PRINCIPLES OF TISSUE ENGINEERING 3RD EDITION - PART 2 (end) doc

I. Introduction II. Cell Types for Pancreatic Substitutes III. Construct Technology IV. In Vivo Implantation V. Concluding Remarks VI. Acknowledgments VII. References Bioartifi cial Pancreas Athanassios Sambanis Principles of Tissue Engineering, 3 rd Edition ed. by Lanza, Langer, and Vacanti Copyright © 2007, Elsevier, Inc. All rights reserved. I. INTRODUCTION Diabetes is a signifi cant health problem, affecting an estimated 20.8 million people in the United States alone, with nearly 1.8 million affl icted with type 1 diabetes [http:// diabetes.niddk.nih.gov/dm/pubs/statistics/index.htm#7]. Type 1 diabetes results from the loss of insulin-producing cell mass (the β-cells of pancreatic islets) due to autoim- mune attack. Type 2 diabetes has a more complicated disease etiology and can be the result of not producing enough insulin and/or the body’s developing a resistance to insulin. Although initially controlled by diet, exercise, and oral medication, type 2 diabetes often progresses toward insulin dependence. It is estimated that insulin-dependent diabetics (both types 1 and 2) exceed 4 million people in the United States. Although insulin-dependent diabetes (IDD) is considered a chronic disease, even the most vigilant insulin therapy cannot reproduce the precise metabolic control present in the nondiseased state. The poor temporal match between glucose load and insulin activity leads to a number of complications, including increased risk of heart disease, kidney failure, blindness, and amputation due to peripheral nerve damage. Providing more physiological control would alleviate many of the diabetes-related health problems, as suggested by fi ndings from the Diabetes Control and Complications Trial (The Diabetes Control and Complications Trial Research Group, 1997) and its continu- ation study (DCCT/EDIC NEJM 353(25):2643–53, 2005). Cell-based therapies, which provide continuous regulation of blood glucose through physiologic secretion of insulin, have the potential to revolutionize diabetes care. Several directions are being considered for cell-based therapies of IDD, including implantation of immunopro- tected allogeneic or xenogeneic islets, of continuous cell lines, or of engineered non-β-cells. For allogeneic islet trans- plantation, a protocol developed by physicians at the Uni- versity of Edmonton (Shapiro et al., 2001a, 2001b, 2001c, Bigam and Shapiro, 2004) has dramatically improved the survivability of grafts. The protocol uses human islets from cadaveric donors, which are implanted in the liver of care- fully selected diabetic recipients via portal vein injection. The success of the Edmonton protocol is attributed to two modifi cations relative to earlier islet transplantation studies: the use of a higher number of islets and the implementation of a more benign, steroid-free immunosuppressive regimen. However, two barriers prevent the widespread application of this therapy. The fi rst is the limited availability of human tissue, because generally more than one cadaveric donor pancreas is needed for the treatment of a single recipient. The second is the need for life-long immunosuppression, which, even with the more benign protocols, results in long- term side effects to the patients. A tissue-engineered pancreatic substitute aims to address these limitations by using alternative cell sources, relaxing the cell availability limitation, and by reducing or eliminating the immunosuppressive regimen necessary for survival of the graft. A number of signifi cant challenges are Chapter Forty-Two Ch042_P370615.indd 619Ch042_P370615.indd 619 6/1/2007 3:01:15 PM6/1/2007 3:01:15 PM 620 CHAPTER FORTY-TWO • BIOARTIFICIAL PANCREAS facing the development of such a substitute, however. These include procuring cells at clinically relevant quantities; the immune acceptance of the cells, which is exacerbated in type 1 diabetes by the resident autoimmunity in the patients; and the fact that diabetes is not an immediately life-threat- ening disease, so any other therapy will have to be more effi cacious and/or less invasive than the current standard treatment of daily blood glucose monitoring and insulin injections. In general, developing a functional living-tissue replace- ment requires advances and integration of several types of technology (Nerem and Sambanis, 1995). These are (1) cell technology, which addresses the procurement of functional cells at the levels needed for clinical applications; (2) con- struct technology, which involves combining the cells with biomaterials in functional three-dimensional confi gura- tions. Construct manufacturing at the appropriate scale, and preservation, as needed for off-the-shelf availability, also fall under this set of technologies; (3) technologies for in vivo integration, which address the issues of construct immune acceptance, in vivo safety and effi cacy, and moni- toring of construct integrity and function postimplantation. The same three types of technology need also be developed for a pancreatic substitute. It should be noted, however, that the critical technologies differ, depending on the type of cells used. With allogeneic or xenogeneic islets or beta-cells, the major challenge is the immune acceptance of the implant. In this case, encapsulation of the cells in permse- lective membranes, which allow passage of low-molecular- weight nutrients and metabolites but exclude larger antibodies and cytotoxic cells of the host, may assist the immune acceptance of the graft. With cell therapies based on potentially autologous nonpancreatic cells, targeted by gene expression vectors in vivo, or retrieved surgically, engineered ex vivo, and returned to the host, the major challenge is engineering insulin secretion in precise response to physiologic stimuli. Lastly, with stem or progenitor cells, the primary hurdle is their reproducible differentiation into cells of the pancreatic β-phenotype. Figure 42.1 shows schematically the two general therapeutic approaches based on allo- or xenogeneic cells (Fig. 42.1A) or autologous cells (Fig. 42.1B). This chapter is therefore organized as follows. We fi rst describe the types of cells that have been used or are of potential use in engineering a pancreatic substitute. We then discuss issues of construct technology, specifi cally encapsulation methods and the relevant biomaterials, man- ufacturing issues, and preservation of the constructs. The challenges of in vivo integration and results from in vivo experiments with pancreatic substitutes are presented next. We conclude by offering a perspective on the current status and the future challenges in developing an effi cacious, clini- cally applicable bioartifi cial pancreas. II. CELL TYPES FOR PANCREATIC SUBSTITUTES Islets Despite several efforts, the in vitro expansion of primary human islets has met with limited success. Adult human islets are diffi cult to propagate in culture, and their expan- sion leads to dedifferentiation, generally manifested as loss of insulin secretory capacity. Although there exist reports on the redifferentiation of expanded islets (Lechner et al., 2005; Ouziel-Yahalom et al., 2006) and of nonislet pancreatic cells, which are discarded after islet isolation (Todorov et al., 2006), the phenotypic stability and the in vivo effi cacy of these cells remain unclear. Additionally, with expanded and A. Allo- or Xenogeneic Cells B. Autologous Cells Implantation Cell retrieval Ex vivo manipulation Cell storage Cell storage Cell amplification Cell encapsulation Implantation Capsule storage Cell procurement In vivo gene therapy A. Allo- or Xenogeneic Cells B. Autologous Cells Implantation Cell retrieval Ex vivo manipulation Cell storage Cell storage Cell amplification Cell encapsulation Implantation Capsule storage Capsule storage Cell procurement In vivo gene therapy FIG. 42.1. Approaches for bioartifi cial pancreas development using allo- or xenogeneic cells (A) and autologous cells (B). In (A), islets are procured from pancreatic tissue, or cell lines are thawed from cryostorage and expanded in culture; cells are encapsulated for immunoprotection before they are implanted to achieve a therapeutic effect; encapsulated cells may also be cryopreserved for inventory management and sterility testing. In (B), cells are retrieved surgi- cally from the patient; manipulated ex vivo phenotypically and/or genetically in order to express β-cell characteristics, and in particular physiologically responsive insulin secretion; the cells are implanted for a therapeutic effect either by themselves or, preferably, after incorporation in a three-dimensional substitute; some of the cells may be cryopreserved for later use by the same individual. In in vivo gene therapy approaches, a transgene for insulin expres- sion is directly introduced into the host and expressed by cells in nonpancreatic tissues. Ch042_P370615.indd 620Ch042_P370615.indd 620 6/1/2007 3:01:16 PM6/1/2007 3:01:16 PM II. CELL TYPES FOR PANCREATIC SUBSTITUTES • 621 redifferentiated islets, it remains unknown whether the insulin-secreting cells arose from the redifferentiation of mature endocrine cells or from an indigenous stem or pro- genitor cell population in the tissue isolate (Todorov et al., 2006). Animal, such as porcine, islets are amply available, and porcine insulin is very similar to human, differing by only one amino acid residue. However, the potential use of porcine tissue is hampered by the unlikely but possible transmission of porcine endogenous retroviruses (PERV) to human hosts as well as by the strong xenograft immuno- genicity that they elicit. Use of closed, PERV-free herds is reasonably expected to alleviate the fi rst problem. With regard to immunogenicity, a combination of less immuno- genic islets, islet encapsulation in permselective barriers, and host immunosuppression may yield long-term survival of the implant. The use of transgenic pigs that do not express the α-Gal (α[1,3]-galactose) epitope is one possible approach for reducing the immunogenicity of the islets. Studies also indicate that neonatal pig islets induce a lower T-cell reac- tivity than adult islets (Bloch et al., 1999), even though the α-Gal epitope is abundant in neonatal islets as well (Rayat et al., 1998). Furthermore, it is possible that the primary antigenic components in islet tissue are the ductal epithelial and vascular endothelial cells, which express prominently the α-Gal epitope; on the other hand, β-cells express the epitope immediately after isolation but not after mainte- nance in culture (Heald et al., 1999). It should also be noted that the large-scale isolation of porcine islets under condi- tions of purity and sterility that will be needed for eventual regulatory approval pose some major technical hurdles, which have not been addressed yet. -Cell Lines Recognizing the substantial diffi culties involved with the procurement and amplifi cation of pancreatic islets, several investigators have pursued the development of con- tinuous cell lines, which can be amplifi ed in culture yet retain key differentiated properties of normal β-cells. One of the fi rst successful developments in this area was the gen- eration of the βTC family of insulinomas, derived from transgenic mice carrying a hybrid insulin-promoted simian virus 40 tumor antigen gene; these cells retained their dif- ferentiated features for about 50 passages in culture (Efrat et al., 1988). The hypersensitive glucose responsiveness of the initial βTC lines was reportedly corrected in subsequent lines by ensuring expression of glucokinase and of the high- K m glucose transporter Glut2, and with no or low expression of hexokinase and of the low-K m transporter Glut1 (Efrat et al., 1993; Knaack et al., 1994). A similar approach was used to develop the mouse MIN-6 cell line that exhibits glucose- responsive secretion of endogenous insulin (Miyazaki et al., 1990). Subsequently, Efrat and coworkers developed the βTC-tet cell line, in which expression of the SV40 T antigen (Tag) oncoprotein is tightly and reversibly regulated by tet- racycline. Thus, cells proliferate when Tag is expressed, and shutting off Tag expression halts cell growth (Efrat et al., 1995; Efrat, 1998). Such reversible transformation is an elegant approach in generating a supply of β-cells via pro- liferation of an inoculum, followed by arrest of the growth of cells when the desirable population size is reached. When retained in capsules, proliferating cells do not grow uncon- trollably, since the dissolved-oxygen concentration in the surrounding milieu can support up to a certain number of viable, metabolically active cells in the capsule volume. This number of viable cells is maintained through equilibration of cell growth and death processes (Papas et al., 1999a, 1999b). Thus, growth arrest is useful primarily in preventing the growth of cells that have escaped from broken capsules in vivo and in reducing the cellular turnover in the capsules. The latter reduces the number of accumulated dead cells in the implant and thus the antigenic load to the host affected by proteins from dead and lysed cells that pass through the capsule material. In a different approach, Newgard and coworkers (Clark et al., 1997) carried out a stepwise introduction of genes related to β-cell performance into a poorly secreting rat insulinoma (RIN) line. In particular, RIN cells were itera- tively engineered to stably express multiple copies of the insulin gene, the glucose transporter Glut2, and the gluco- kinase gene, which are deemed essential for proper expres- sion of β-cell function. Although this is an interesting methodology, it is doubtful that all genes necessary for reproducing β-cell function can be identifi ed and stably expressed in a host cell. Recently, signifi cant progress was made toward establishing a human pancreatic β-cell line that appears functionally equivalent to normal β-cells (Narushima et al., 2005). This was accomplished through a complicated procedure involving retroviral transfection of primary β cells with the SV40 large T antigen and cDNAs of human telomerase reverse transcriptase. This resulted in a reversibly immortalized human β-cell clone, which secreted insulin in response to glucose, expressed β-cell transcrip- tional factors, prohormone convertases 1/3 and 2, which process proinsulin to mature insulin, and restored normo- glycemia upon implantation in diabetic immunodefi cient mice (Narushima et al., 2005). With regard to β-cell lines capable of proliferation under the appropriate conditions, key issues that remain to be addressed include (1) their long-term phenotypic stability, in vitro and in vivo; (2) their potential tumorigenicity, if cells escape from an encapsulation device, especially when these cells are allografts that may evade the hosts’ immune defenses for a longer period of time than acutely rejected xenografts; and (3) their possible recognition by the auto- immune rejection mechanism in type 1 diabetic hosts. Engineered Non– Pancreatic Cells The use of non–β pancreatic cells from the same patient, engineered for insulin secretion, relaxes both the cell availability and immune acceptance limitations that exist with other types of cells. It has been shown that the A-chain/ Ch042_P370615.indd 621Ch042_P370615.indd 621 6/1/2007 3:01:16 PM6/1/2007 3:01:16 PM 622 CHAPTER FORTY-TWO • BIOARTIFICIAL PANCREAS C-peptide and B-chain/C-peptide cleavage sites on the pro- insulin gene can be mutated so that the ubiquitous endo- peptidase furin recognizes and completely processes proinsulin into mature insulin absent of any intermediates (Yanagita et al., 1992). Based on this concept, several non- endocrine cell lines have been successfully transfected to produce immunoreactive insulin, including hepatocytes, myoblasts, and fi broblasts (Yanagita et al., 1993). In a differ- ent approach, Lee and coworkers (2000) expressed a syn- thetic single-chain insulin analog, which does not require posttranslational processing, in hepatocytes. Although recombinant insulin expression is relatively straightforward, a key remaining challenge is achieving the tight regulation of insulin secretion in response to physiologic stimuli, which is needed for achieving normoglycemia in higher animals and, eventually, humans. One approach for achieving regulation of insulin secre- tion is through regulation of biosynthesis at the gene tran- scription level, as realized in hepatocytes by Thule et al. (Thule et al., 2000; Thule and Liu, 2000) and Lee et al. (2000). Besides the ability to confer transcriptional-level regulation, hepatocytes are particularly attractive as producers of recombinant insulin due to their high synthetic and secre- tory capacity and their expression of glucokinase and Glut2 (Cha et al., 2000; Lannoy et al., 2002). Hepatic delivery by viral vectors and expression of the glucose-responsive insulin transgene in diabetic rats controlled the hypergly- cemic state for extended periods of time (Lee et al., 2000; Thule and Liu, 2000; Olson et al., 2003). Nevertheless, trans- criptional regulation is sluggish, involving long time lags between stimulation of cells with a secretagogue and induced insulin secretion as well as between removal of the secretagogue and down-regulation of the secretory response (Tang and Sambanis, 2003). The latter is physiologically more important, because it means that the cells continue to secrete insulin after glucose has been down-regulated, thus resulting in potentially serious hypoglycemic excursions. Increasing the number of stimulatory glucose elements in a promoter enhances the cellular metabolic responsiveness in vitro (Thule et al., 2000). With regard to secretion down- regulation, Tang and Sambanis (2003) hypothesized that the slow kinetics of this process following removal of the tran- scriptional activator are due to the stability of the preproin- sulin mRNA, which continues to become translated after transcription has been turned off. Using a modifi ed prepro- insulin cDNA that produced an mRNA with two more copies of the insulin gene downstream of the stop codon resulted in preproinsulin mRNA subjected to nonsense mediated decay and thus destabilized. This signifi cantly expedited the kinetics of secretion down-regulation on turning off tran- scription (Tang and Sambanis, 2003). Thus, the combina- tion of optimal transcriptional regulation with transgene message destabilization promises further improvements in insulin secretion dynamics from transcriptionally regulated hepatic cells. It should be noted, however, that despite the time delays inherent in transcriptional regulation, hepatic insulin gene therapy is suffi cient to sustain vascular nitric oxide production and inhibit acute development of diabetes-associated endothelial dysfunction in diabetic rats (Thule et al., 2006). Hence, many aspects of the thera- peutic potential of hepatic insulin expression remain to be explored. Another appealing target cell type is endocrine cells, which possess a regulated secretory pathway and the enzymatic machinery needed to process authentic proinsu- lin into insulin. Early work in this area involved expression of recombinant insulin in the anterior pituitary mouse AtT-20 cell line (Moore et al., 1983), which can be sub - jected to repeated episodes of induced insulin secretion using nonmetabolic secretagogues (Sambanis et al., 1990). Cotransfection with genes encoding the glucose transporter Glut-2 and glucokinase resulted in glucose-responsive insulin secretion (Hughes et al., 1992, 1993). However, limi- tations of this approach include possible instabilities in the cellular phenotype and the continued secretion of endoge- nous hormones, such as adrenocorticotropic hormone from AtT-20 cells, which are not compatible with prandial metabolism. In this regard, endocrine cells of the intestinal epithe- lium, or enteroendocrine cells, are especially promising. Enteroendocrine cells secrete their incretin products in a tightly controlled manner that closely parallels the secretion of insulin following oral glucose load in human subjects; incretin hormones are fully compatible with prandial metabolism and glucose regulation (Schirra et al., 1996; Kieffer and Habener, 1999). As with β-cells, enteroendocrine cells are polar, with sensing microvilli on their luminal side and secretory granules docked at the basolateral side, adja- cent to capillaries. Released incretin hormones include the glucagon-like peptides (GLP-1 and GLP-2) from intestinal L-cells and glucose-dependent insulinotropic polypeptide (GIP) from K-cells, which potentiate insulin production from the pancreas after a meal (Drucker, 2002). The impor- tance of enteroendocrine cells (and, in particular, L-cells) was fi rst put forward by Creutzfeldt (1974), whose primary interest in these cells was for the prospect of using GLP-1 for the treatment of type 2 diabetes. Furthermore, ground- breaking work by Cheung et al. (2000) demonstrated that insulin produced and secreted by genetically modifi ed intestinal K-cells of transgenic mice prevented the animals from becoming diabetic after injection with streptozotocin (STZ), which specifi cally kills the β-cells of the pancreas. This is an important proof-of-concept study, which showed that enteroendocrine cell–produced insulin can provide regulation of blood glucose levels. Subsequent work with a human intestinal L-cell line demonstrated that these cells can be effectively transduced to express recombinant human insulin, which colocalizes in secretory vesicles with endog- enous GLP-1 and thus is secreted with identical kinetics to GLP-1 in response to stimuli (Tang and Sambanis, 2003, Ch042_P370615.indd 622Ch042_P370615.indd 622 6/1/2007 3:01:16 PM6/1/2007 3:01:16 PM 2004). The intestinal tract could be considered an attractive target for gene therapy because of its large size, making it the largest endocrine organ in the body (Wang et al., 2004); however, enteroendocrine cell gene therapy faces serious diffi culties due to anatomic complexity, with the entero- endocrine cells being located at the base of invaginations of the gut mucosa called crypts, the very harsh conditions in the stomach and intestine, and the rapid turnover of the intestinal epithelium. Contrary to direct in vivo gene delivery, ex vivo gene therapy involves retrieving the target cells surgically, culturing them and possibly expanding them in vitro, genetically engineering them to express the desired proper- ties, and then returning them to the host, either as such or in a three-dimensional tissue substitute. It is generally thought that the ex vivo approach is advantageous, for it allows for the thorough characterization of the genetically engineered cells prior to implantation, possibly for the preservation of some of the cells for later use by the same individual and, importantly, for localization and retrievability of the implant. However, the challenges imposed by the ex vivo approach, including the surgical retrieval, culturing, and in vitro genetic engineering of the target cells are signifi cant, so such methods are currently under development. Differentiated Stem or Progenitor Cells Naturally, throughout life, islets turn over slowly, and new, small islets are continually generated from ductal pro- genitors (Finegood et al., 1995; Bonner-Weir and Sharma, 2002). There is also considerable evidence that adult plu- ripotent stem cells may be a possible source of new islets (Bonner-Weir et al., 2000; Ramiya et al., 2000; Kojima et al., 2003). However, efforts to regenerate β-cells in vitro or in vivo by differentiation of embryonic or adult stem or pan- creatic progenitor cells have produced mixed results. Insulin- producing, glucose-responsive cells, as well as other pan- creatic endocrine cells, have been generated from mouse embryonic stem cells (Lumelsky et al., 2001). Insulin-secret- ing cells obtained from embryonic stem cells reversed hyperglycemia when implanted in mice rendered diabetic by STZ injection (Soria et al., 2000). In another study, mouse embryonic stem cells transfected to express constitutively Pax4, a transcription factor essential for β-cell development, differentiated into insulin-producing cells and normalized blood glucose when implanted in STZ-diabetic mice (Blyszczuk et al., 2003). On the other hand, other studies do not support differentiation of embryonic stem cells into the β-cell phenotype (Rajagopal et al., 2003). Overall, the mixed and somewhat inconsistent results point to the consider- able work that needs to be done before stem or progenitor cells can be reliably differentiated into β cells at a clinically relevant scale. Harnessing the in vivo regenerative capacity of the pancreatic endocrine system may present a promis- ing alternative approach. Engineering of Cells for Enhanced Survival In Vivo Because islets and other insulin-secreting cells experi- ence stressful conditions during in vitro handling and in vivo postimplantation, several strategies have been imple- mented to enhance islet or nonislet cell survival in pancre- atic substitutes. Strategies generally focus on improving the immune acceptance of the graft, enhancing its resistance to cytokines, and reducing its susceptibility to apoptosis. Phe- notypic manipulations include extended culturing of neo- natal and pig islets at 37°C, which apparently reduces their immunogenicity, possibly by down-regulating the major histocompatibility class 1 antigens on the islet surface; islet pretreatment with TGF-β1; and enzymatic treatment of pig islets with α-galactosidase to reduce the a-galactosyl epitope on islets (Prokop, 2001). However, the permanency of these modifi cations is unknown. For instance, a-galactosyl epi- topes reappear on islets 48 hours after treatment with α- galactosidase. With proliferative cell lines destined for recombinant insulin expression, selection of clones resis- tant to cytokines appears feasible (Chen et al., 2000). Gene chip analysis of resistant cells may then be used to identify the genes responsible for conferring cytokine resistance. Genetic modifi cations for improving survival in vivo may offer prolonged expression of the desired properties relative to phenotypic manipulations, but they also present the possibility of modifying the islets in additional, undesir- able ways. Notable among the various proposed approaches, reviewed in Jun and Yoon (2005), are the expression of the immunomodulating cytokines IL-4 or a combination of IL- 10 and TGF-β, which promoted graft survival by preventing immune attack in mice; and the expression of the antiapop- totic bcl-2 gene using a replication defective herpes simplex virus, which resulted in protection of β-cells from a cytokine mixture of interleukin-1β, TNF-α, and IFN-γ in vitro. III. CONSTRUCT TECHNOLOGY Construct technology focuses on associating cells with biocompatible materials in functional three-dimensional confi gurations. Depending on the type of cells used, the primary function of the construct can be one or more of the following: to immunoprotect the cells postimplantation, to enable cell function, to localize insulin delivery in vivo, or to provide retrievability of the implanted cells. Encapsulated Cell Systems Encapsulation for immunoprotection involves sur- rounding the cells with a permselective barrier, in essence an ultrafi ltration membrane, which allows passage of low- molecular-weight nutrients and metabolites, including insulin, but excludes larger antibodies and cytototoxic cells of the host. Figure 42.2 summarizes the common types of encapsulation devices, which include spherical microcap- sules, tubular or planar diffusion chambers, thin sheets, and vascular devices. III. CONSTRUCT TECHNOLOGY • 623 Ch042_P370615.indd 623Ch042_P370615.indd 623 6/1/2007 3:01:16 PM6/1/2007 3:01:16 PM 624 CHAPTER FORTY-TWO • BIOARTIFICIAL PANCREAS Encapsulation can be pursued via one of two general approaches. With capsules fabricated using water-based chemistry, cells are fi rst suspended in un-cross-linked polymer, which is then extruded as droplets into a solution of the cross-linking agent. A typical example here is the very commonly used alginate encapsulation. Alginate is a complex mixture of polysaccharides obtained from sea- weeds, which forms a viscous solution in physiologic saline. Islets or other insulin-secreting cells are suspended in sodium alginate, and droplets are extruded into a solution of calcium chloride. Calcium cross-links alginate, instanta- neously trapping the cells within the gel. The size of the droplets, hence also of the cross-linked beads, can be con- trolled by fl owing air parallel to the extrusion needle so that droplets detach at a smaller size than if they were allowed to fall by gravity; or by using an electrostatic droplet genera- tor, in which droplets are detached from the needle by adjusting the electrostatic potential between the needle and the calcium chloride bath. Capsules generated this way can have diameters from a few hundred micrometers to more than one millimeter. Alginate by itself is relatively perme- able; to generate the permselective barrier, beads are treated with a polycationic solution, such as poly-l-lysine or poly- l-ornithine. The reaction time between alginate and the polycation determines the molecular weight cutoff of the resulting membrane. Poly-l-lysine is highly infl ammatory in vivo, however, so beads are coated with a fi nal layer of alginate to improve their biocompatibility. Hence, calcium alginate/poly-l-lysine/alginate (APA) beads are fi nally formed. Treating the beads with a calcium chelator, such as citrate, presumably liquefi es the inner core, forming APA membranes. Other materials that have been used for cell microencapsulation include agarose, photo-cross-linked poly(ethylene glycol), and (ethyl methacrylate, methyl methacrylate, and dimethylaminoethyl methacrylate) copolymers (Mikos et al., 1994; Sefton and Kharlip, 1994). Advantages of hydrogel microcapsules include a high surface-to-volume ratio, and thus good transport proper- ties, as well as ease of handling and implantation. Small beads can be implanted in the peritoneal cavity of animals simply by injection, without the need for incision. Other common implantation sites include the subcutaneous space and the kidney capsules. Disadvantages include the fragility of the beads, especially if the cross-linking cation becomes chelated by compounds present in the surrounding milieu or released by lysed cells, and the lack of easy retrievability once the beads have been dispersed in the peritoneal cavity of a host. Earlier problems caused by the variable composi- tion of alginates and the presence of endotoxins have been resolved through the development and commercial avail- ability of ultrapure alginates of well-defi ned molecular weight and composition (Sambanis, 2000; Stabler et al., 2001). Hydrogels impose little diffusional resistance to solutes, and indeed effective diffusivities in calcium alginate and agarose hydrogels are in the range of 50–100% of the corre- sponding diffusivities in water (Tziampazis and Sambanis, 1995; Lundberg and Kuchel, 1997). However, with conven- tional microencapsulation, the volume of the hydrogel con- tributes signifi cantly to the total volume of the implant. For example, with a 500-µm microcapsule containing a 300-µm islet, the polymer volume constitutes 78% of the total capsule volume. Additionally, conventional microcapsules may not be appropriate for hepatic portal vein implantation because, besides their higher implant volume relative to the same number of naked islets, they may result in higher portal vein pressure and more incidences of blood coagulation in the liver. To address this problem, methods have been devel- oped for islet encapsulation in thin conformal polymeric coats. Materials that have been used for conformal coating include photopolymerized poly(ethylene glycol) diacrylate (Hill et al., 1997; Cruise et al., 1999) and hydroxyethyl methacrylate-methyl methacrylate (HEMA-MMA) (May and Sefton, 1999; Sefton et al., 2000). Encapsulated cell systems can also be fabricated by pre- forming the permselective membrane in a tubular or disc- Vascular device Microcapsules Membrane chambers Titanium housing Immunobarrier membranes Silicone rubber spacer Tubular Planar Sheet Vascular device Microcapsules Membrane chambers Titanium housing Immunobarrier membranes Silicone rubber spacer Tubular Planar Sheet FIG. 42.2. Schematics of commonly used encapsulation devices for insulin-secreting cells. Vascular devices and membrane chambers of tubular, planar, or sheet architectures are gener- ally referred to as macrocapsules, in distinction from the much smaller microcapsules. Ch042_P370615.indd 624Ch042_P370615.indd 624 6/1/2007 3:01:16 PM6/1/2007 3:01:16 PM shaped confi guration, fi lling the construct with a suspension of islets or other insulin-secreting cells in an appropriate extracellular matrix, and then sealing the device. This approach is particularly useful when organic solvents or other chemicals harsh to the cells are needed for the fabrica- tion of the membranes. Membrane chambers can be of tubular or planar geometry (Fig. 42.2). The cells are sur- rounded by the semipermeable membrane and can be implanted intraperitoneally, subcutaneously, or at other sites. Membrane materials used in fabricating these devices include polyacrylonitrile-polyvinyl chloride (PAN-PVC) copolymers, polypropylene, polycarbonate, cellulose nitrate, and polyacrylonitrile-sodium methallylsulfonate (AN69) (Lanza et al., 1992; Mikos et al., 1994; Prevost et al., 1997; Delaunay et al., 1998; Sambanis, 2000). Typical values of device thickness or fi ber diameter are 0.5–1 mm. Advan- tages of membrane chambers are the relative ease of han- dling, the fl exibility with regard to the matrix in which the cells are embedded, and retrievability after implantation. A major disadvantage is their inferior transport properties, since the surface-to-volume ratio is smaller than that of microcapsules and diffusional distances are longer. Constructs connected to the vasculature via an arterio- venus shunt consist of a semipermeable tube sur- rounded by the cell compartment (Fig. 42.2). The tube is connected to the vasculature, and transport of solutes between the blood and the cell compartment occurs via the pores in the tube wall. A distinct advantage of the vascular device is the improved transport of nutrients and metabolites, which occurs by both diffusion and con- vection. However, the major surgery that is needed for implantation and problems of blood coagulation at the anastomosis sites have considerably reduced enthusiasm for these devices. Other Construct Systems A common approach for improving the oxygenation of cells in diffusion chambers is to encourage the formation of neovasculature around the implant. This is discussed in the following “In Vivo Implantation” section. Other innovative approaches that have been proposed include the electro- chemical generation of oxygen in a device adjacent to a planar immunobarrier diffusion chamber containing the insulin-secreting cells (Wu et al., 1999); and the coencapsu- lation of islets with algae, where the latter produce oxygen photosynthetically upon illumination (Bloch et al., 2006). These were in vitro studies, however, and the ability to translate these approaches to effective in vivo confi gura- tions remains unknown. In a different design, Cheng et al. (2004, 2006) combined constitutive insulin-secreting cells with a glucose- responsive material in a disc-shaped construct. As indicated earlier, it is straightforward to genetically engineer non- β-cells for constitutive insulin secretion; the challenge is in engineering appropriate cellular responsiveness to physiologic stimuli. In this proposed device, a concanavalin A (con A)–glycogen material, sandwiched between two ultrafi ltration membranes, acted as a control barrier to insulin release from an adjacent compartment containing the cells. Specifi cally, con A–glycogen formed a gel at a low concentration of glucose, which was reversibly con- verted to sol at a high glucose concentration, as glucose displaced glycogen from the gel network. Since insulin dif- fusivity is higher through the sol than through the gel, insu- lin released by the cells during low-glucose periods diffused slowly through the gel material; when switched to high glucose, the insulin accumulated in the cell compartment during the previous cycle was released at a faster rate through the sol-state polymer. Overall, this approach converted the constitutive secretion of insulin by the cells to a glucose-responsive insulin release by the device (Cheng et al., 2006). Again, these were in vitro studies, and the in vivo effi cacy of this approach remains to be evaluated. Construct Design and In Vitro Evaluation Design of three-dimensional encapsulated systems can be signifi cantly enabled using mathematical models of solute transport through the tissue and of nutrient consumption and metabolite production by the cells. Beyond the microvasculature surrounding the construct, transport of solutes occurs by diffusion, unless the construct is placed in a fl ow environment, in which case convective transport may also occur. Due to its low solubility, transport of oxygen to the cells is the critical issue. Models can be used to evaluate the dimensions and the cell density within the construct so that all cells are suffi ciently nourished and the capsule as a whole is rapidly responsive to changes in the surrounding glucose concentration (Tziampazis and Sambanis, 1995). Experimental and modeling methods for determining transport properties and reaction kinetics have been described previously (Sambanis and Tan, 1999). Furthermore, models can be developed to account for the cellular reorganization that occurs in constructs with time as a result of cell growth, death, and possibly migration processes. Such reorganization is especially signifi cant when proliferating insulin-secreting cell lines are encapsu- lated in hydrogel matrices (Stabler et al., 2001; Simpson et al., 2005; Gross et al., 2007). Pancreatic tissue substitutes should be evaluated in vitro prior to implantation, in terms of their ability to support the cells within over prolonged periods of time and to exhibit and maintain their overall secretory properties. Long-term cultures can be performed in perfusion bioreactors under conditions simulating aspects of the in vivo environment. In certain studies, the bioreactors and support perfusion circuits were made compatible with a nuclear magnetic resonance spectrometer. This allowed measuring intracel- lular metabolites, such as nucleotide triphosphates, as a function of culture conditions and time, without the need III. CONSTRUCT TECHNOLOGY • 625 Ch042_P370615.indd 625Ch042_P370615.indd 625 6/1/2007 3:01:17 PM6/1/2007 3:01:17 PM 626 CHAPTER FORTY-TWO • BIOARTIFICIAL PANCREAS to extract the encapsulated cells (Papas et al., 1999a, 1999b). Such studies produce a comprehensive understanding of the intrinsic tissue function in a well defi ned and controlled environment prior to introducing the additional complexity of host–implant interactions in in vivo experiments. The secretory properties of tissue constructs can be evaluated with low time resolution in simple static culture experiments by changing the concentration of glucose in the medium and measuring the secreted insulin. In general, a square wave of insulin concentration is implemented, from basal to inducing basal conditions for insulin secre- tion. To evaluate the secretory response with a higher time resolution, perfusion experiments need to be performed, in which medium is fl owed around the tissue and secreted insulin is assayed in the effl uent. Again, a square wave of glucose concentration is generally implemented. By com- paring the secretory dynamics of free and encapsulated cells, one can ensure that the encapsulation material does not introduce excessive time lags that might compro- mise the secretory properties of the construct. Indeed, properly designed hydrogel microcapsules introduce only minimal secretory time lags (Tziampazis and Sambanis, 1995; Sambanis et al., 2002). Manufacturing Considerations Fabrication of pancreatic substitutes of consistent quality requires the use of cells that are also of consistent quality. Although with clonal, expandable cells this is a rather straightforward issue, with islets isolated from human and animal tissues there can be signifi cant variability in the quantity and quality of the cells in the preparations. With islets from cadaveric human donors, the quality of the iso- lates is assessed by microscopic observation, viability stain- ing, and possibly a static insulin secretion test. It is generally recognized, however, that a quantitative, objective assess- ment of islet quality would help improve the consistency of the preparations and thus, possibly, the transplantation outcome. It is conceivable that encapsulated cell systems could be fabricated at a central location from which they are dis- tributed to clinical facilities for implantation. In this scheme, preservation of the constructs for long-term storage, inven- tory management, and, importantly, sterility control would be essential. Cryopreservation appears to be a promising method for maintaining fabricated constructs for prolonged time periods. Although there have been signifi cant studies on the cryopreservation of single cells and some tissues, the problems pertaining to cryopreserving artifi cial tissues are only beginning to be addressed. Cryopreservation of mac- roencapsulated systems is expected to be particularly chal- lenging and has not been reported in the literature. However, βTC-cells encapsulated in alginate beads have been pre- served successfully (Mukherjee et al., 2005; Song et al., 2005). An especially promising approach involves using high concentrations of cryoprotective agents so that water is converted to a glassy, or vitrifi ed, state at low temperatures; the absence of ice crystals in both the intracellular and extracellular domains appears helpful in maintaining not only cellular viability but also the structure and function of the surrounding matrix (Mukherjee et al., 2005; Song et al., 2005). IV. IN VIVO IMPLANTATION This section highlights results from in vivo experiments using the different confi gurations outlined earlier. Results with encapsulated cell systems are presented fi rst. Since in vivo experiments with non-β-cells engineered for insulin secretion are at present based mostly on in vivo gene therapy approaches, these are described next. Technologies for the in vivo monitoring of cells and constructs and the issue of implant retrievability are then discussed. Encapsulated Cell Systems In vivo experiments with pancreatic substitutes are numerous in small animals, limited in large animals, and few in humans. Allogeneic and xenogeneic islets in hydrogel microcapsules implanted in diabetic mice and rats have generally restored normoglycemia for prolonged periods of time. In the early study of O’Shea et al. (1984), islet allografts encapsulated in APA membranes were implanted intraperi- toneally in streptozotocin-induced diabetic rats. Of the fi ve animals that received transplants, three remained normo- glycemic for more than 100 days. One of these three animals remained normoglycemic 368 days postimplantation. In the later study of Lum et al. (1992), rat islets encapsulated in APA membranes and implanted in streptozotocin diabetic mice restored normoglycemia for up to 308 days, with a mean xenograft survival time of 220 days. With all recipients, nor- moglycemia was restored within two days postimplanta- tion. Control animals receiving single injections of unencapsulated islets remained normoglycemic for less than two weeks (O’Shea et al., 1984; Lum et al., 1992). More recently, APA-encapsulated βTC6-F7 insulinomas restored normoglycemia in diabetic rats for up to 60 days (Mamujee et al., 1997), and APA-encapsulated βTC-tet insulinomas in NOD mice for at least eight weeks (Black et al., 2006). In the latter study, it was also observed that no host cell adherence occurred to microcapsules, and there were no signifi cant immune responses to the implant, with cytokine levels being similar to those of sham-operated controls. These results are thus indicative of the potential use of an immu- noisolated continuous β-cell line for the treatment of diabe- tes. With the recently developed human cell line (Narushima et al., 2005), experiments were performed with unencapsu- lated cells transplanted into streptozotocin-induced dia- betic severe combined immunodefi ciency mice. Control of blood glucose levels started within two weeks postimplanta- tion, and mice remained normoglycemic for longer than 30 weeks (Narushima et al., 2005). Besides rodents, long-term restoration of normoglycemia with microencapsulated islets Ch042_P370615.indd 626Ch042_P370615.indd 626 6/1/2007 3:01:17 PM6/1/2007 3:01:17 PM has been demonstrated in large animals, including sponta- neously diabetic dogs, where normoglycemia was achieved with canine islet allografts for up to 172 days (Soon-Shiong et al., 1992), and monkeys, where in one animal porcine islet xenografts normalized hyperglycemia for more than 150 days (Sun et al., 1992). More recently, one of the companies working on islet encapsulation technology announced that primate subjects in ongoing studies have continued to exhibit improved glycemic regulation over a six-month period after receiving microencapsulated porcine islet transplants (MicroIslet Inc. Press Release, August 7, 2006). In vivo results with vascular devices are reportedly mixed. Implantation of devices containing allogeneic islets as arteriovenous shunts in pancreatectomized dogs resulted in 20–50% of the dogs becoming normoglycemic up to 10 weeks postimplantation without exogenous insulin admin- istration. When xenogeneic bovine or porcine islets were used, only 10% of the dogs remained normoglycemic 10 weeks postimplantation. All dogs were reported diabetic or dead after 15 weeks (Sullivan et al., 1991). Recently, a hollow- fi ber device composed of polyethylene-vinyl alcohol fi bers and a poly-amino-urethane-coated, nonwoven polytetra- fl uoroethylene fabric seeded with porcine islets provided normalization of the blood glucose levels in totally pancre- atectomized pigs when connected to the vasculature of the animals (Ikeda et al., 2006). It should be noted, however, that the overall interest in vascular devices has faded, due to the surgical and blood coagulation challenges they pose. Although several hypotheses exist, the precise cause of the eventual in vivo failure of encapsulated cell systems remains unclear. Encapsulation does not completely prevent the immune recognition of the implant. Although direct cel- lular recognition is prevented, antigens shed by the cells as a result of secretion and, more importantly, lysis in the cap- sules eventually pass through the permselective barrier and are recognized by the antigen-presenting cells of the host. For example, in one study, antibodies against islets in a tubular diffusion chamber were detected in plasma two to six weeks postimplantation, suggesting that islet antigens crossed the membrane and stimulated antibody formation in the host (Lanza et al., 1994). In another study, alginate- encapsulated islets were lysed in vitro by nitric oxide pro- duced by activated macrophages (Wiegand et al., 1993). Passage of low-molecular-weight molecules cannot be prevented by immunoprotective membranes imposing a molecular weight cutoff on the order of 50 kDa. It should be noted that in one human study involving encapsulated allo- geneic islets, the patient had to be provided with low levels of immunosuppression (Soon-Shiong et al., 1994). In a more recent report, also with encapsulated allografts implanted peritoneally, type 1 diabetic patients remained nonimmu- nosuppressed but were unable to withdraw exogenous insulin (Calafi ore et al., 2006). Nonspecifi c infl ammation may also occur around the implant and develop into a fi brous capsule, reducing the oxygen available to the cells within. The fi brotic layer has been found to consist of several layers of fi broblasts and collagen with polymorphonuclear leukocytes, macrophages, and lymphocytes. The surface roughness of the membrane may also trigger infl ammatory responses. In one study, membranes with smooth outside surfaces exhibited a minimal fi brotic reaction 10 weeks postimplantation, regardless of the type of encapsulated cells, whereas rough surfaces elicited a fi brotic response even one week postim- plantation (Lanza et al., 1991). Use of high-purity materials also helps to minimize infl ammatory reactions. If a material is intrinsically infl ammatory, such as poly-l-lysine, it can be coated with a layer of noninfl ammatory material, such as alginate, to minimize the host’s reaction. Such coverage may not be suffi ciently permanent, though, resulting in the eventual fi brosis of the implant. Indeed, several investi- gators report improved results with plain alginate beads without a poly-l-lysine layer, especially when allogeneic cells are used in the capsules. Provision of nutrients to and removal of metabolites from encapsulated cells can be especially challenging in vivo. Normal pancreatic islets are highly vascularized and thus well oxygenated. There exists evidence that unencapsulated islets injected in the portal system of the liver become revascularized, which enhances their engraft- ment and function. On the other hand, encapsulation prevents revascularization, so the implanted tissue is nour- ished by diffusion alone. Promotion of vascularization around the immunoprotective membrane increases the oxygenation of the implanted islets (Prokop et al., 2001). Interestingly, transformed cells, such as the βTC3 line of mouse insulinomas, are more tolerant of hypoxic conditions than intact islets; such cells may thus function better than islets in implanted capsules (Papas et al., 1996). However, with transformed cells, too, enhanced oxygen- ation increases the density of functional cells that can be effectively maintained within the implant volume. Vascular- ization is dependent on the microarchitecture of the mate- rial, which should have pores 0.8–8.0 µm in size, allowing permeation of host endothelial cells (Brauker et al., 1992, 1995). Vascularization is also enhanced by the delivery of angiogenic agents, such as FGF-2 and VEGF, possibly with controlled-release devices (Sakurai et al., 2003). Although vascularization can be promoted around a cell-seeded device, improved success has been reported if a cell-free device is fi rst implanted and vascularized and the cells are then introduced. One example of this procedure involved placing a cylindrical stainless steel mesh in the subcutane- ous space of rats, with the islets introduced 40 days later (Pileggi et al., 2006). Replacement of a vascularized implant is challenging, however, due to the bleeding that occurs. A solution to this problem may entail the design of a device that can be emptied and refi lled with a suspension of cells in an extracellular matrix without disturbing the housing and the associated vascular network. IV. IN VIVO IMPLANTATION • 627 Ch042_P370615.indd 627Ch042_P370615.indd 627 6/1/2007 3:01:17 PM6/1/2007 3:01:17 PM 628 CHAPTER FORTY-TWO • BIOARTIFICIAL PANCREAS Gene and Cell-Based Therapies In vivo effi cacy studies with gene therapy and non-β- cells genetically engineered for insulin secretion are gener- ally limited to small animals. Intraportal injection of recombinant adenovirus expressing furin-compatible insulin under the control of a glucose-responsive promoter containing elements of the rat liver pyruvate kinase gene restored near-normal glycemia in streptozotocin diabetic rats for periods of 1–12 weeks (Thule and Liu, 2000). With hepatic delivery of a recombinant adeno-associated virus expressing a single-chain insulin analog under the control of an l-type pyruvate kinase promoter, Lee and coworkers (2000) controlled blood glucose levels in streptozotocin dia- betic rats and NOD mice for periods longer than 20 weeks. However, transiently low blood glucose levels observed three to fi ve hours after glucose loading indicated a draw- back of the transcriptional regulation of insulin expression, which may result in hypoglycemic episodes (Lee et al., 2000). Possible approaches toward ameliorating this problem include optimizing the number of glucose-regulatory and insulin-sensing elements in the promoter (Jun and Yoon, 2005) and destabilizing the preproinsulin mRNA; the latter has been shown to expedite signifi cantly the down-regula- tion of secretion dynamics from transcriptionally controlled cells on removal of the secretory stimulus (Tang and Sam- banis, 2003). In vivo gene therapy with small animals has also shown success when the target cells for insulin expression were intestinal endocrine K- or gastric G-cells. Using a transgene expressing human insulin under the control of the glucose- dependent insulinotropic peptide (GIP) promoter, Cheung et al. (2000) expressed insulin specifi cally in gut K-cells of transgenic mice, which protected them from developing diabetes following STZ-mediated destruction of the native β-cells. Similarly, use of a tissue-specifi c promoter to express insulin in gastric G-cells of mice resulted in insulin release into circulation in response to meal-associated stimuli, sug- gesting that G-cell insulin expression is benefi cial in the amelioration of diabetes (Lu et al., 2005). Translation of these approaches to adult animals and, eventually, humans, requires the development of effective methods of gene delivery to intestinal endocrine or gastric cells in vivo or the development of effective ex vivo gene therapy approaches. In Vivo Monitoring Monitoring of the number and function of insulin- secreting cells in vivo would provide valuable information directly on the implant and possibly offer early indications of implant failure. Additionally, in animal experiments, the ability to monitor an implant noninvasively reduces the number of animals that are needed in the experimental design and helps establish a critical link between implanta- tion and endpoint physiologic effects, the latter commonly being blood glucose levels and animal weight. Imaging techniques can provide unique insight into the structure/function relationship of a construct in vitro and in vivo. There are several imaging modalities that have been applied to monitor tissue-engineered constructs, including computed tomography (CT), positron emission tomogra- phy (PET), optical techniques, and nuclear magnetic reso- nance (NMR) imaging and spectroscopy. Among these, NMR offers the unique advantage of providing information on both construct integrity and function, without the need to modify the cells genetically (e.g., through the expression of green fl uorescent protein, used in optical methods) or the introduction of radioactive labels (e.g., PET agents). Furthermore, since magnetic fi elds penetrate uniformly throughout the sample, NMR is ideally suited to monitor constructs implanted at deep-seated locations. Its disad- vantage is its low sensitivity. Whereas optical and radionu- clide techniques can detect tracer quantities, NMR detects metabolites that are available in the millimolar or, in some cases, submillimolar range. The ability to monitor noninvasively in vivo a pancreatic substitute by NMR was reported recently (Stabler et al., 2005). Agarose disc-shaped constructs containing mouse insulinoma βTC3-cells were implanted in the peritoneal cavity of mice. Construct integrity was visualized by MR imaging and the metabolic activity of the cells within by water-suppressed 1 H NMR spectroscopy (Fig. 42.3). Control experiments established that the total choline (TCho) reso- nance at 3.2 ppm, which is attributed to three choline metabolites, correlated positively and linearly with the number of viable cells within the construct, measured with an independent assay. To obtain the TCho signal in vivo without interference from the surrounding host tissue, such as peritoneal fat, the central agarose disc containing the cells had to be surrounded by cell-free agarose buffer zones. This ensured that the MR signal arose only from the implanted cells, even as the construct moved due to animal breathing. A second problem that had to be resolved was that glucose diffusing into the construct produced a resonance that inter- fered with the TCho resonance at 3.2 ppm. For this, a unique glucose resonance at 3.85 ppm was used to correct for the interference at 3.2 ppm so that a corrected signal, uniquely attributed to TCho, could be obtained. The latter correlated positively and linearly with the number of viable cells, mea- sured with an independent assay, on the constructs postex- plantation (Fig. 42.3). Hence, with the appropriate implanted construct architecture and signal processing, the number of viable cells in an implant could be followed in the same animal as a function of time (Stabler et al., 2005). Labeling of cells with magnetic nanoparticles, which can be detected by magnetic resonance, and genetically engineering cells so that they express a fl uorescent or lumi- nescent marker that can be optically detected are other methods being pursued to track the location and possibly function of implanted cells in vivo. It is expected that devel- opment of robust monitoring methodologies will be helpful Ch042_P370615.indd 628Ch042_P370615.indd 628 6/1/2007 3:01:17 PM6/1/2007 3:01:17 PM [...]... NCC of Fgf8 hypomorphs show increased levels of apoptosis (Abu-Issa et al., 20 02; D U Frank et al., 20 02) and reduced expression of Fgf10 (Frank et al., 20 02) , a factor that may mediate proliferation of the pharyngeal endoderm (D U Frank et al., 20 02) There is also a mild reduction in the expression of genes associated with differentiated NCC (Abu-Issa et al., 20 02) , suggesting that the maintenance of. .. data II ENGINEERING TO GENERATE INSULIN-PRODUCING CELLS Engineering Pancreatic Beta-Cells The pancreas is composed of endocrine and exocrine tissues The endocrine pancreas occupies less than 5% of the pancreatic tissue mass and is composed of cell clusters called the islets of Langerhans The islets of Langerhans contain insulin-producing beta-cells (about 80% of cells in the islets), glucagon-producing... (1998) Glucose-insulin kinetics of a bioartificial pancreas made of an AN69 hydrogel hollow fiber containing porcine islets and implanted in diabetic mice Artif Organs 22 , 29 1 29 9 Drucker, D J (20 02) Biological actions and therapeutic potential of the glucagon-like peptides Gastroenterology 122 , 531–544 6/1 /20 07 3:01:17 PM VII REFERENCES • Efrat, S (1998) Cell-based therapy for insulin-dependent diabetes... commences, with the first evidence of differentiation into cortical and medullary cell types appearing by E 12. 5 (Bennett et al., 20 02) De- 6/1 /20 07 3: 02: 43 PM IV THYMUS ORGANOGENESIS • velopment of the two compartments then proceeds in a lymphocyte-independent manner until E15.5 (Klug et al., 20 02; Jenkinson et al., 20 05) The expression of MHC II and MHC I on the surface of thymic epithelial cells is first... MTS20 +24 + cells within the fetal mouse thymus directly Ontogenic analysis has demonstrated that the proportion of MTS20 +24 + epithelial cells was highest in the early thymus primordium, decreasing to less than 1% in the postnatal thymus (Bennett et al., 20 02) , consistent with the expression profile expected of markers of fetal tissue progenitor cells Phenotypic analysis of the MTS20 +24 + and MTS20 24 −... populations of the E 12. 5 thymus indicated that all cells in the MTS20 +24 + population coexpressed K5 and K8, while none expressed TEC differentiation markers, including MHC II (Bennett et al., 20 02) Importantly, the functional capacity of isolated MTS20 +24 + cells and MTS20 24 − cells was then determined via ectopic transplantation This analysis demonstrated that 6/1 /20 07 3: 02: 43 PM 6 52 C H A P T E R F O R T Y -. .. REMARKS • 629 A B Glucose Lactate Surface Coil In Vivo TCho Glucose-corrected TCho y = 22 44x – 24 R2 = 0.87 3000 25 00 C 20 00 1500 1000 500 0 0 0 .2 0.4 0.6 0.8 1 1 .2 MTT Absorbance FIG 42. 3 Magnetic resonance imaging and localized spectroscopy of a disc-shaped agarose construct containing βTC3 mouse insulinoma cells at an initial density of 7 × 107 cells/mL agarose implanted in the peritoneal cavity of a... Brunetti, P (20 06) Microencapsulated pancreatic islet allografts into nonimmunosuppressed patients with type 1 diabetes: first two cases Diabetes Care 29 , 137–138 Ch0 42_ P370615.indd 630 Cha, J.-Y., Kim, H., Kim, K.-S., Hur, M.-W., and Ahn, Y (20 00) Identification of transacting factors responsible for the tissue- specific expression of human glucose transporter type 2 isoform gene J Biol Chem 27 5, 18358–18365... Pharm Des 11, 29 27 29 40 Walther, W., and Stein, U (20 00) Viral vectors for gene transfer: a review of their use in the treatment of human diseases Drugs 60, 24 9 27 1 Wu, L., Nicholson, W., Wu, C Y., Xu, M., McGaha, A., Shiota, M., and Powers, A C (20 03) Engineering physiologically regulated insulin secretion in non-beta cells by expressing glucagon-like peptide 1 receptor Gene Ther 10, 17 12 1 720 Ch043_P370615.indd... C B (1993) Transfection of AtT -2 0 ins cells with GLUT -2 but not GLUT-1 confers glucose-stimulated insulin secretion Relationship to glucose metabolism J Biol Chem 26 8, 1 520 5–1 521 2 Ikeda, H., Kobayashi, N., Tanaka, Y., Nakaji, S., Yong, C., Okitsu, T., Oshita, M., Matsumoto, S., Noguchi, H., Narushima, M., Tanaka, K., Miki, A., Rivas-Carrillo, J D., Soto-Gutierrez, A., Navarro-Alvarez, N., Tanaka, K., . REMARKS • 629 Surface Coil y = 22 44x – 24 R 2 = 0.87 0 500 1000 1500 20 00 25 00 3000 00 .20 .40.60.811 .2 MTT Absorbance In Vivo TCho Glucose-corrected C Surface Coil y = 22 44x – 24 R 2 = 0.87 0 500 1000 1500 20 00 25 00 3000 00 .20 .40.60.811 .2 MTT. endog- enous GLP-1 and thus is secreted with identical kinetics to GLP-1 in response to stimuli (Tang and Sambanis, 20 03, Ch0 42_ P370615.indd 622 Ch0 42_ P370615.indd 622 6/1 /20 07 3:01:16 PM6/1 /20 07. types of cells. It has been shown that the A-chain/ Ch0 42_ P370615.indd 621 Ch0 42_ P370615.indd 621 6/1 /20 07 3:01:16 PM6/1 /20 07 3:01:16 PM 622 CHAPTER FORTY-TWO • BIOARTIFICIAL PANCREAS C-peptide