Generation of Islets form Stem Cells

PRINCIPLES OF TISSUE ENGINEERING Generation of Islets from Stem Cells: 605-618: 2007
















I. Introduction II. Islet Transplantation III. Alternative Sources of Islet Cells

IV. Biomaterials V. Acknowledgments VI. References

Generation of Islets from Stem Cells

Bernat Soria, Abdelkrim Hmadcha, Francisco J. Bedoya, and Juan R. Tejedo

Principles of Tissue Engineering, 3rd Editioned. by Lanza, Langer, and Vacanti

Copyright © 2007, Elsevier, Inc.All rights reserved.

I. INTRODUCTIONDiabetes is a devastating disease affecting millions of

people around the world. Islet transplantation has demon-strated that cell therapy works. Although this technique needs further improvements (amelioration islet isolation and sur-vival, new immunosuppressive regimens, etc.), the “proof of principle” for future cell therapies has been established. However, the lack of suffi cient donors, together with the need for immunosuppression, limits clinical applications of these techniques. New sources of insulin-producing cells are needed. The possibilities are xenogenic islets, surrogate beta-cells, adult stem cells from the pancreas and other tissues, and bioengineered embryonic stem cells. Macro- and microislet encapsulation may improve islet survival, avoid rejection, and permit immunosuppressive regimen-free transplantations. This chapter discusses these approaches.

Diabetes mellitus as a devastating metabolic disease affects around 2–5% of the world’s population. While type 1 diabetes mellitus is characterized by autoimmune destruc-tion of islets, it is now well recognized that reduced pancre-atic beta-cell mass and insulin secretion failure play a pivotal role in the development and progression of type 2 diabetes mellitus (Roche et al., 2005).

Daily insulin injections are necessary for patient sur-vival, mainly in the case of type 1 diabetes. However, dia-betic people very often develop late complications, such

as neuropathy, nephropathy, retinopathy, and cardiovascu-lar disorders, because the insulin injection does not mimic β-cell function exactly. The Diabetes Control and Complica-tions Trial (DCCT, 1993) has demonstrated that intensive insulin therapy restores blood glucose homeostasis and subsequently reduces the appearance of diabetic complica-tions. However, it requires educated and motivated patients; long-term follow-up of patients resulted in no clear differ-ences. As a matter of fact, in both type 1 and type 2 diabetes mellitus, impaired blood glucose due to lack or death of β-cells is a common feature; thus, the best perspective for its treatment is beta-cell replacement.

Pancreas transplantation provides good glycemic con-trol and insulin independence, together with an improve-ment in diabetic complications (Ryan et al., 2006), but it has the associated morbidity of major surgery. Unfortunately, it requires long-term immunosuppression, with its attendant risks. Simultaneous pancreas and kidney transplantation is a worthwhile option to employ when renal transplantation is needed (Ryan et al., 2006).

Clinical islet transplantation trials have been estab-lished as proof for beta-cell replacement therapy. Injection of islets isolated from cadaveric organ donor pancreata into the portal vein of type 1 diabetic patients results in insulin independence (Ryan et al., 2005). However, this procedure most often requires the use of islets from more

Chapter Forty-One

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606 C H A P T E R F O R T Y - O N E • G E N E R A T I O N O F I S L E T S F R O M S T E M C E L L S

than one donor, and preserved graft function is based on the use of immunosuppressive medication, which has to counteract both immunorejection and recurrent beta-cell autoimmunity.

Lack of donors, the low yield of islet isolation proce-dures, and the need for immunosuppression are the most important caveats for future applications of cell therapy for diabetic patients.

II. ISLET TRANSPLANTATIONIslet transplantation from cadaveric donors has been

shown to improve the quality of life of severe diabetic patients. The Edmonton protocol (Shapiro et al., 2000) reported that insulin independence can be reached in type 1 diabetes subjects. The success was attributed mainly to two aspects: (1) the application of a steroid-free new immu-nosuppressive regime and (2) subsequent transplantation of freshly prepared islets from two or more donors (Shapiro et al., 2000). Five-year follow-up reported that 80% of trans-planted patients are C-peptide positive, but only 5–10% maintained insulin independence (Ryan et al., 2005). The side effects of immunosuppressant drugs (mostly diabeto-genic), together with recurrent autoimmune attacks, may explain the limited success. In order to achieve successful cell therapies, effi cient islet isolation procedures, less toxic immunosuppressive regimes, and the exploration of new places for transplant have to be developed.

Islet Isolation and SurvivalOne of the bottlenecks in pancreatic islet transplanta-

tion is reaching a high number of functional engrafted islets. It is estimated that only 15–30% of the allogeneic implanted islets are functional, thus forcing the use of two to three subsequent donors to achieve insulin independence. Islet isolation using the semiautomatic Ricordi method remains, with slight modifi cations, the best method for obtaining high amounts of viable islets.

The yield from islet isolation depends basically on the donor and processing parameters. Several studies have con-cluded that the islets from normal, overweight, and obese nondiabetic cadaver donors are also suitable (I. Matsumoto et al., 2004). In addition, islets from donors with a low body-mass index non-heart-beating were used successfully in an islet transplantation (Goto et al., 2005). Unfortunately, brain-dead donors promote a rapid anti-infl ammatory reac-tion that predisposes the islets to a subsequent immuno-logic reaction in the recipient after transplantation (Takada et al., 2004). Limited experience with islets from living donors (S. Matsumoto et al., 2005) suggests better results. Islet transplant from a single donor is possible and results in more uniform islet preparation and makes possible a lower level immunosuppression therapy by searching for a compatible recipient (B. W. Lee et al., 2005).

The Ricordi method may be summarized as follows: organ extraction and transport, perfusion, digestion, and

purifi cation of islets. The maintenance of isolated islets prior to transplant and increasing islet engraftment may be instru-mental to increasing the chance of success. Organ extraction and transport has requirements similar to those for pancreas transplantation. The use of a two-layer method (perfl uoro-carbon/University of Wisconsin solution) has been seen to increase the yield and quality of isolated islets (Hering et al., 2004; Papas et al., 2005). However, Papas et al. recently have shown that the two-layer method can ameliorate oxygen-ation in only around 15% of the pancreas (Papas et al., 2005). Preservation solutions, such as M-Kyoto solution, containing trehalose as a cytoprotector and ulinistatin as a trypsin inhibitor improve islet quality signifi cantly as compared to University of Wisconsin solution in the two-layer method (Noguchi et al., 2006). In addition, intraductal glutamine administration improves islet yield, because it protects from lipoperoxidation and apoptosis (Avila et al., 2005).

During perfusion and digestion it is necessary to opti-mize enzymatic digestion by means of liberase H1 and to preserve islet integrity. Excess exposure to enzyme can produce islet fragmentation, decreased insulin secretory ability, and apoptosis (Balamurugan et al., 2005). On the other hand, the automatic method in the Ricordi chamber results in a strong mechanical shaking.

The difference in density between the islets and the rest of the pancreatic tissues allows for islet purifi cation by means of separation on continuous density-gradient systems, using the COBE 2991 cell separator. The issues in the purifi cation step are related to the purity and effi ciency of the continuous gradient systems. Iodixanol gradients have clear advantages over other gradient systems. When purity is high, the islet fraction is small, whereas a great number of islets may be found in less pure fractionation methods. Recently the additional step named rescue gradi-ent purifi cation (RGP) has been described; islets recovered from these fractions were equivalent to islets obtained in a high-purity fraction (Ichii et al., 2005).

With regard to islet maintenance, the possibility of increasing viability via subsequent islet culture is being explored by many groups, for example, coculturing islets with Sertoli cells (Teng et al., 2005) as well as with small intestinal submucosa (Tian et al., 2005), the protective effect of l-glutamine and nitric oxide on the islet culture (Tejedo et al., 2004; Brandhorst et al., 2005) has also been described.

Furthermore, in recent years, several experimental strategies have been developed to enhance islet engraft-ment. For instance, antioxidant therapy with nicotinamide (Moberg et al., 2003) as well as sulforaphane (Solowiej et al., 2006), vitamin D3 (Riachy et al., 2006), 17 beta-estradiol (Eckhoff et al., 2003), pentoxiphylline (Juang et al., 2000), caspase inhibitors (Brandhorst et al., 2003), or cholesterol-lowering agents such as simvastatin (Contreras et al., 2002) have all demonstrated a positive impact in the preclinical setting and suggest a potential role in future clinical trials designed to improve islet engraftment. Recently it has been



I I . I S L E T T R A N S P L A N T A T I O N • 607

shown that after intraportal islet transplant, a lipotoxic destruction of islets is induced by hepatic lipids, an effect that can be prevented when the islets have previously been covered with leptin (Unger, 2005).

Additionally, pancreatic islets express tissue factor, the major in vivo initiator of coagulation. During clinical islet transplantation, when islets come into direct contact with blood in the portal vein, islet-produced tissue factor triggers a detrimental clotting reaction, referred to as instant blood-mediated infl ammatory reaction (IBMIR), which is charac-terized by activation of the coagulation and complement systems, rapid binding and activation of platelets, and infi l-tration of leukocytes to islets. Together, these effects cause a disruption of islet morphology, islet dysfunction, and death. At present, several forms to inhibit it or to counteract the effects have been proposed (Johansson et al., 2006). Islet surface modifi cation with poly(ethylene glycol) (PEG) was proposed as a strategy to prevent rejection (Contreras et al., 2004; D. Y. Lee et al., 2006). The rationale for this strategy is based on the concept that proteins and enzymes modifi ed with PEG are nonimmunogenic. Incorporation of PEG into the islet surface has been proposed to “camoufl age” surface antigens and prevent immunogenic reactions. In addition, PEG has been used successfully to reduce plasma protein adsorption and platelet adhesion to blood vessels and vas-cular devices because of its low interfacial free energy with water, high surface mobility, and steric stabilization effects. Contreras et al. (2004) using xenogenic porcine islets with their camoufl aged surface with PEG and additional plus genetic modifi cation to overexpress Bcl-2, and additional surface islet coverage by incorporation of albumin decreas-ing cytotoxicity mediated by XNA and complement. PEG incorporation onto the islet surface presumably will decrease the binding of platelets on its surface, thereby decreasing IBMIR. The combination of PEG camoufl age with immu-nosuppressive medication would be highly effective in clini-cal islet transplantation (D. Y. Lee et al., 2006).

ImmunosuppressionMost immunosuppressant agents are diabetogenic. Min-

imizing these effects while maintaining adequate potency to contend with both allograft rejection and autoimmune recur-rence has been instrumental in making islet transplantation a clinical reality. A combination of sirolimus, a low dose of tacrolimus, and daclizumab avoided graft rejection and improved implant survival. Previous reports indicated that the combination of sirolimus and tacrolimus-based trials reported low rates of rejection in liver, kidney, and whole pancreas transplantation (McAlister et al., 2000). Both com-pounds block T-cell activation. Daclizumab is a monoclonal antibody against IL-2 receptor, allowing the suppression of glucocorticoid administration in patients, which is very harmful to islet cells. The result of this immunosuppressor combination is the prevention of immune response activa-tion and clonal expansion of T-lymphocytes. Sirolimus has

facilitated clinical islet transplant success by providing effec-tive immunosuppression to contend with both auto- and alloimmunity. However, the agent is also responsible for many of the side effects encountered after islet transplanta-tion, including nausea, vomiting, anemia, ovarian cysts, mouth ulcers, diarrhea, optic neuropathy, proteinuria, hy-pereosinophilic syndrome, parvovirus infection, aspiration pneumonia, severe depression, and hypertension (Hafi z et al., 2005; Ryan et al., 2005). It has also been suggested that sirolimus could still have a detrimental effect on islet engraft-ment and neovascularization as well as potential detrimental direct toxicity to islets (Hafi z et al., 2005). On balance, however, this agent has proven to be advantageous as compared to former therapies based on steroid and high-dose calcineurin inhibitor. In contrast, combining glucocorticoid-free immu-nosuppressive strategy with low-dose FK506 tacrolimus and mycophenolate mofetil (MMF) could protect islet grafts in islet transplantation without diabetogenic side effects (Du and Xu, 2006). Pretransplant immunosuppression induction with sirolimus and humanized anti-CD-3 antibody, hOKT3γ1 (Ala-Ala), in recipients has resulted in engraftment and insulin independence after single-donor islet transplant (Hering et al., 2004).

Site of TransplantationThe liver is the most commonly used site; islet allografts

are infused percutaneously into the portal vein (Shapiro et al., 2000). Potential complications of an infusion into the liver include bleeding, portal venous thrombosis, and portal hypertension (Ryan et al., 2005). Although portal blood pressure is monitored during the procedure and anticoagu-lant agents are used to prevent clotting, anticoagulation can promote hepatic bleeding at the sites of the percutaneous needle punctures. Furthermore, intrahepatic islets may be exposed to environmental toxins and potentially toxic pre-scribed medications, such as the immunosuppressive drugs, absorbed from the gastrointestinal tract and delivered into the portal vein. Thus islets transplanted are unable to release glucagon during hypoglycemia. In view of these problems, the use of nonhepatic sites for islet transplantation has been suggested.

The optimal site for transplantation of islets has not yet been defi ned. The implant site should provide an adequate microenvironment, vascularization, and nutritional support to maximize chances for the best engraftment of cells and to minimize morbidity. Several transplantation sites for islet engraftment have been reported in various experimental animal models, including intraperitoneal, intravenous, intrathecal, intrapancreatic, intrasalivary gland, intracere-bral, muscle, spleen, liver via the portal vein, mammary fat pad, anterior eye chamber, omental pouch, testis, and renal capsule (Roche et al., 2005). We used both spleen and kidney capsules as the recipient organ for the bioengi-neered insulin-positive aggregates (Soria et al., 2000; Leon-Quinto et al., 2004). Transplantation into the spleen is an



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experimental technique fast enough to minimize the risk of death in diabetic animals. Although the implanted cells could be traced by means of the expression of a reporter gene (i.e., h-galactosidase), graft monitoring could not be followed properly under these conditions because a portion of the cells migrated to the liver and the animal had to be sacrifi ced (Soria et al., 2001). Kidney capsule appears to be a better place because the graft can easily be obtained via nephrectomy and then analyzed, which allows one to deter-mine in vivo differentiation processes and structural changes in the implanted tissue

III. ALTERNATIVE SOURCES OF ISLET CELLS

Organ procurement from cadaveric donors, even in countries like Spain, which ranks number one in the world in organ donation, will always be a limited source of islets. Search for alternative sources becomes a necessity. Xenogenic islets, bioengineered beta-cells, and directed dif-ferentiation of embryonic and adult stem cells could be considered.

Xenogenic IsletsXenogenic sources have been proposed, but they have

problems related to immunological and physiological incompatibility, to the identity of insulin as compared to human insulin, and to the risk of zoonotic disease transmis-sion, mainly retrovirus. Porcine islets are the most studied source. They have a control of glucose similar to that of humans, and the insulin has been used for more than 70 years in diabetic humans. Rejection of porcine islets is sub-stantially greater than that of human islets. However, follow-ing the development of transgenic humanized pigs lacking xenoantigens, these are more resistant to immune attack (Phelps et al., 2003), and the possibility exists of producing transgenic pigs with individualized matching for recipient HLA types. Zoonotic disease represents, however, a major drawback. Xenogenic islet encapsulation has been explored in detail (Lanza et al., 1999; de Groot et al., 2004; Qi et al., 2004; Schaffellner et al., 2005). In vitro experiments suggest that encapsulation is an effective barrier to diffusion of zoo-notic disease (Petersen et al., 2002).

Bioengineered b-cellsBeta-cell surrogates have to achieve expression, pro-

cessing, packaging, storing, and secretion of insulin in a glucose-dependent manner (Samson and Chan, 2006). Dif-ferent cell types have been genetically modifi ed to express GLUT 2, glucokinase, and insulin (Faradji et al., 2001), although the results obtained so far are not satisfactory. Coexpression of GLUT 2 and glucokinase in intermediate lobe pituitary cells causes death by apoptosis due to an increment of cytotoxic effects in the presence of 3 mm of glucose (Faradji et al., 2001). Nevertheless, the principal

limitation to the use of transformed insulin-producing cell lines is the uncontrolled proliferation of these cells.

Hepatocytes are good candidates to be used as tem-plates to obtain surrogate beta-cells. Hepatocytes possess similar glucose-sensing machinery to that of beta-cells and express glucokinase and glucose transporter 2 (GLUT2). Rat hepatocytes transfected with PDX1-VP16, a superactive version of PDX1, can be transdifferentiated into insulin-producing cells in the presence of high levels of glucose (Cao et al., 2004). Human liver cells transfected with PDX-1 are able to transdifferentiate into insulin-producing cells in the presence of nicotinamide and epidermal growth factor (Sapir et al., 2005). Hepatocytes transformed with human insulin cDNA express and secrete insulin that is regulated by low glucose and nitric oxide production (Qian et al., 2005). These cells can modulate the hyperglycemia when they are transplanted in diabetic animal models. The expres-sion of PDX1 human fetal liver cells induces the activation of beta-cell genes and has functional beta-cell characteris-tics. When these cells were cultured in starved-serum condi-tions in the presence of activin, they differentiated into insulin-producing cells that produce approximately 60% of the insulin content of normal beta-cells and regulate the hyperglycemia in diabetic animal models (Zalzman et al., 2005). Taken together these results suggest that the liver is a potential autologous source of insulin-producing cells.

Another approach has exploited the characteristics of the gut-associated K-cell. This cell possesses a peptide secretory pathway, since one of its functions is to enhance insulin release through secretion of the glucose-dependent insulinotropic polypeptide (GIP). Intestinal mucosal K-cells transfected with the human preproinsulin gene have been injected into mouse embryos. Implanted mice not only produced human insulin within the gut, but also maintained suffi cient synthesis and secretory capacity to protect the mice from diabetes following deliberate destruction of the endogenous beta-cell mass (Cheung et al., 2000).

Pancreatic Stem CellsThe pancreas is an endoderm-derived organ, consisting

of exocrine and endocrine cells. Development of the endo-crine cells begins with a common multipotent precursor that is directed along divergent pathways to form the differ-ent cell types contained in the islets of Langerhans: α-cells (glucagon), β-cells (insulin), δ-cells (somatostatin), and PP cells (pancreatic polypeptide). This process is dependent on a set of transcription factors (Samson and Chan, 2006).

A cascade of these factors coordinates the stepwise changes in gene expression that guide pluripotent pancre-atic progenitor cells along the pathway to mature pancreatic cells (Fig. 41.1). During embryonic development these cells derived from a common stem cell, characterized by the expression of hepatocyte nuclear factor 6 (HFN-6) (Poll et al., 2006) and Pdx-1 transcription factor (Wilding and



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Gannon, 2004). Pdx-1 leads downstream to Ngn3 factor expression, which is a member of the basic helix-loop-helix (bHLH) family. This factor specifi es an endocrine-cell fate (Watada, 2004). Beta2/NeuroD, NeuroD/BETA2 is another basic helix-loop-helix pro-endocrine factor activated by Ngn3 downstream of PDX-1 in the endocrine developmen-tal cascade. Beta2/NeuroD heterodimerizes with ubiquitous bHLH proteins of the E2A family to regulate transcription of the insulin gene and other β-cell-specifi c genes (Huang et al., 2002). Isl 1 is a LIM homeodomain-family member that controls the differentiation of postmitotic endocrine pro-genitors. Isl 1 interacts with Beta2 to promote insulin gene transcriptional activation (Peng et al., 2005). The activity of the NK-family member and homeodomain protein Nkx2.2 is necessary for the maturation of β-cells. Nkx2.2 expression is dependent on the NeuroD 1 only in β-cells (Sander et al., 2000; Itkin-Ansari et al., 2005), whereas its distant homolog Nkx6.1 controls their expansion (Sander et al., 2000). The Pax-gene family encodes a group of transcription factors that are key regulators of vertebrate organogenesis, since

they play major roles in embryonic pattern formation, cell proliferation, and cell differentiation. Pax4 is a paired-box homeoprotein whose expression is restricted to the central nervous system and the developing pancreas (Sosa-Pineda, 2004). Thus, Pax4 functions early in the development of islet cells to promote the differentiation of β- and α-cells. Pax6, also important for β-cell development, regulates the pro-moters of insulin, glucagon, and somastotatin genes and molecules of adhesion (Sosa-Pineda, 2004).

Embryonic Stem CellsEmbryonic stem cells (ESC) have self-renewal capacity

in defi ned culture conditions and potential to differentiate into any cellular types present in the developed organism. ESC are derived from the inner mass of blastocyst (Fig. 41.2) and can be induced to differentiate into different lineages in vitro by means of the specifi c differentiation protocols to generate cardiomyocytes, endothelial cells, glial precursors, neurons cells, and oligodendrocytes (Wobus and Boheler, 2005).

I I I . A L T E R N A T I V E S O U R C E S O F I S L E T C E L L S • 609

Endoderm Pre-pancreaticendoderm

Endocrineprecursor

NeuroD1

α cell

?PP+ cell PP cell

δ cell

β cell

Ductcell

Acinar Exocrine cell

Ngn3

Hes1

Notch+

Hb9

PDX-1

Isl1

Ptf1/p48

Mist1 Foxa2HNF3g

Brn4Arx1Nkx6.2

MafB Pax6

Nkx2.2

Pax4

Nkx6.1MafA

Pax6PDX-1Isl1Hb9Sox4Pet 1HNF 1b, HNF4a,

HNF6, Foxa1, Foxa2,GATA4/5/6

Sox-2HNF-6FOX 1HNF1β

BrachyurySox 17

ESC

Meso-endoderm

Oct-4NANOGSOX-2FOX D3

FIG. 41.1. Hierarchical cascade of transcription factor in the development of pancreatic tissue from embryonic stem cells. Adapted from Samson and Chan (2006).



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Mouse ESC can be induced to differentiate into insulin-producing cells (Soria et al., 2000; Soria et al., 2001). Using a cell-trapping system we have been able to develop methods to obtain insulin-producing cells from embryonic stem cells. Insulin-producing cells correct hyperglycemia when transplanted into diabetic animal models (Soria et al., 2000; Leon-Quinto et al., 2004; Vaca et al., 2006). ESC are trans-fected with a vector that contains two selection cassettes, one controlling the expression of antibiotic resistance that permits the selection of transfected cells, and another a chi-meric gene containing a functional specifi c promoter driving the expression of a structural gene that codifi es for antibi-otic resistance allowing the selection of cells that express the selected gene (Fig. 41.3). This method has been shown to work with insulin and Nkx 6.1 promoters (Soria et al., 2000; Leon-Quinto et al., 2004; Vaca et al., 2006).

Recently we published a protocol in which fetal soluble factor from pancreatic buds were used to direct ESC differen-tiation into insulin-producing cells further selected with a human insulin promoter — βgeo/PGK-hygro construction (Vaca et al., 2006). Further improvements in the differentia-tion and selection procedure will result in better results.

Furthermore, expression of some β-cell develop ment transcription factors may promote differentiation of ESC into betalike cells, i.e., Pax-4 constitutive expression (Sosa-Pineda, 2004), Pdx-1 overexpression regulated by a Tet-off regulation system (Miyazaki et al., 2004), or Nkx2.2-transfected ESC (Shiroi et al., 2005). Preliminary results suggest the potential use of human embryonic stem cells to obtain betalike-cells (Brolen et al., 2005).

Adult Stem CellsTransdifferentiation of adult cells into insulin-produc-

ing cells also provides another exciting opportunity for β-cell expansion (Ruhnke et al., 2005; Seeberger et al., 2006). Recently our group has published the about obtaining beta-like-cells from white blood cells by reprogramming blood monocytes in the presence of macrophage colony-stimuling factor and interleukin 3, followed by incubation with epi-dermal and hepatic growth factor and nicotinamide. These cells showed an in vitro glucose-dependent insulin secre-tion and normalized blood glucose levels in diabetic mice (Ruhnke et al., 2005). While these strategies seem promising, more studies are needed.

FIG. 41.2. Derivation of embryonic stem cells from the inner mass of the blastocyst.



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Generation of betalike cells from human adult pancre-atic tissues has also been explored, such as development of differentiated islets from pancreatic duct cells and mesen-chymal stem cells from human pancreatic ductal epithe-lium (Gershengorn et al., 2004; Seeberger et al., 2006), human adult pancreatic tissues (Lechner et al., 2005), nonendocrine pancreatic epithelial cells (Hao et al., 2006), and proliferating human islet–derived cells (Ouziel-Yahalom et al., 2006). It is interesting that nonendocrine pancreatic epithelial cells were capable of endocrine differentiation when they were incubated with factors present in human fetal pancreas cells.

The strategy of redifferentiation with proliferating human islet–derived cells includes environmental activa-tion of transcription factors such as Pdx-1, Neuro D, Nkx 2.2, and Nkx 6.1 by betacellulin, a member of the epidermal growth factor family (Ouziel-Yahalom et al., 2006).

IV. BIOMATERIALSImmunological Considerations

Transplanted islets are recognized as antigens by a host, triggering the process of recruiting and activating of immune cells, such as macrophages, fi broblasts, granulocytes, and

FIG. 41.3. Protocol for differentiation of ESC into insulin-producing cells. Adapted from Soria et al. (2001).

I V . B I O M A T E R I A L S • 611



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lymphocytes. The activated immune cells secrete various cytokines and cytotoxic molecules, which can induce func-tional and structural damage to islets (Sigrist et al., 2005). In addition, infl ammatory cytokines such as interferon-γ stimu late the production and release of chemokines by transplanted islets. These chemokines promote the activa-tion of macrophages and have been implicated in a series of biochemical, metabolic, and functional changes, such as increased phagocytosis capacity, chemotaxis, and secretion of chemoattractants, that result in the hampering of islet engraftment (Sigrist et al., 2004). Interleukin (IL)-1 activates T-cells, resulting in the increased production and expression of IL-2 and IL-2 receptor (IL-2R), which are considered the major components of immune rejection. Selective preven-tion of the IL-2/IL-2R interaction using the IL-2R binding agent prolongs allograft survival. TNF-α increases adhesion of molecules on the grafted tissue, thereby stimulating graft rejection as well as inducing cell death by apoptosis. Recently it has been described that the granzyme B produced by allo-genic cytotoxic T-lymphocytes participates actively in the rejection (Sutton et al., 2006). In addition, nitric oxide and reactive oxygen species generated by activated macrophages act as a cytotoxic factor (Chae et al., 2004; Sigrist et al., 2005).

EncapsulationEncapsulation of pancreatic islets for transplantation

into diabetic patients may solve two major obstacles in clin-ical applications. (1) Immunoisolation may allow for trans-plantation of islets in the absence of immunosuppression; (2) it may permit grafting of xenogenic islets, insulin-producing cells differentiated from stem cells, or surrogate beta-cell lines, thereby overcoming the logistical problems associated with the limited supply of human pancreases.

Encapsulation of cells or artifi cial organs consists of placing them inside bioactive materials (usually polymeric membranes, such as alginate), whose physicochemical properties facilitate free diffusion of nutrients, oxygen, electrolytes, and therapeutic bioactive secretory and cellu-lar waste products produced in their inner while avoiding the intake of proteins of high molecular weight, such as the immunoglobulins, and cells of the immune system (Fig. 41.4). Nevertheless, cytokines and chemokines possess a molecular weight close to insulin, so a molecular fi lter cutoff will not exclude the cytokines and chemokines responsible for the immune response (Sigrist et al., 2005). Encapsulation has not yet been applied in clinical practice, mainly because survival of encapsulated islet grafts is limited. The principal causes for the failure of microencap-sulated islet grafts relate to lack of biocompatibility, limited immunoprotective properties, and hypoxia (de Groot et al., 2004).

Encapsulated allogenic and xenogenic islets differ regarding the specifi c immunologic response when they are transplanted. Allograft rejection occurs as a result of the

activation of cellular immunity by interactions of host T-cells with the islet graft, as previously described, while humoral immunity, including antibodies and complement proteins, is mostly responsible for the rejection of xeno-grafts. It is important to consider that xenografts are less capable of binding and responding to human cytokines. Immunoisolation may also protect against antigens of allo-geneic or xenogeneic cells. Such antigens could be cell surface molecules and cell components, including those released on cell death. Shedding of antigens from encapsu-lated cells would initiate a molecular tissue response around the implant, which could affect the viability and function of the encapsulated cells. The recognition of antigens through this indirect pathway may lead to the activation of T helper cells, which then secrete cytokines and regulate the cell-mediated immune response and infl ammation (Kizilel et al., 2005). Other strategies that complement encapsulation should be explored to avoid the damage that free diffusion molecules such as nitric oxide and free-radical oxygen mol-ecules can cause (Chae et al., 2004).

Islets could be either macro- or microencapsulated. Macroencapsulation can use either extravascular or intra-vascular devices (Kim et al., 2005; Kizilel et al., 2005). Micro-encapsulation envelops islets in an alginate-based membrane and other biomaterials (Lanza et al., 1999; Kim et al., 2005).

FIG. 41.4. Immunoisolation concept and microcapsule models. The islets are being enveloped in alginate-based microcapsules (alginate, poly-lysine, alginate), which permit the free diffusion of glucose, insulin, nutrients, wastes, and chemokines. The microcapsule protects from the immune system reactions and can protect from nitric oxide and reactive oxygen species. The size of the pore retains the xenogeneic virus.



The advantages and disadvantages of both are shown in Table 41.1.

MacroencapsulationMacroencapsulation refers to the enveloping of a large

mass of islets in a chamber of a perm-selective membrane, forming either an intravascular macrocapsule (connected as a shunt to the systemic circulation) or an extravascular macrocapsule (transplanted subcutaneously or intraperito-neally) (Fig. 41.5). Intravascular macrocapsules are usu -ally perfusion chambers of microporous or nanoporous material that are directly connected to the blood circulation. Extravascular macrocapsules are usually diffusion cham-bers in the shape of a tube or sphere. The typical dimension of macrocapsules is in the range of 0.5–1.5 mm inner diameter and 1–10 cm length (Kizilel et al., 2005).

Polymers for macroencapsulation are mechanically more stable and the wall capsule generally thicker than those used in microencapsulation. So this technique gives greater long-term stability to the implant. However, the thicker wall and the larger diameter of the capsule can impair diffusion, threatening the viability of the islet and slowing the insulin kinetic release as a result of diffusion limitations of nutrients and oxygen. To guarantee adequate feeding of the cells, the islet density of the macrocapsules is kept quite low and never exceeds 5–10% of the volume frac-tion. Optimal results have been obtained by immobilization of the islets in a matrix before fi nal macroencapsulation. As a consequence, large devices have to be implanted to provide suffi cient masses of insulin-producing islets. These large graft volumes are impractical and cannot be implanted in conventional sites for transplantation of

islets such as the liver, kidney capsule, or spleen. Even the relatively large space in the peritoneal cavity does not suffi ce for the large volume required for the long-term function of an islet graft in macrocapsules. Also, the relatively large surface-to-volume ratio of the macrocapsules interferes with adequate regulation of the glucose levels, because exchange of glucose and insulin occurs rather slowly. One advantage of the implantation of macrocapsules is the ease of retrieval in case of complications (Kizilel et al., 2005). Macroencapsulated rat islets implanted in the peritoneal cavity of diabetic mice maintained an effective insulin secre-tory response (Qi et al., 2004).

Bioartifi cal pancreases should avoid the possibility of contamination with virus from xenogenic islets while allow-ing insulin and nutrient exchanges. Hydroxymethylated polysulphone macrocapsules display insulin release kinet-ics very similar to those of free-fl oating islets and retain retrovirus release (Petersen et al., 2002).

VascularizationPostimplantation conditions such as the design of the

capsule, the environment at the implantation site, and the development of fi brosis around the construct can produce hypoxia-induced death of islet (Papas et al., 1999). Enhanc-ing the oxygenation of the capsule will increase the number of viable cells within and thus its overall secretory capacity. In this context, neovascularization around the capsule may be benefi cial for the overall effi cacy of such tissue substitutes in vivo. Encapsulated islets promote vascular-ization after transplantation due to VEGF release (Lembert et al., 2005). A prevascularized site in the intermuscular space may be created by implanting a polyethylene

I V . B I O M A T E R I A L S • 613

Table 41.1. Advantages and disadvantages of transplantation of unencapsulated, microencapsulated, and macro-

encapsulated islets (adapted from Emerich et al. 1992)

Unencapsulated Microencapsulation MacroencapsulationAdvantagesAnatomical integration between Use of allo- and xenoislets without Use of allo- and xenoislets without host and transplanted islet immunosuppression immunosuppression Thin wall and spherical shape are Good mechanical stability optimal for cell viability and free Good cell viability and free diffusion of diffusion of nutrients and insulin nutrients and insulin RetrievableDisadvantagesRequires immunosuppression Mechanically and chemically fragile Internal characteristics (i.e., diameter) may potentially limit free diffusion of nutrients/ insulin and cell viabilityTissue availability limited Limited retrievability Need for multiple implants may produce signifi cant tissue displacement/damageTissue survival often poorLimited retrievability



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terephthalate (PET) mesh bag with a collagen sponge and gelatin microspheres containing basic fi broblast growth factor (bFGF), before the implantation of islet macroencap-sulates (Balamurugan et al., 2003).

BiocapsulesNanoporous biocapsules are bulk and surface micro-

machined to present uniform and well-controlled pore sizes as small as 7 nm, tailored surface chemistries, and precise microarchitectures, in order to provide immunoisolating microenvironments for cells. Such a design may overcome some of the limitations associated with conventional encap-sulation and delivery technologies, including chemical instabilities, material degradation or fracture, and broad membrane pore sizes (Desai et al., 2004).

MicroencapsulationIslet microencapsulation was fi rst proposed in 1964

(Chang, 1964) and has been shown to be useful in maintain-

ing glucose homeostasis in rodents, dogs, and primates (Elliot et al., 2005; Kizilel et al., 2005; Calafi ore et al., 2006; Dufrane et al., 2006).

Alginate–polylysine-alginate (Fig. 41.4) is most fre-quently used because it has been found not to interfere with cellular function and to be stable for years in small and large animals as well as in human beings (Lanza et al., 1999; de Groot et al., 2004; Elliot et al., 2005; Calafi ore et al., 2006). Alginate is a polysaccharide extracted from seaweed. In solu-tion it has a high viscosity, but in the presence of polyvalent cations, such as CaCl2, it forms gels less insoluble in water. Impurities present in alginate polymers may contribute to the failure of the encapsulated islet implants. Alginates con-taining higher fractions of α-l-guluronic acid (G blocks) resi-dues are more biocompatible (i.e., they do not induce a cytokine response from monocytes) than those containing a larger fraction of α-d-mannuronic acid (M blocks) residues. Omer et al. (2005) obtained smaller microcapsules with better stability and biocompatibility using highly purifi ed

FIG. 41.5. Models of macrocapsules. Extravascular macrocapsule and intravascular macrocapsule.



alginate containing high G or M blocks with low endotoxin levels and BaCl2 as cross-linked agent. In addition, simulta-neous incorporation of alginate, BaCl2 and human serum albumin improves the biocompatibility and protects adult rat and human islets against xenorejection for long periods after transplantation (Omer et al., 2005). Several other strate-gies for increasing biocompatibility have been assayed, such as the use of alginate-poly-l-lysine-alginate modifi ed with polyethylenglycol (Desai et al., 2000), polyacrylates (Isayeva et al., 2003), agarose (Kobayashi et al., 2003), sodium cellu-lose sulfate (Schaffellner et al., 2005), and the polymerization of acrylamide monomers on islet cells encapsulated in agarose microspheres (Dupuy et al., 1988). Also, surface coating with polyethylene oxide improves the viability of microencapsulated islets by promoting oxygen supply and reduces the absorption of proteins associated with fi brotic reaction (Kim et al., 2005). Despite all these efforts to increase biocompatibility, it has not been possible to eliminate the formation of fi brotic outgrowths.

An encapsulation system that improves strength and mechanical stability has been obtained using a double cross-linked simple physical mixture of sodium alginate (ionically cross-linked) and a polyethylene glycol-acrylate (PEGA) (covalently cross-linked) to form AP capsules, where the porosity and permeability depend on the ratio of alginate to PEGA. Tests in vitro have shown that the AP capsules provide a biocompatible and nontoxic environment for islets and are capable of retaining normal functions (Desai et al., 2000).

The immunoprotective properties of the microcapsules are related to the physicochemical characteristics. Polymers containing sulfonic acid or sulfate groups, which have a strong affi nity for complement proteins, improve these properties. With xenogenic islets, where the complement reaction is the principal humoral immune reaction, the poly(styrene sulfonic acid) mixed with agarose protects xenogenic islets in mice (Petersen et al., 2002; Lembert et al., 2005).

Since all polymers used for immunoisolation are not completely inert, several researchers are now studying the use of condrocytes and their matrix for islet encapsulation, to prevent immunorecognition and destruction of trans-planted islets (Pollok et al., 2001).

Optimal Site for TransplantationSelecting the optimal transplantation site for mic ro-

encapsulated islets is an important consideration. Trans-plantation of microencapsulated xenogenic islets into intraperitoneal sites has been studied. Pig islets encapsu-

lated in an alginate-based matrix survive for over 200 days in chemically diabetic animal models (Omer et al., 2005), However, a high number of encapsulated pig islets and graft failures were also observed in most mice after 42 days of implantation. The variation observed is usually attributed to insuffi cient biocompatibility of the microcap-sule, which causes an accumulation of macrophages and fi broblasts on the microcapsule, provoking necrosis of the islets. Dufrane et al. (2006) investigated the impact of implantation sites on the biocompatibility of alginate-encapsulated pig islets in nondiabetic rats. Thirty days after transplantation, explanted capsules from intraperitoneal sites demonstrated a higher degree of broken capsules and capsules with severe cellular overgrowth. On the other hand, capsules removed from subcutaneous and kidney subcap-sule sites showed no such damage. They concluded that kidney subcapsular and subcutaneous spaces represent an interesting alternative.

Recently the Calafi ore team (University of Perugia, Italy) published their results of pilot phase-1 clinical trials with the human allograft islet microencapsulated in 1.6% sodium alginate and sequentially double-coated with 0.12% and 0.06% poly-l-ornithine and fi nally with 0.04% sodium algi-nate. The islet microencapsulates were intraperitoneally (IP) implanted into two selected nonimmunosuppressed patients with type 1 diabetes. Both patients showed a rise in sCPR levels several weeks posttransplant, amelioration of their mean daily blood glucose levels, and a progressive decline in exogenous insulin consumption. GHb also decreased throughout months of posttransplant follow-up. At 60 days posttransplant, an OGTT in patient 1 showed a biphasic C-peptide response, compatible with the presence of differentiated islet β-cells. At 1 year (patient 1) and 6 months (patient 2) of posttransplant follow-up, sCPR was still being detected in these recipients (Calafi ore et al., 2006).

On the other hand, cytotoxic molecules produced by activated macrophages such as IL-1β, TNF-α, NO, and reactive oxygen species may also pass through the polymer membrane and damage the transplanted tissue (Chae et al., 2004). The strategies used to protect the islet from these molecules are diverse. As an approach to preventing NO-induced damage, the rat islet and insulinoma cells (RINm5F) microencapsulated in alginate–poly-l-lysine-alginate were coencapsulated with cross-linked hemoglobin (Hb-C)–poly(ethylene glycol). These strategies protect from damage induced by nitric oxide and prevent islet death by hypoxia (Chae et al., 2004).

V. ACKNOWLEDGMENTSWe thank Giuseppe Pettinato and Sergio Mora for the artwork. This work has been partially supported by grants from Instituto de Salud Carlos III (PI0521/06), Ministerio de

Educación y Ciencia (SAF 2003-367), and Fundación Pro-greso y Salud (Junta de Andalucía).

V . A C K N O W L E D G M E N T S • 615



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Generation of Islets form Stem Cells