretion.     

Effect of thyrotropin-releasing hormone on development of the pituitary-thyroid system in fetal rats in organ culture

Six groups of thyroid glands from 18-day fetal rats were explanted to organ culture for 2 days. In one group, thyroid was cultured alone and in the remaining five groups thyroid was cocultured with pituitaries from fetuses ranging in age from 17 to 21 days. In each of the groups, half of the cultures had thyrotropin-releasing hormone (TRH) added to the medium. Histometric parameters of the thyroid follicle such as diameter and cell height were used as indicators of development of the thyroid gland. When 18-day thyroid was cultured alone, addition of TRH did not accelerate development. When either one 18-day or two 17-day pituitaries were cocultured with thyroid, a significant increase in diameter and cell height was seen. Addition of TRH to the medium induced little or no further change. When the thyroid was cultured with 19- to 21-day pituitaries, a marked increase in thyroid development was observed; and the addition of TRH caused further acceleration in thyroid development. These results suggest that, in organ culture, 17- to 18-day pituitary glands can release some thyrotropin (TSH) with or without additional TRH. Older pituitaries (19- to 21-day) apparently can release an amount of TSH in the presence of TRH that is greater than their own spontaneous TSH secretion.     

Inherent beta-cell dysfunction induced by transgenic expression of allogeneic major histocompatibility complex class I antigen in islet cells.

Insulin-dependent diabetes mellitus (IDDM) is generally believed to be an autoimmune disease resulting from T-cell dysfunction that produces beta-cell damage, but it is conceivable that some forms of IDDM are not immunologically mediated. The effect of the expression of a foreign transgenic MHC class I antigen (H-2Kb), restricted to pancreatic islet beta-cells, was tested in vitro and in nude (athymic) mice to determine whether beta-cell dysfunction was due to non-immune mechanisms. The models used clearly excluded immune involvement in beta-cell damage. Fetal pancreas from transgenic and littermate control mice was maintained in organ culture for up to 18 days and insulin secretion into the medium assessed. For the initial 3-4 days in vitro, fetal control and transgenic pancreas secreted similar amounts of insulin, but thereafter insulin secretion by the transgenic tissue decreased in comparison with the controls. When the cultured pancreas was transplanted into nude mice, the transgenic issue produced smaller grafts than the control pancreas, but there was wide variation in graft size. Expression of H-2Kb antigens in beta-cells of nude transgenic mice also resulted in early-onset diabetes. The insulin content in the pancreas of young H-2Kb transgenic euthymic mice, (previously shown not to have insulitis), was reduced but glucagon content was normal. The reduction in in vivo insulin production was similar chronologically to the reduced insulin production by transgenic islets in vitro. These data confirm the non-immune loss of beta-cell function in MHC-transgenic mice and they may be a model for atypical Type I diabetes.     

Effect of dexamethasone on the fetal mouse small intestine in organ culture

The formation of intestinal villi (organogenesis phase) may be studied in organ culture with a completely synthetic medium in 15-day fetal mouse duodenal explants. However, in these explants absorptive cells remained poorly differentiated with all the hormones studied except with epidermal growth factor. In order to elucidate the role of hormones and other factors on the maturation of absorptive cells (maturation phase) in the fetal rodent in organ culture, we have taken the explants after the organogenesis phase. We have studied different culture conditions and have found that 17-day mouse duodenal explants can be cultured during 48 hours with Leibovitz L-15 medium in a 95% O2-5% CO2 atmosphere provided that the explants are relatively large (5 X 2 mm). With this method, dexamethasone (Dx) has been shown to have a direct effect on the maturation of the fetal duodenal mucosa. The addition of Dx (300 ng/ml) to the completely synthetic medium 1) improves the morphology of the explants, 2) induces a significant increase in maltase activity in the tissues, and 3) reduces significantly the labeling index of the duodenal explants after 48 hours of culture. Direct action of Dx on the duodenal mucosa is shown for the first time in organ culture using a completely synthetic medium. This method will permit us to study the effects of other intrinsic and extrinsic factors on the regulation of enzymatic maturation in fetal small intestine.     

Fetal and neonatal rat pancreas in organ culture age-related effects of corticosterone on the acinar cell component

Fetal rat pancreas (ages 16 to 22 days postcoitum) and neonatal pancreas (4 days postnatal) were grown in organ culture for four days. The medium consisted of chick embryo extract and rooster serum either with or without the addition of corticosterone (3 × 10^?5 M). Acinar cell differentiation was assessed using quantitative light microscopic linear scanning of tissue sections and enzymatic analysis of amylase in the culture media and in the explants. In the younger fetal tissue of 16 and 18 days postcoitum exocrine differentiation continued in vitro. The effect of corticosterone was an enhancement of the degree of in vitro differentiation. Even with corticosterone, however, the degree of differentiation in vitro was less than that observed during a similar period in vivo. In differentiated pancreas (20- and 22-day fetal and 4-day neonatal) the acinar pancreas degenerated under control conditions and a selective growth of the endocrine pancreas was observed. The addition of corticosterone to the media resulted in a maintenance of the differentiated state of the acini except in 22-day fetal tissue in which the acini were not preserved. The differences between these results and the work of other investigators and the possible in vivo role of adreno corticosteroids in exocrine pancreatic differentiation is discussed.     

Synthesis of soluble and insoluble elastins in cultures of chick aortic cells

Aortic cells were isolated from 9- and 12-day embryonic chick aortas and cultured for varying periods after first passage. Cells obtained from 9-day tissue remained indefinitely as monolayers and possessed a relatively low rate of tropoelastin synthesis. Cells obtained from 12-day tissue remained monolayers for 4 to 8 days, after which time portions of the culture contracted into matrix containing chemically definable insoluble elastin and forming desmosine cross-links. The rate of tropoelastin synthesis was significantly higher in the 12-day derived cells suggesting that these cells had been committed to elastogenesis in vivo and retained this commitment in vitro. A chick tropoelastin cDNA was obtained and partially characterized from a lambda gt11 expression cDNA library. Using the tropoelastin cDNA probe, measurement of the steady-state level of tropoelastin revealed that the increased rate of tropoelastin synthesis in the 12-day cells was accompanied by a significant increase in the level of tropoelastin mRNA steady-state levels. The aortic cell cultures present an important model system for extending studies of chick aortic elastogenesis. The aortic cell cultures synthesize tropoelastin at a rate 10% less than the corresponding organ culture. Significantly, the production of tropoelastin is productive in formation of insoluble, chemically definable elastin. The definition of insoluble includes both amino acid composition and desmosine formation.     

Abnormalities of dendritic cell precursors in the pancreas of the NOD mouse model of diabetes

The non-obese diabetic (NOD) mouse is a widely used animal model for the study of human diabetes. Before the start of lymphocytic insulitis, DC accumulation around islets of Langerhans is a hallmark for autoimmune diabetes development in this model. Previous experiments indicated that an inflammatory influx of these DCs in the pancreas is less plausible. Here, we investigated whether the pancreas contains DC precursors and whether these precursors contribute to DC accumulation in the NOD pancreas. Fetal pancreases of NOD and control mice were isolated followed by FACS using ER-MP58, Ly6G, CD11b and Ly6C. Sorted fetal pancreatic ER-MP58(+) cells were cultured with GM-CSF and tested for DC markers and antigen processing. CFSE labeling and Ki-67 staining were used to determine cell proliferation in cultures and tissues. Ly6C(hi) and Ly6C(low) precursors were present in fetal pancreases of NOD and control mice. These precursors developed into CD11c(+) MHCII(+) CD86(+) DCs capable of processing DQ-OVA. ER-MP58(+) cells in the embryonic and pre-diabetic NOD pancreas had a higher proliferation capacity. Our observations support a novel concept that pre-diabetic DC accumulation in the NOD pancreas is due to aberrant enhanced proliferation of local precursors, rather than to aberrant "inflammatory infiltration" from the circulation.     

Development of intrahepatic bile ducts in rat foetal liver explants in vitro.

The origin of intrahepatic bile ducts was investigated in organ cultures of 13, 16 and 19-day foetal rat livers embedded for up to 10 days in a semi-solid agar gel on a filter-raft assembly. Following 10 days in culture, 13-day foetal explants consisted of liver cell trabeculae lined by endothelial cells. Although maturation of the liver cells and bile canaliculi was observed, duct development was found in less than 10% of the explants. Supplementation of the media with putative inducers of bile duct development or culture of explants adjacent to other tissues did not induce regular duct development. By contrast, explants from the porta hepatis of 19-day foetuses cultured for 4 days, but not 10 days, regularly contained duct-like structures. The formation of the few ducts in cultures of 13-day foetal liver explants indicates that these cells can arise by transformation of hepatoblasts but that specific inducers of development are required for predictable and continuous differentiation of biliary epithelial cells.     

Ultrastructural evidence for blood microvessels devoid of an endothelial cell lining in transplanted pancreatic islets.

The aim of the present study was to investigate, at the ultrastructural level, the process of revascularization of freshly isolated islets or cultured islets after transplantation under the kidney capsule of syngeneic mice. Native islets in adult pancreases from mice, pigs, and humans contained only capillaries with fenestrated endothelium. However, the endothelial cell lining was disrupted in both freshly isolated and cultured mouse islets. Shortly after transplantation (6 weeks) approximately 80% of graft microvessels contained no endothelial cell lining. Similar data on microvessel morphology were found when fetal porcine islet-like cell clusters were implanted into athymic nude mice. Re-endothelialization was a slow process, with 25% of the microvessels still lacking endothelium 6 months after transplantation of cultured mouse islets or islet-like cell cluster. However, when freshly isolated mouse islets are used only 25% of microvessels within the islet graft lacked endothelium 6 weeks after implantation. We suggest that capillaries damaged during islet isolation may provide a preformed channel, serving as a scaffold for newly formed islet graft blood vessels. The presence of non-endothelialized microvessels, with an associated lack of barrier function, might make transplanted islets more prone to thrombosis or an attack by the immune system. This provides a tentative explanation for the increased vulnerability of islet grafts when compared with whole pancreas transplants.     

The effect of tensional load on isolated embryonic chick tendons in organ culture

Digital flexor tendons isolated from 17-18 day embryonic chickens were cultured intact, either on steel mesh grids, or in an apparatus designed to apply a mechanical load to the tissue. Tendons cultured without an applied load continued to synthesize protein and glycosaminoglycans throughout a 7-day period, but DNA synthesis decreased during this time. Increases in both protein and DNA synthesis were observed in tendons experimentally loaded for 48-72 h. Glycosaminoglycan production by tendons isolated from 17-day embryos was also increased in loaded tendons, sulfated GAG being increased more than hyaluronic acid. The same loading regime applied to tendons from 18-day embryos produced a smaller, yet significant increase in sulfated glycosaminoglycans but hyaluronate production was reduced. These investigations demonstrate that embryonic chicken tendons can be maintained in a viable state in organ culture and may provide a useful model for studies of the effects of mechanical forces on the synthetic capability and structure of connective tissue cells.     

Recent observations on the regulation of fetal metabolism by glucose

Glucose is the principal energy substrate for the the fetus and is essential for normal fetal metabolism and growth. Fetal glucose metabolism is directly dependent on the fetal plasma glucose concentration. Fetal glucose utilization is augmented by insulin produced by the developing fetal pancreas in increasing amounts as gestation proceeds, which enhances glucose utilization among the insulin-sensitive tissues (skeletal muscle, liver, heart, adipose tissue) that increase in mass and thus glucose need during late gestation. Glucose-stimulated insulin secretion increases over gestation. Both insulin secretion and insulin action are affected by prevailing glucose concentrations and the amount and activity of tissue glucose transporters. In cases of intrauterine growth restriction (IUGR), fetal weight-specific tissue glucose uptake rates and glucose transporters are maintained or increased, while synthesis of amino acids into protein and corresponding insulin–IGF signal transduction proteins are decreased. These observations demonstrate the mixed phenotype of the IUGR fetus that includes enhanced glucose utilization capacity, but diminished protein synthesis and growth. Thus, the fetus has considerable capacity to adapt to changes in glucose supply by relatively common and understandable mechanisms that regulate fetal metabolism and growth and could underlie certain later life metabolic disorders such as insulin resistance, obesity and diabetes mellitus.     

Conophylline与尼克酰胺联合诱导脂肪源干细胞为功能性类胰岛细胞

背景：1型糖尿病的胰岛移植治疗一直面临供体来源不足与免疫排斥两大关键问题,寻找一种自体来源的种子细胞通过组织工程方法制备类胰岛组织可以提供充足新型供体、降低异基因供体移植的不良反应。 目的：分析成人脂肪干细胞体外分化为对葡萄糖敏感、可分泌胰岛素的功能性胰岛样细胞团的能力,探索体外制备类胰岛组织的技术路线。 方法：首先分离纯化人体脂肪干细胞,采用新型植物诱导剂Conophylline与其他诱导因子的不同组合将脂肪干细胞诱导分化为胰岛样细胞团,观察不同组合的诱导分化效率,并利用特异性染色、RT-PCR,免疫细胞化学等方法对诱导分化后的细胞团在基因水平与蛋白水平上进行鉴定,最后用ELISA法检测细胞团在不同浓度葡萄糖刺激下胰岛素的分泌情况。 结果与结论：脂肪干细胞具有多能干细胞特性,可诱导分化为具有胰岛素分泌和葡萄糖浓度反应性类胰岛细胞团;Conophylline与尼克酰胺联合诱导可大幅度提高诱导分化效率。     

Abnormalities in the functioning of adipocytes from R6/2 mice that are transgenic for the Huntington's disease mutation

In an effort to characterize the basis of abnormalities in body weight regulation (i.e. wasting) in Huntington's disease (HD), we examined adipocytes in a transgenic model of HD, the R6/2 mouse. These mice typically show severe wasting beginning at approximately 12 weeks of age and die between 12 and 15 weeks. Despite an overall growth retardation compared with wild-type littermates, we observed an enhanced accumulation of body fat at 8-9 weeks of age in R6/2 mice fed laboratory chow or a synthetic high fat, high sugar diet. The obesity was not accompanied by symptoms associated with diabetes, as there were no abnormalities in serum glucose, serum insulin or the ability of insulin to stimulate glucose metabolism in epididymal adipose tissue. As expected, the obesity in the high fat, high sugar-fed R6/2 mice was accompanied by increased serum leptin. The ability of insulin to stimulate leptin release from isolated epididymal adipose tissue was also enhanced in R6/2 mice. In contrast, the ability of isoproterenol to inhibit leptin release was reduced in adipose tissue from R6/2 mice, as was the lipolytic effect of isoproterenol. These data suggest that the obesity observed at 8-9 weeks in R6/2 mice may stem from a defect in fat breakdown by adipocytes.     

Wearable and implantable pancreas substitutes

A lifelong-implanted and completely automated artificial or bioartificial pancreas (BAP) is the holy grail for type 1 diabetes treatment, and could be a definitive solution even for other severe pathologies, such as pancreatitis and pancreas cancer. Technology has made several important steps forward in the last years, providing new hope for the realization of such devices, whose feasibility is strictly connected to advances in glucose sensor technology, subcutaneous and intraperitoneal insulin pump development, the design of closed-loop control algorithms for mechatronic pancreases, as well as cell and tissue engineering and cell encapsulation for biohybrid pancreases. Furthermore, smart integration of the mentioned components and biocompatibility issues must be addressed, bearing in mind that, for mechatronic pancreases, it is most important to consider how to recharge implanted batteries and refill implanted insulin reservoirs without requiring periodic surgical interventions. This review describes recent advancements in technologies and concepts related to artificial and bioartificial pancreases, and assesses how far we are from a lifelong-implanted and self-working pancreas substitute that can fully restore the quality of life of a diabetic (or other type of) patient.     

Endocrine pancreas in cystic fibrosis: an immunohistochemical study

The pancreases of 17 patients who had cystic fibrosis with and without diabetes mellitus were evaluated at autopsy by routine staining and immunohistochemical methods for insulin, glucagon, somatostatin, and pancreatic polypeptide. Qualitative assessment of the number of islets of Langerhans and the degrees of exocrine pancreatic atrophy, fibrosis, and fat replacement was made for each pancreas. Quantitative assessment of islet composition was performed in 15 of the 17 based on the immunochemical reactivity of each cell type. Nondiabetic patients with cystic fibrosis in the latter part of the first decade of life have classic fibrocystic changes of the pancreas, with some persisting exocrine tissue, islets that appear normal, and prominent nesidioblastosis. The latter process may protect these patients from glucose intolerance. Young adult diabetic patients with cystic fibrosis have total loss of exocrine pancreas with fat replacement, lack of nesidioblastosis, a qualitative decrease in the number of islets, fibrosis of and amyloid deposits in islets, decreased numbers of insulin-containing cells in each islet, and atrophy of islet cells, probably resulting from progressive ischemia. Although the potential exists for an increasing incidence of diabetes mellitus in patients with cystic fibrosis as their life spans increase, individual variation occurs in this disease.     

Extracts of rat amniotic fluid contain a potent inducer of intestinal crypt formation

The effect of acid, basic, and organic extracts from Long Evans rat amniotic fluid (RAF) and from Swiss ICR mouse amniotic fluid (MAF) was studied on 15-day fetal mouse duodenal mucosa in organ culture. Amniotic fluids (20 ml) were lyophilized and extracted (1) with CHCl₃:MeOH and the organic phase was evaporated; then (2), the residue was acidified with a solution of 0.1 N HCl in 10% acetic acid and the liquid phase was lyophilized; finally (3), 0.01 M NH₄OH was added to the residue and the liquid phase was lyophilized. The product of each extraction was added to 20 ml of Trowell T8 medium. Acid and basic extracts of RAF and MAF have no effect on the formation of duodenal villi and crypts after 48 hours of culture. With the organic extract of MAF, small villi are present after 48 hours of culture and absorptive cells are poorly differentiated. With the organic extract of RAF, well-developed villi have differentiated after 48 hours of culture; moreover, crypts are present at the same stage and Paneth cells are identified within these crypts. During the 8–10 hour period, the explants cultured with the Trowell T8 medium supplemented with RAF or MAF organic extracts show a 35% increase in ³H-thymidine incorporation over the controls cultured with Trowell T8 medium alone. These results indicate that organic extracts from MAF and RAF are able to promote villus formation in undifferentiated explants from 15-day fetal mouse duodenum in organ culture. Furthermore, RAF organic extract contains a factor that can induce the formation of duodenal crypts and the differentiation of Paneth cells in culture at least 2 days before their normal appearance.