Bernard E. Tuch

Safety and Viability of Microencapsulated Human Islets Transplanted Into Diabetic Humans

OBJECTIVE Transplantation of insulin-producing cells placed inside microcapsules is being trialled to overcome the need for immunosuppressive therapy. RESEARCH DESIGN AND METHODS Four type 1 diabetic patients with no detectable C-peptide received an intraperitoneal infusion of islets inside microcapsules of barium alginate (mean 178,200 islet equivalents on each of eight occasions). RESULTS C-peptide was detected on day 1 post-transplantation, and blood glucose levels and insulin requirements decreased. C-peptide was undetectable by 1–4 weeks. In a multi-islet recipient, C-peptide was detected at 6 weeks after the third infusion and remains detectable at 2.5 years. Neither insulin requirements nor glycemic control was affected. Capsules recovered at 16 months were surrounded by fibrous tissue and contained necrotic islets. No major side effects or infection occurred. CONCLUSIONS While allografting of encapsulated human islets is safe, efficacy of the cells needs to improve for the therapy to make an impact on the clinical scene.

Effects of High-Density Lipoproteins on Pancreatic β-Cell Insulin Secretion

Objective—Type 2 diabetes is characterized by impaired &bgr;-cell secretory function, insulin resistance, reduced high-density lipoprotein (HDL) levels, and increased cardiovascular risk. Given the current interest in therapeutic interventions that raise HDLs levels, this study investigates the effects of HDLs on insulin secretion from &bgr;-cells. Methods and Results—Incubation of Min6 cells and primary islets under basal or high-glucose conditions with either apolipoprotein (apo) A-I or apoA-II in the lipid-free form, as a constituent of discoidal reconstituted HDLs (rHDLs), or with HDLs isolated from human plasma increased insulin secretion up to 5-fold in a calcium-dependent manner. The increase was time and concentration dependent. It was also KATP channel and glucose metabolism dependent under high-glucose, but not low-glucose, conditions. The lipid-free apolipoprotein-mediated increase in insulin secretion was ATP binding cassette (ABC) transporter A1 and scavenger receptor-B1 dependent. The rHDL-mediated increase in insulin secretion was ABCG1 dependent. Exposure of &bgr;-cells to lipid-free apolipoproteins also increased insulin mRNA expression and insulin secretion without significantly depleting intracellular insulin or cholesterol levels. Conclusion—These results establish that lipid-free and lipid-associated apoA-I and apoA-II increase &bgr;-cell insulin secretion and indicate that interventions that raise HDLs levels may be beneficial in type 2 diabetes.

Transmission of porcine endogenous retroviruses in severe combined immunodeficient mice xenotransplanted with fetal porcine pancreatic cells.

Background. Xenotransplantationusing pig organs or tissues may alleviate the human donor organ shortage. However, one concern is the potential transmission of pig pathogens to humans, especially pig endogenous retroviruses (PERV), which infect human cell lines in vitro. In this report, the cross-species in vivo transmission of PERV by xenotransplantation was studied using a severe combined immunodeficient (SCID) mouse model. Methods. Twenty-one SCID mice were transplanted with fetal pig pancreatic cells and left for periods from three to 41 weeks before being killed. DNA and RNA were extracted from liver, spleen, and brain of these mice, and examined for PERV using nested polymerase chain reaction (PCR) and reverse transcriptase-PCR. The pig mitochondrial cytochrome oxidase II subunit gene (COII) was also amplified to monitor the presence of pig cell microchimerism in xenotransplanted tissues, and a housekeeping gene was included to monitor the DNA quality and quantity. Results. Examination of 39 DNA samples from tissues of the 21 xenografted mice identified two mouse tissues (M4-liver and M19-spleen) that were positive for PERV but negative for COII. A total of 23 (59%) of the mouse tissues were positive for both PERV and COII, 6 (16%) were negative for both, and 8 (20%) were positive for COII only. PCR and direct sequencing of the PCR products identified three PERV variants, which were different from the PERV sequence detected by PCR direct sequencing from the pig donor cells. Conclusions. The PERV+/COII− results from M4-liver and M19-spleen indicated the presence of PERV transmission from pig to mouse tissue. The PERV variants detected in the mouse tissues indicated that different PERVs were transmissible from the pig to mouse tissue during xenotransplantation. The negative reverse transcriptase-PCR results for PERV from three mouse samples including M4-liver and M19-spleen suggest there was no active PERV transcription in the mouse tissues, although this would need to be studied further.

Alginate microcapsule for propagation and directed differentiation of hESCs to definitive endoderm.

Human embryonic stem cells (hESCs) are potential renewable sources of cells in replacement therapies for many diseases including type 1 diabetes. We have established a three dimensional (3D) model to culture and differentiate hESCs that are encapsulated in calcium alginate microcapsules. This system promotes cellular interactions that are essential for both maintaining pluripotency and differentiation. This 3D model also provides opportunity to separate out hESCs from fibroblasts used as feeder layer during culture. In this study, we compared the viability and proliferation of the encapsulated hESCs cultured in serum replacement (SR) medium, human fetal fibroblast-conditioned medium (hFF-CM), in the presence and absence of Y-27632, a ROCK inhibitor. Treatment of hESCs with Y-27632 promoted cell survival, cell cluster formation and proliferation rate in both SR medium and hFF-CM. These encapsulated hESC clusters were then directly differentiated to definitive endoderm cells that expressed mesendoderm (Brachyury 70-fold), definitive endoderm (SOX17>300-fold, FOXA2>800-fold, and CXCR4>100-fold) and primitive gut tube (HNF1beta>120-fold) as compared to the undifferentiated hESCs. These data show that microcapsules can be used for differentiation of hESCs into definitive endoderm in 3D and could have potential application for immune-isolation and prevention of teratomas formation of hESCs during transplantation.

Growth and Differentiation of Embryoid Bodies Derived from Human Embryonic Stem Cells: Effect of Glucose and Basic Fibroblast Growth Factor

Abstract Differentiation of embryonic stem (ES) cells generally occurs after formation of three-dimensional cell aggregates, known as embryoid bodies (EBs). This differentiation occurs following suspension culturing of EBs in media containing a high (25 mM) glucose concentration. Although high-glucose-containing media is used for maintenance and proliferation of ES cells, it has not been demonstrated whether this is a necessary requirement for EB development. To address this, we examined the growth and differentiation of EBs established in 0-mM, 5.5-mM (physiological), and 25-mM (high) glucose concentrations, through morphometric analysis and examination of gene and protein expression. The effect on EB development of supplementation with basic fibroblast growth factor (FGF2) was also studied. We report that the greatest rate of EB growth occurs in 5.5 mM glucose media. A morphological study of EBs over 104 days duration under glucose-containing conditions demonstrated the development of all three major embryonic cell types. The difference from normal human development was obvious in the lack of rostrocaudal control by the notochord. In the latest stages of development, the main tissue observed appeared to be cartilage and cells of a mesodermal lineage. We conclude that physiological glucose concentrations are suitable for the culturing of EBs, that the addition of FGF2 enhances the temporal expression of genes including POU5F1, nestin, FOXA2, ONECUT1, NEUROD1, PAX6, and insulin, and that EBs can be cultured in vitro for long periods, allowing for further examination of developmental processes.

Transplantation of 3D scaffolds seeded with human embryonic stem cells: biological features of surrogate tissue and teratoma-forming potential

AIM To generate complex surrogate tissue by transplanting 3D scaffolds seeded with human embryonic stem cells (hESCs) between the liver lobules of severe combined immunodeficient (SCID) mice and to assess the teratoma-forming potential. MATERIALS & METHODS 3D poly-(lactic-co-glycolic acid) (PLGA) scaffolds coated with laminin were seeded with hESCs and then transplanted between the liver lobules of SCID mice. After a period of in vivo differentiation, the scaffolds were retrieved and analyzed using reverse transcription polymerase chain reaction, immunofluorescent staining and scanning electron microscopy. RESULTS A proportion of the hESCs within the scaffolds differentiated into cells that produced proteins characteristic of specific tissues, including endoderm and pancreatic markers glucogon-like peptide-1 receptor, islet amyloid polypeptide and Insulin. Markers of hepatic and neuronal lineages were also investigated. Major matrix proteins abundant in multiple tissue types, including collagen I, laminin and collagen IV, were found to be profuse within the scaffold pores. Transplantation of the seeded scaffolds between liver lobules also resulted in extensive vascularization both from host blood vessel incursion and the differentiation of hESCs into endothelial progenitor cells. An investigation of teratoma-forming potential demonstrated that transplantation of 3D scaffolds seeded with hESCs will, under certain conditions, lead to the growth of teratomas. DISCUSSION Transplantation of 3D scaffolds seeded with hESCs between liver lobules resulted in the development of surrogate tissue containing cells that produced proteins representing the pancreatic, hepatic and neuronal lineages, the assembly of an extracellular matrix structure and the formation of a vasculature. hESCs seeded within 3D scaffolds and transplanted into SCID mice were capable of forming teratomas. However, the formation and progression of teratoma growth is shown to be dependant on both the site of transplantation and the treatment of cells prior to transplantation.

Differentiation of encapsulated embryonic stem cells after transplantation.

Background. Embryonic stem cells (ESC) when transplanted into recipients with different major histocompatibility antigens may be rejected, especially as cells differentiate and expression of these antigens increases. One method to prevent rejection is to place the developing ESC in microcapsules. It is currently unknown what effect encapsulation has on the ability of ESC to differentiate. Methods. Human ESC (hESC; hES03 line) and mouse ESC (mESC; R1 line) were encapsulated in 2.2% barium alginate and transplanted intraperitoneally in SCID and BALB/c mice respectively. Cell morphology, viability, and gene characterization were assessed after retrieving the capsules up to four weeks from SCID mice and three months from BALB/c mice. Results. Encapsulation prevented hESC and mESC from forming teratomas up to four weeks and three months, respectively. mESC but not hESC formed aggregates within the capsules, which remained free of fibrosis. Some but not all the transplanted encapsulated hESC differentiated towards all three lineages, but more so towards an endodermal lineage as shown by increased expression of alpha fetoprotein. This was similar to what occurred when encapsulated and non-encapsulated hESC were cultured in vitro for two weeks. In contrast to the hESC, transplanted encapsulated mESC differentiated mostly towards an ectodermal lineage as shown by increased expression of nestin and glial fibrillary acidic protein. In vitro, encapsulated and nonencapsulated mESC also began to differentiate, but not down any specific lineage. Conclusions. Encapsulated ESC do differentiate, although along multiple pathways, both when transplanted and maintained in culture, just as nonencapsulated ESC do when removed from their feeder layer.

Mechanisms of arginine-induced increase in cytosolic calcium concentration in the beta-cell line NIT-1

Summary The effects of l-arginine and its analogues NG-nitro-l-arginine, NG-methyl-l-arginine, l-homoarginine and d-arginine on cytosolic calcium concentration were investigated to characterise the mechanisms of arginine-induced stimulation and to determine if nitric oxide production played a role in this stimulation. NIT-1 cells, a transgenic beta-cell line, were used for this purpose since they release insulin in response to typical beta-cell stimuli. Our data demonstrate that the arginine-induced increase in cytosolic calcium concentration was completely dependent on the influx of extracellular Ca2 + via verapamil-sensitive voltage-activated Ca2 + channels and that arginine stimulation requires the presence of a nutrient in order to cause an increase in cytosolic calcium concentration. The nutrient likely acted by closing the K +ATP channels, since its effect could be inhibited by activation of these channels with diazoxide. l-arginine, as well as nitro-arginine and methyl-arginine which are not substrates for the production of nitric oxide, caused similar increases in cytosolic calcium concentration. Non-metabolisable arginine analogues homoarginine and d-arginine also caused increases in the cytosolic calcium concentration although not to the same extent. Insulin secretion was enhanced to the same extent by all analogues of arginine. It can be concluded that the arginine-induced increase in cytosolic calcium concentration in NIT-1 cells is attributable to an electrogenic effect following the transport of arginine leading to depolarisation of the plasma membrane potential, although metabolism of the amino acid itself may also partially contribute to the response. [Diabetologia (1997) 40: 374–382]

Gene therapy of diabetes: glucose-stimulated insulin secretion in a human hepatoma cell line (HEP G2ins/g).

In order to design a feasible somatic cell gene delivery system for the treatment of type I diabetes, a suitable cell type needs to be determined. We have previously shown that the stable transfection of the full-length insulin cDNA into the human liver cell line, (HEP G2ins) resulted in synthesis, storage and acute regulated release of insulin to analogues of cAMP, but not to the physiological stimulus glucose. In attempting to explain the lack of glucose responsiveness of the HEP G2ins cells we have stably transfected these cells with the human islet glucose transporter GLUT 2 (HEP G2ins/g cells). The HEP G2ins/g cell clones exhibit glucose-stimulated insulin secretion and glucose potentiation of the secretory response to nonglucose secreta- gogues. While glucose responsiveness commenced at a lower concentration than normal islets, a secretion curve approaching normal physiological conditions was generated. Immunoelectron microscopy revealed the presence of insulin-containing granules, similar in size and appearance to those of the normal beta cell. These results demonstrate that while it is most likely that the HEP G2ins/g cell line predominantly secretes insulin via the constitutive pathway, significant acute regulated release was seen in response to glucose, and thus represents significant progress in the creation of a genetically engineered ‘artificial beta cell’ from a human hepatocyte cell line.

Function of a genetically modified human liver cell line that stores, processes and secretes insulin

An alternative approach to the treatment of type I diabetes is the use of genetically altered neoplastic liver cells to synthesize, store and secrete insulin. To try and achieve this goal we modified a human liver cell line, HUH7, by transfecting it with human insulin cDNA under the control of the cytomegalovirus promoter. The HUH7-ins cells created were able to synthesize insulin in a similar manner to that which occurs in pancreatic β cells. They secreted insulin in a regulated manner in response to glucose, calcium and theophylline, the dose–response curve for glucose being near-physiological. Perifusion studies showed that secretion was rapid and tightly controlled. Removal of calcium resulted in loss of glucose stimulation while addition of brefeldin A resulted in a 30% diminution of effect, indicating that constitutive release of insulin occurred to a small extent. Insulin was stored in granules within the cytoplasm. When transplanted into diabetic immunoincompetent mice, the cells synthesized, processed, stored and secreted diarginyl insulin in a rapid regulated manner in response to glucose. Constitutive release of insulin also occurred and was greater than regulated secretion. Blood glucose levels of the mice were normalized but ultimately became subnormal due to continued proliferation of cells. Examination of the HUH7-ins cells as well as the parent cell line for β cell transcription factors showed the presence of NeuroD but not PDX-1. PC1 and PC2 were also present in both cell types. Thus, the parent HUH7 cell line possessed a number of endocrine pancreatic features that reflect the common endodermal ancestry of liver and pancreas, perhaps as a result of ontogenetic regression of the neoplastic liver cell from which the line was derived. Introduction of the insulin gene under the control of the CMV promoter induced changes in these cells to make them function to some extent like pancreatic β cells. Our results support the view that neoplastic liver cells can be induced to become substitute pancreatic β cells and become a therapy for the treatment of type I diabetes.