Diabetes mellitus, the metabolic disorder is rapidly on the rise, becoming one of the main threats to human health and imposing large socio-economic burden on the society in the 21st century (Dall et al.; 2010). International Diabetes Federation in 2011 estimated that over 300 million people around the world have diabetes and is expected to rise to 500 million within next 20 years. The global prevalence of diabetes is shifting significantly from the developed countries to the developing countries. Current therapy for diabetes involves oral antidiabetic drugs and insulin administration; these approaches do not mimic the pulsatile insulin secretory patterns of native β islets for the regulation of glucose in real-time nor provide tight control of blood glucose to avoid late complications of the disease. Whole pancreas transplantation holds promise towards a cure for diabetes, but this procedure requires major surgery and lifelong immunosuppression to prevent graft rejection. Transplantation of islet cells isolated from a donor pancreas has been shown to control glucose levels successfully. Being less invasive, it is a better alternative to pancreas transplantation yet scarcity of donors, maintenance of islet functions such as cell growth and survival
One approach to overcome these obstacles of immune rejection is islet encapsulation (Kizilel et al., 2005; Mikos et al; 1994) that uses an immuno-protective biomaterial to create a permselective membrane around a group of islet cells. Transplanted islet cells are separated from the immunological system of host by means of an artificial selectively permeable membrane which allows passage of metabolites and nutrients, while excluding based on size, the larger proteins and cells of the immune system. Thus, encapsulation is designed to limit, and ideally eliminate, an immunological response to the non-host islet cells. Isolation of the islet cells from the human immune system may also make xeno-transplants such as porcine islets, stem cells derived insulin producing cells possible, eliminating the supply problem that exists and the usage of immune suppressive drugs.
Current research is directed towards exploration of alternative sources of pancreatic islet cells. Pancreatic β- cell lines, embryonic stem cells (ESC), adult progenitor cells (APC), and regenerating native islet cells, generation of β cells by therapeutic cloning and differentiation of islets from pancreatic duct cells as well as stem cells are being explored for their potential to serve as β-cell sources. Large numbers of cells can be generated from β cell lines, although their unrestricted proliferation is also a serious concern in the context of cellular therapies. Unlike stem and progenitor cells, β islet cells have limited ability to multiply under normal conditions, although investigations into factors that stimulate β-cell regeneration have yielded promising results. Therapeutic molecules capable of increasing β-cell mass in vivo may eliminate the need for invasive surgical procedures. However, the extent of adult β cell regenerative capacity is unclear. Ex-vivo expansion of islets is indispensable prior to transplantation regardless of the source of islets. Monolayer culture of islets fail to maintain the three dimensional shape and integrity of islets resulting in consequent apoptosis and necrosis.
2. Islet transplantation
Islet cell transplantation is an effectual treatment for improving glycemic condition in diabetic patients thereby reducing the late complications of disease (Shapiro, 2003). The procedure involves isolating islets from a deceased organ donor, purifying, processing and infusing them into diabetic patients.
In the early 1970’s, Dr. Clyde Barker at the University of Pennsylvania and the late Paul Lacy at the Washington University in St. Louis were the pioneers in exploring the concept of islet transplantation as a means to cure diabetes. In 1972, Ballinger and Lacy reported amelioration of diabetes in islet recipient rats (Ballinger & Lacy, 1972). In 1973, Reckard and Barker were the first to show that islet transplantation could completely and permanently restore normoglycemia in rodent models of chemically induced diabetes (Reckard & Barker, 1973).
Human islets isolation procedure is more complex than rodent islets isolation (Gray et al., 1984). Ricordi’s automated isolation method had given hope for the production of abundant islets for the clinical use (Ricordi et al., 1989). Scharp et al. performed the islets transplantation under immunosuppression in diabetes patients and patients were insulin independent at the period of 22 days (Scharp et al., 1990). This was followed by several other cases, but success rates continued to be low (International Islet Transplant Registry). In 1999, Bretzel et al reported a markedly improved 3-month islet graft function rate of at least 75% in 24 consecutive patients (Bretzel et al., 1999). In the 1-year follow-up of 37 patients, 24% had achieved insulin independence (Bretzel et al., 2000). Between 1999 and 2005 about 650 patients were treated worldwide (Bretzel et al., 2007).Unfortunately, long-term results did not prove that promising.
The first successful islet allograft was reported in 1990 with steroid free immunosuppressant tacrolimus (Tzakis et al., 1990). The success rate of islet transplantation became outstanding after the Edmonton trial in 2000, which described successful intraportal alloislet transplantation, defined as insulin independence, in 7 consecutive patients with hyperlabile diabetes and frequent episodes of hypoglycemia (Shapiro et al., 2000). The success was partly ascribed to the usage of a steroid free immunosuppressive regimen which was a new combination of immunosuppressive drugs, consisting of sirolimus, tacrolimus and daclizumab, excluding the diabetogenic glucocorticoids and large numbers of donor islets (Shapiro et al., 2006).
The short-term results of islet transplantation with Edmonton trial were promising, with insulin independence in approximately 80% of patients at 1 year post-transplant but the proportion of insulin independent recipients declined after the first year post-transplant. Five year follow-up study after transplantation revealed that only 10% of the patients remained insulin independent (Ryan et al., 2005; Shapiro et al., 2006). The suggested reasons attributing for this decline include alloimmune rejection, autoimmune recurrence, and/or toxicity of immunosuppressive medications (Monti et al., 2008). However, about 80% were still C-peptide positive, indicating functioning grafts. Now, a slightly modified Edmonton protocol is used worldwide with reproducible results (Shapiro AM et al., 2006).
Though islet transplantation research has made significant progress, concern over toxicity as well as cost of immunosuppressive therapy still remains. Insulin independence and long term graft survival were achieved for more than three years through a modified immunosuppressive protocol (Bellin et al., 2008) even so the requirement of multiple donors to obtain 10000 islet equivalents per kilogram of patient’s weight remains unsolved. Although insulin independence remains the ultimate goal, today, stabilization of glucose levels and avoidance of hypoglycemia are considered to be the main indications for islet transplantation.
3. Alternate sources of pancreatic β cells
The scarcity of donor pancreas for islet transplantation is a major obstacle to the widespread use of islet transplantation which urged the focus towards alternate sources of β cells for future transplants. Several alternative means have been suggested which include use of xenogenic islets and immortalized beta cell lines (Narushima et al., 2005). Recent advances in the field of stem cell differentiation and regeneration therapy have focused on new ways to generate insulin-producing beta cells that can be used for transplantation. Several candidate cells have been identified including embryonic stem cells (ESC) and adult stem cells or progenitor cells residing in the pancreas or other organs. The differentiated beta cells have shown to regenerate by replication, which opens the possibility to generate novel beta cells
3.1. Xenogenic islets
In a xenogenic approach, islets from different species are used for transplantation purpose. Porcine islets serve as a potential source in view of the fact that porcine insulin differ from human insulin by 1 amino acid. Neonatal porcine islets were also induced to mature endocrine phenotype under
3.2. Stem cells to treat diabetes
Stem cells are non specialized cells which have the ability to self regenerate and differentiate into specialized cell types depending on the niche or external signaling cues (Smith, 2006). Stem cells offer a limitless supply source for islets as well reduces the graft rejection problems (Street et al., 2004). Ideally stem cells used for cell based therapy should meet the following criteria (Gimble, 2003) : It should be available in abundant quantities (millions to billions of cells), harvest procedure should be less invasive, have multilineage differentiation potential and could be efficiently transplanted to the host.
3.2.1. Embryonic Stem Cells (ESC)
Embryonic stem cells which are derived from the inner cell mass of pre-implantation blastocysts have gained the attention of researchers due to its pluripotent nature. Human embryonic stem cells (hES) hold promise for research and clinical applications. hES have some unique abilities as compared to all sources of adult cells:
the expansion of ESC in the undifferentiated state is nearly unlimited; and
ESC can give rise to all cell types including pancreatic insulin-producing beta cells.
Attempts of directed differentiation of hES to cardiomyocytes (Klug et al., 1996), and neurons (Li et al., 1998) have been reported. Many studies have reported the differentiation of mouse and human embryonic stem cells to islet like clusters (Segev et al., 2004; Vaca et al., 2006) either by modifying the culture conditions or by genetic manipulation. Lineage tracing experiments revealed that beta cells are derived from the embryonic endoderm followed by a sequential and transient activation of specific transcription factors like Pdx1, NeuroD / Beta 2, Isl1, Nkx6.1, Nkx2.2, MafA, Pax4, and Pax6. Most published protocols aim to mimic these differentiation factors for the stepwise development of the endo and exocrine pancreas during the embryonic phase. In one approach ESC were induced to generate embryoid bodies under
Insulin immunoreactivity was demonstrated to occur as the consequence of insulin uptake from medium (Rajagopal, J., 2003). Few authors proved that nestin positive cells tend to differentiate towards neuronal lineage rather than pancreatic lineage (Sipione et al., 2004). Kania et al explained the cause for the controversial results in generation of pancreatic cells. It was suggested that insulin producing cells derived from embryonic stem cells without applying lineage selection is dependent on the differentiation factors and protocols (Kania et al., 2004).
Though hES are versatile cells, ethical concerns on the use of human hES, and chances of teratoma formation (Fujikawa et al., 2005) limits their usage. Direct transplantation of embryonic stem cells has reported to culminate in teratoma formation (Nussbaum et al., 2007) from contaminating undifferentiated ESCs. Safe transplantation of hES could be attempted by viral vector mediated transfection
3.2.2. Adult Stem Cells
The potential use of adult stem cells offers the advantage of an autologous model whereby a patient’s own cells can be used, thereby circumventing immune rejection. Adult stem cells (ASC) are multipotent cells capable of self renewal. They have been reported to be present in almost every tissue like bone marrow, blood, heart, liver, pancreas, adipose tissue and could be transplanted directly without genetic modification or pre-treatments. They exhibit high degree of genomic stability during culture conditions. ASC lack tissue specific characteristics but it could be differentiated to specialized cell types under the influence of appropriate signaling cues (Barry & Murphy, 2004). The stem cell microenvironment plays an important role in its differentiation to committed cells (Galli et al., 2000; Zhao et al., 2002). The potential of adult human stem cells from various sources to differentiate to insulin producing cells have been explored by various research groups. The relative ease of isolating adult stem cells and their expansion makes it an ideal source for cell based therapy.
126.96.36.199. Pancreatic stem cells
Pancreatic progenitor/stem cells which are closely related with beta cell lineage represent an attractive source for generation of beta cells (Serup et al., 2001). Human pancreatic ductal cells and islet stem cells have been expanded and differentiated to islet like clusters capable of producing insulin
188.8.131.52. Bone marrow stem cells (BMSC)
Bone marrow stem cells were induced to differentiate to mature endocrine pancreatic lineage
184.108.40.206. Adipose stem cells
Human subcutaneous adipose tissue, abundant and easily accessible serves as a potential source of adult mesenchymal stem cells. The harvest procedure by lipoaspiration / liposuction is less invasive. Adipose stem cells have been reported to exhibit an increased
220.127.116.11. Progenitor cells and stem cells from other tissues
The mechanisms involved in the generation of new beta cells in postnatal life remains controversial. Lineage tracing experiments suggest that after partial pancreatectomy, the majority of novel beta cells derive from pre-existing beta cells by beta cell proliferation rather than stem cell differentiation (Dor et al., 2004; Georgia and Bhushan, 2004). These findings raise new hope that it may be possible to use beta cells as source for the
It is important to note that the above mentioned experiments do not exclude the existence of pancreatic stem cells. There is convincing evidence that under specific experimental conditions adult stem cells, which reside in the pancreatic ducts, within islets, or exocrine tissue, can develop into beta cells. Bonner-Weir and colleagues as well as other groups provide evidence in several studies that murine and human stem cells or progenitor cells reside in the epithelium of the small pancreatic ducts, expressing the marker cytokeratin-19 (CK19). The ductal cells can expand and give rise to novel beta cells after 90% pancreatectomy or treatment with streptozotocin (Bonner-Weir et al., 1993; Wang et al., 1995; Bonner-Weir S et al., 2000; Gao et al., 2003). This suggests that still unknown cellular or soluble factors are needed to induce terminal differentiation into the endocrine fate. The identification of these critical factors is one focus of current research.
Amnion epithelial cells (Hou et al., 2008; Kadam et al., 2010a), and stem cells from liver (Yang et al., 2002), wharton’s jelly of umbilical cord (Chao et al., 2008), umbilical cord blood (Phuc et al., 2010), placenta (Kadam et al., 2010b), gall bladder (Sahu et al., 2009) etc were also differentiated to insulin producing cells. In every case the differentiated islets expressed islet specific proteins and were capable of secreting insulin in response to glucose stimulation, albeit less than native islets. A better understanding of the events that control stem cell commitment and the signaling pathways for differentiation to pancreatic lineage is required to improve the culture conditions for
4. Tissue engineering based strategies
Tissue engineering applies the principles of cell transplantation, material science, and engineering towards the development of biologic substitutes that can restore and maintain normal function. The success of tissue engineering relies on the development of a suitable scaffold, cell source and defined culture conditions. Tissue engineering strategies employed for islet transplantation could be categorized into use of scaffolds for simulation of the native ECM and immunoisolation via encapsulation of islets.
The biomaterial chosen to synthesize the scaffold should be biocompatible and biodegradable (Mohan & Nair, 2005). The scaffold should reproduce an extracellular environment for supporting cell function (Dufour et al., 2005). Neither the scaffold nor its degradation products should be toxic to host. Biocompatibility of chosen biomaterial plays an important role in controlling the rate of protein adsorption and fibrosis. The scaffold should provide three dimensional space for neotisssue formation and the mechanical strength should match the native tissue to withstand
4.1. Extracellular matrix - Mimicking nature
A major limitation with two dimensional cultures is the lack of microenvironment indispensable for appropriate spatial organization and function of cells which in turn is provided by extracellular matrix (ECM) in native tissue. The ECM of native islets is mainly constituted by type IV collagen, laminin although fibronectin, collagen I, collagen V also have been detected (Stendahl et al., 2009). The effect of ECM proteins on adhesion and proliferation of rat islets have been studied. The results indicated that there were strong interactions between islets and ECM proteins via integrins (Chen et al., 2008). ECM protein coated scaffolds have shown improved graft efficacy at implanted site (Salvay et al., 2008).
Earlier reports of islets cultured under two dimensional conditions exhibited low survival rate and reduced insulin secretion (Paraskevas et al., 1997). Progress in survivability of islets and increased insulin secretion has been achieved by adopting 3D culture conditions. Polyglycolic acid scaffolds coated with lysine were shown to promote islet cell adhesion and survival. The control cells grown in 2D culture underwent apoptosis by day 7 due to accumulation of metabolites and shortage of nutrients (Chun et al., 2008).
Pancreatic islets cultured on agarose cryogel sponge (Bloch et al., 2005) were reported to secrete 15 fold higher insulin at 3mM glucose than islets cultured on polystyrene cell culture dish but failed to respond to stimulation at 16.7mM glucose. The failure was due to limited oxygen and nutrient diffusion across agarose cryogels. Adequate oxygen is a critical parameter to islet cell function and survival (Sweet et al., 2002). The decreased insulin response of pancreatic islets cultured on scaffold for prolonged period due to inefficient oxygen transfer were also reported (Cui et al., 2001).
A novel 3D culture system comprising a semi-interpenetrating polymer network of gelatin and polyvinyl pyrrolidone was designed in our laboratory for pancreatic islets and stem cells to promote their survival and function. The porous nature of the scaffold facilitated efficient nutrient and metabolite exchange. Islets seeded on this scaffold maintained their morphology for more than 30 days whereas control islets cultured on cell culture dish underwent apoptosis by 7th day. The test islets secreted insulin on stimulation with glucose which was comparable to that of freshly isolated mouse islets (Muthyala et al.,2010).
Zhao et al demonstrated the use of three dimensional self assembling peptide nanofiber hydrogel scaffold for islet culture. The peptides formed two beta sheet structures with hydrophilic and hydrophobic surfaces in aqueous solution. The hydrophobic moiety facilitated its self assembly in water and the nanofiber structures were flexible for fabrication to different geometrical shapes that allowed for efficient nutrient and metabolite transfer. The nanofiber scaffold simulated the microenvironment
4.2. Immunoisolation strategies for islet transplantation
The principle behind immunoisolation is protection of islets from host immune system using a selectively permeable membrane as a barrier. Low molecular weight substances which include nutrients, oxygen, secretory molecules and cell signaling molecules freely diffuse through the membrane, but passage of immune cells and its products which have high molecular weight is prevented. Immunoisolation mechanism encourages the use of allogenic/ xenogenic sources of islets for transplantation and holds promise towards use of autologous stem cell derived islets in type I diabetic patients. Immunoisolation mechanism includes macroencapsulation and microencapsulation (Narang & Mahato, 2006) of cells.
Microencapsulation is the encapsulation of single islets or small groups of islets. These capsules are usually spherical in shape (Chang, 1964). Microcapsules offer the advantage of increased oxygen and nutrient transport due to the large surface area to volume ratio. Microcapsules are advantageous due to several reasons like greater surface to volume ratio, and ease of implantation. The spherical shapes owe to better diffusion capacity and are mechanically stable. The primary drawback of microencapsulation is the difficulty in removing the implants if necessary. Moreover the implantation could be achieved by simple injection procedure (De Vos et al., 2002). Porcine islets microencapsulated in alginate-polylysine-alginate transplanted to diabetic monkeys could achieve normoglycemia without immunosuppression for more than 800 days (Y. Sun et al., 1996). Human and rat islets encapsulated in alginate gels when transplanted in mice survived for 7 months (Schneider et al., 2005). Xenogenic islets immobilized in microcapsules fabricated from alginate –PLL when implanted into peritoneum of non immunosuppressed diabetic rats remained in excellent condition for more than 40 weeks (Lanza et al., 1999). Despite of these advantages some authors have reported reduced functionality of microencapsulated islets in response to glucose challenge (Sandler et al., 1997).
Macroencapsules contain a large mass of islet cells within a diffusion chamber, which are usually formed from spun coat membranes or spun drawn hollow fibers. The advantages of macrocapsules are they could be easily retrieved when required and can be shaped in required geometries such as tubes or discs. Two approaches such as intravascular and extravascular have been tried out in macroencapsulation. Intravascular approach utilizes the principle of perfusion chambers which consists of microporous tubular structures perfused with blood and enclosed within another tube. Islets were seeded in the space between the hollow fibers and the device is anastomised to the host vasculature (Chick et al, 1975). Polyacrylonitrile and polyvinylchloride copolymers have been chosen as materials for creating artificial microcapillaries. Results from implantation of intravascular macrocapsules of islets have shown restoration of normoglycemia in various animal models (AM. Sun et al., 1977). Due to the direct contact of device with the blood, intense anticoagulation is required to prevent thrombus formation, consequently the material chosen should be highly blood compatible and thromboresistant. These concerns have shifted the attention towards extravascular devices.
Extravascular devices are based on the principle of diffusion chambers which does not require anastomosis to host vasculature. The geometry could be planar in the form of flat or hollow fiber model (Scharp et al., 1984). This approach does not pose severe biocompatibility issues and risks to the patient as that of intravascular devices. Extravascular devices can be implanted to different sites such as peritoneal cavity (Lanza et al., 1999), subcutaneously or under kidney capsules (Siebers et al., 1990) with minimal surgical risks. Most commonly used biomaterials for macrocapsule fabrication are nitrocellulose acetate, 2-hydroxyethyl methacrylate (HEMA), acrylonitrile, polyacrylonitrile and polyvinylchloride copolymer, and alginate.
The biocompatibility of immunoisolation membrane depends on several factors like geometry of the device, implantation site and material chosen. Hollow fiber geometry is preferred because of its reduced surface area of contact with the host per islet and reduced foreign body response. Higher density of islet cells often results in reduced viability and necrosis at the center due to nutrient limitation. Smooth outer surface and hydrogels have been reported to improve the biocompatibility by the absence of interfacial tension, thus reducing protein adsorption, cell adhesion and fibrosis (Burczak et al., 1996). Nair et al studied the effect of degree of hydrophilicity on tissue response of polyurethane interpenetrating networks (IPN) (Nair et al., 1992).The results indicated that an increase in hydrophilicity of polyurethane –polyvinyl pyrrolidone IPN’s elicited an inert tissue response.
George et al., (2002), Nair (2009, Indian Patent 230740) demonstrated the use of non porous polyurethane membranes and porous polyurethane IPN macrocapsules as islet immunoisolative matrices. Islet cell morphology remained intact and insulin secretion ability was also retained within the immunoislation membranes. Membranes allowed diffusion of glucose and insulin while retained transplant rejection factors like antibodies, immunoglobulins and immune cells. Reduced protein adsorption and cell adhesion on polyurethane membranes contributed to improve the biocompatibility which made them ideal for immunoisolation. The IPN macrocapsules also served as an
4.3. Combined approach of tissue engineering and immunoisolation
Muthyala et al (2010) employed a combination approach utilising the properties of scaffold to mimic the native ECM and macroencapsulation for immunoisolation to protect the islets from immune cell responses. Pancreatic progenitor derived islets were seeded on gelatin-polyvinylpyrrolidone scaffolds and further macroencapsulated in a polyurethane–polyvinylpyrrolidone semi IPN macrocapsule. The construct when implanted into peritoneal cavity of diabetic rats normalized glycemic condition all through the study period of 3 months. Animals implanted with tissue engineered islets without macroencapsulation showed no reversal of hyperglycemia and died within 15-20 days due to infiltration of host immune cells. Hence the combination approach was found to be very effective in achieving euglycemia by maintaining islet survival and functionality as well as protecting the cells from host immune attack.
5. Site for transplantation
The optimal site should be chosen for transplanting islets inorder to meet its high energy requirement and metabolic rate (Hardy et al., 2010). The implantation site has effect on hypoxic conditions which determines islet survival. Safety considerations have been raised regarding the optimal site for transplantation so as to improve islet engraftment and survival (Dufrane et al., 2006; Pillegi et al., 2001). Graft vascularization is an important criterion in islet survival and function (Jansson & Carlsson, 2002). Although immunoisolation prevents the integration of host blood vessels with transplanted islets effective diffusion of nutrients and oxygen can occur within 200μm distances hence highly vascularised sites should be chosen for transplantation. Islet transplantation into prevascularized sites dramatically improves graft survival and function relative to transplantation into non-modified tissue (Balamurugan et al., 2003). Vascularization can be introduced in graft by incorporation of angiogenic growth factors like VEGF (Stendahl et al., 2008) or endothelial cells (Miki et al., 2006). Insulin independence have been achieved by intra-portal islet transplantation in diabetic patients (Shapiro et al., 2005), however liver could not be considered as an optimal site since islets in liver will be exposed to high nutrient concentration and other factors that are toxic and may result in impairment of beta cells(Hiller et al., 1991; Wilson & Chaikof, 2008). Peritoneal cavity has also been tried for implantation of islets which requires 200%-400% more islets (Siebers et al., 1993). Subcutaneous site (Pillegi et al., 2006) have been chosen in diabetic athymic mice for transplantation of islets cultured on plasma –fibroblast gel scaffold (Perez-Basterrechea et al., 2009). Normoglycemia was achieved over 60 day period and vascularization was observed in and around islets. Kidney subcapsular spaces have also been chosen as implantation site to improve biocompatibility of tissue engineered constructs. Islets cultured on biodegradable polymer scaffold transplanted to omental pouch of diabetic dogs resulted in achievement of euglycemia upto 152 days till graft was taken out (Kin et al., 2008).
Curative therapy for diabetes mellitus mainly implies replacement of functional insulin-producing pancreatic cells, with pancreas or islet-cell transplants. Shortage of donor organs spurs research into alternative means of generating cells from islet expansion, encapsulated islet xenografts, human islet cell-lines, and stem cells. Embryonic and adult stem cells are potential sources for cell replacement and merit further scientific investigation. The expense of the benefit of cell transplantation is the need for immunosuppressive treatment of the recipient, with all its potential risks. Biocompatible macrocapsules for transplantation of islets and islet-like cell clusters differentiated from stem cells help overcome the immune rejection without Immunosuppressive drug therapy. A tissue engineering approach aims to mimic the natural extracellular matrix environment for supporting the transplanted islet cells without sacrificing form and function. A combination approach of tissue engineering, immunoisolation and most appropriately differentiated islet may propel clinical trials involving engineered strategies for cell replacement in diabetic patients, in the not too distant future.
Authors are grateful to Director, SCTIMST and Head BMT Wing for permissions to publish this work and funding from the DBT, Govt. of India. N. Aloysious also acknowledges the CSIR for the JRF fellowship.