Summary
Restoration of insulin independence and normoglycemia has been the overarching goal in diabetes research and therapy. While whole-organ and islet transplantation have become gold-standard procedures in achieving glucose control in diabetic patients, the profound lack of suitable donor tissues severely hampers the broad application of these therapies. Here, we describe current efforts aimed at generating a sustainable source of functional human stem cell-derived insulin-producing islet cells for cell transplantation and present state-of-the-art efforts to protect such cells via immune modulation and encapsulation strategies.
Main Text
Over the last century, improvements in the synthesis and delivery of recombinant insulin have substantially decreased the morbidity and mortality associated with diabetes mellitus. Despite these advances, more than 400 million people across the world who are affected by diabetes mellitus continue to suffer from devastating secondary complications, including diabetic nephropathy, retinopathy, and neuropathy. Intensified metabolic control has reduced or prevented the development and progression of secondary complications in two landmark trials in patients with type I (Nathan et al., 1993) and type 2 (Holman et al., 2008, UK Prospective Diabetes Study (UKPDS) Group, 1998a, UK Prospective Diabetes Study (UKPDS) Group, 1998b) diabetes mellitus. Unfortunately, the tighter control associated with intensified regimens has been limited by the inherent risks of hypoglycemia. Excellent metabolic control without the need for exogenous insulin can be achieved with beta cell replacement, either through solid organ pancreas transplantation or pancreatic islet transplantation. Both strategies for beta cell replacement stabilize or minimize progression of the secondary complications associated with diabetes mellitus, providing stable long-term allograft function as demonstrated by insulin independence and normalization of glycated hemoglobin (HbA1C) levels.
Despite the increasing success of both strategies for beta cell replacement, broader application of islet and pancreas transplantation is severely limited by the number of available donor pancreases and the need for lifelong immunosuppression; as a result, only a small fraction of people with diabetes mellitus can currently benefit from these therapies. Creating an unlimited source of insulin-producing cells from stem cells will permit widespread application of beta cell replacement to achieve insulin independence. As this source of beta cells moves closer to clinical translation, it is important to review the current state of the art in beta cell replacement, with a focus on successful encapsulation and immune modulation strategies that can be applied to stem cell-derived cells.
Clinically Viable Transplantation Strategies for Treating Diabetes
Whole Pancreas Organ Transplantation
Advances in surgical techniques and refinement of immunosuppression have dramatically improved the success of pancreas transplantation performed for diabetes mellitus. The traditional indication for solid organ pancreas transplant has been in recipients with type I diabetes (T1D) and end-stage renal disease, and the procedure is most commonly performed simultaneously with a kidney transplant (SPK). One-year allograft success, as defined by insulin independence, is approximately 90% at most centers performing this operation. Long-term results continue to improve with the evolution of better immunosuppressive regimens, with five- and ten-year pancreas graft survival rates at 73% and 56%, respectively (Gruessner and Gruessner, 2016). Marked improvements in successful transplantation have increased the indications for pancreas transplantation to include pre-uremic T1D recipients with life-threatening diabetes secondary to hypoglycemic unawareness. Type 2 diabetic (T2D) recipients now represent 9% of all SPK recipients, and their early allograft success is comparable to the T1D SPK recipients (Kandaswamy et al., 2018).
Pancreas transplantation requires a strong cardiovascular system to tolerate both the initial procedure as well as the potential complications associated with transplantation of a fragile organ containing digestive enzymes. Pancreas transplants involve the intraperitoneal placement of the pancreas in a heterotopic location. The reconstructed donor pancreas most often receives its arterial blood supply from recipient iliac vessels, portal (superior mesenteric vein) or systemic (iliac vein) venous drainage, and enteric exocrine drainage of the donor pancreas through an anastomosis between the donor duodenal segment and the recipient ileum. The technical success of pancreatic transplants is around 90%–95%. Early loss of the pancreas allograft is related to thrombosis of the pancreas allograft or leaks of pancreatic enzymes resulting in infection, necessitating removal of the allograft. A technically successful allograft results in almost immediate insulin independence. Furthermore, pre-transplant insulin requirements and body mass index (BMI) do not impact the ability to achieve insulin independence, and the vast majority of pancreas transplant recipients with technically successful allografts achieve insulin independence (Kandaswamy et al., 2018). Stable function of the pancreas allograft prevents recurrence of diabetic nephropathy in the simultaneously transplanted kidney and can prevent progression of retinopathy and neuropathy (Fioretto et al., 1998). For that reason, solid organ pancreas transplantation has become the gold standard for beta cell replacement for patients healthy enough to sustain this taxing procedure. Still, the widespread application of the technique is limited by the rigors of the procedure as well as the scarce availability of suitable donor pancreases.
Pancreatic Islet Transplantation
Transplantation of isolated islets provides a gentler alternative to whole-organ transplantation. A brief history of the events leading to this success is important for understanding future strategies for beta cell replacement using stem cell-derived beta cells. Although pancreatic islet transplantation had been conducted in animal models of diabetes since the early 1980s, successful clinical trials in T1D recipients were elusive until the Edmonton protocol was published in 2000 (Shapiro et al., 2000). To establish insulin independence in seven consecutive patients, islets were isolated from deceased donor pancreases and infused into the portal vein of the liver. Two to three infusions of islets from different donors were required to obtain insulin independence, which was initially achieved in all seven patients. The success was attributed to a regimen that minimized exposure to immunosuppressive agents toxic to beta cells, such as steroids or lymphodepleting induction therapy. Instead, the immunosuppressive regimens consisted of low-dose calcineurin inhibitors (CNIs) to minimize nephron and beta cell toxicity. Despite their initial insulin independence, all seven patients lost islet function within 2 years.
This initial experience revealed some of the problems associated with intraportal infusion of islets, as well as the necessity for more potent immunosuppression. The requirement for two or three donor infusions to create an early state of insulin independence was likely related to poor engraftment of the donor islets. The instant blood-mediated inflammatory reaction (IBMIR) and ischemia contributed to islet loss shortly after infusion into the portal system (Eich et al., 2007). The marginal islet mass that survives and engrafts following intraportal infusion is consistent with the finding that insulin independence has been more common in recipients with low BMIs and lower insulin requirements (Barton et al., 2012, Shapiro et al., 2017).
The problems with marginal mass following islet transplantation are further exacerbated by the alloimmune and recurrent autoimmune responses against the graft. In fact, significant improvements in long-term insulin independence have been achieved by using more potent immunosuppression. Recent international registry data have reported three-year insulin independence rates following intraportal infusion of islets at nearly 50% (Shapiro et al., 2017). Improvements in long-term insulin independence required the same potent lymphodepleting induction regimens that were found to be successful in solid organ pancreas transplants (Barton et al., 2012). Novel immunosuppressive regimens involving maintenance based on blocking immune costimulation (CTLA-4Ig, Belatacept) or leukocyte adhesion (anti-LFA1, Raptiva) have further increased the three-year insulin independence rates to 70% (Posselt et al., 2010). This type of immunosuppression is attractive in that these agents specifically target immune cells without toxic effects on beta cells or kidneys. Importantly, insulin independence was achieved in several cases with a single infusion of donor islets. A cost comparison between solid organ pancreas transplantation and pancreatic islet transplantation has demonstrated comparable costs to achieve insulin independence (Moassesfar et al., 2016). Unfortunately, the very limited number of pancreatic islets available for transplantation severely restricts the widespread use of this technology.
Stem Cell-Derived Therapies for the Treatment of Diabetes
Recent advances in the differentiation of human stem cells towards pancreatic islet cells now suggest clear and tangible alternatives to the more conventional treatment options for T1D and T2D described above. Remarkable progress has been made with regard to the generation of functional beta cells from human stem cell populations over the last decade. The underlying strategy is to closely recapitulate the path that pluripotent stem cells take during embryogenesis, from the formation of definitive endoderm, to pancreatic endoderm, to endocrine progenitors, and finally to pancreatic islet cells (Figure 1) (D’Amour et al., 2005, D’Amour et al., 2006, Guo et al., 2013, Kroon et al., 2008, Rezania et al., 2011, Rezania et al., 2012). More recently, efforts have been placed on generating single hormone-positive beta cells capable of glucose-stimulated insulin secretion (GSIS) (Millman et al., 2016, Pagliuca et al., 2014, Rezania et al., 2014, Russ et al., 2015, Zhu et al., 2016). Efforts in several laboratories are focused on generating fully functional beta and islet cells that could soon replace human islets as a source for insulin-producing cells. Indeed, clinical trials using stem cell-derived pancreas progenitors capable of developing into functional beta and islet cells are currently underway. Here, we review the current state of generating human embryonic stem cell (hESC)-derived pancreatic islet cell types.
Optimizing the Stem Cell Niche to Generate Islet-like Structures
Despite the rapid progress in generating insulin-producing cells from hESC populations, the formation of fully functional cells perfectly replicating all aspects of endogenous beta cells has remained elusive. Considering that beta cells in vivo do not exist in isolation, but rather in the context of intricate, 3D structures of the islets of Langerhans, generation of a “full complement” of islet cells should be considered beneficial (Figure 1). Indeed, the maintenance of beta cell function has been reported to be highly dependent on the complex islet cytoarchitecture, with isolated beta cells behaving differently from those within intact islets (Cabrera et al., 2006, Halban et al., 1982, Ravier et al., 2005, Wojtusciszyn et al., 2008). In single, isolated beta cells, for instance, glucose-stimulated insulin secretion is compromised compared to intact islets, due to both elevated basal insulin secretion and reduced maximal insulin secretion in response to glucose (Benninger et al., 2011, Halban et al., 1982). Along with other mechanisms for coordinating cell-cell communication, gap junctions such as Connexin-36 play an important role in maintaining beta cell coupling and islet synchronization of insulin oscillations (Benninger et al., 2011, Ravier et al., 2005). As we consider the methods for generating the most optimally functioning hESC-derived unit for diabetic rescue, it will also be important to consider methods for recapitulating endogenous islet structure, cell-cell contacts, and communication with the islet microenvironment.
In rodent models, pancreatic islets typically consist of a beta cell core surrounded by a mantle of alpha, delta, and pancreatic polypeptide (PP) cells, which secrete other hormones important for proper glucose homeostasis (Bosco et al., 2010, Schaeffer et al., 2011). This architectural arrangement favors homologous cell-cell contacts and is important for normal islet function; rodent models of diabetes exhibit abnormal islet architecture, with alpha, delta, and PP cells intermingling with beta cells within the islet core (Wojtusciszyn et al., 2008). In contrast to rodent islets, human islets favor heterologous cell-cell contacts, with more evenly distributed endocrine types found across an islet and less compartmentalization into peripheral and central compartments (Bosco et al., 2010). The relative proportions of the endocrine subtypes can also differ between murine and human islets, with the former dominated by beta cells (∼80%) and the latter more evenly composed of alpha (40%) and beta (50%) cells (Pan and Wright, 2011), although different proportions have also been noted (Bonner-Weir et al., 2015, Kilimnik et al., 2012). Disparities in composition and structure between mouse and human islets have been linked to functional differences. In murine islets, for instance, beta-beta intercellular contacts are believed to coordinate homogeneous and synchronous release of insulin, whereas human islets, which lack as many homologous beta cell contacts, display asynchronous insulin release following glucose stimulation (Wojtusciszyn et al., 2008). Exciting work is emerging from single-cell sorting and sequencing that supports the notion of distinct beta cell subtypes in human islets (Dorrell et al., 2016).
Another determinant of beta cell function is communication with cells in the islet niche, which is composed of multiple non-endocrine cell types, including endothelial, perivascular, neuronal, and mesenchymal cells (Hayden et al., 2008, Lammert et al., 2003a, Reinert et al., 2014). In particular, pancreatic islets are highly vascularized; each beta cell is believed to make one or more contacts with an endothelial cell, and together they form specialized ultrastructural features at their interface (Bonner-Weir and Orci, 1982, Henderson and Moss, 1985). During pancreatic development, endothelial cells are initially recruited into the burgeoning islet by vascular endothelial growth factor A (VEGF-A), which is secreted by endocrine cells (Lammert et al., 2003b). It is the endothelial cells that then synthesize and secrete the components of the islet basement membrane, which is believed to serve as a critical modulator of beta cell growth, survival, and function (Lammert et al., 2003a, Nikolova et al., 2006, Oberg-Welsh, 2001, Stendahl et al., 2009, Wang and Rosenberg, 1999). Work by the Otonkoski group demonstrated the presence of two basement membranes around the endothelium and beta cells in human islets (Otonkoski et al., 2008, Virtanen et al., 2008). Given the specialized relationship between beta cells and supporting cells in their natural environment, it stands to reason that recapitulation of some elements of the endogenous beta cell niche may lead to improved survival and function of hESC-derived beta cells in vitro.
The vascular islet niche consists not only of endothelial cells, but also of mural cells called pericytes. Pericytes wrap around endothelial cell tubes to provide structural support and generate functional, mature blood vessels (Díaz-Flores et al., 2009) and modulate endothelial cell proliferation, survival, and function (Jain, 2003). The role of pericytes in normal islet function is not completely understood, but one study using genetic ablation suggested that their loss in the adult murine islet results in impairments in insulin expression and glucose-clearing (Sasson et al., 2016).
Given the importance of islet cytoarchitecture for endocrine function, culture systems can be designed to recapitulate elements of the intricate 3D structure of the developing islet. Various methods have been used to generate engineered pancreatic islets, or so-called “pseudo-islets,” in vitro (Kojima, 2014). Multiple studies have reported improved function of reaggregated pseudo-islets, either in vitro or in vivo after transplantation. There is also evidence that beta cells reaggregated with endothelial progenitor cells demonstrate improved GSIS compared to pseudo-islets composed only of beta cells (Penko et al., 2011).
In summary, more work is needed to determine the cellular composition and architectural arrangement of human islets that is optimal for function. Challenges will likely include decisions regarding how to source the desired human niche cells, particularly given that there is a lack of truly specific markers for some of these cell types, and that the supply may be limiting. One option is the generation of the niche cells themselves from hESCs or induced pluripotent stem cells (iPSCs). Another challenge is that current techniques for creating pseudo-islets rely on the property of self-organization among endocrine and non-endocrine cells; 3D printing of islet tissues may someday be possible and could instead allow the enforcement of a desired architecture. Future work aimed at incorporating flow in microfluidic devices, as has been done in various other systems with “organ-on-a-chip” approaches, may allow even more sophisticated optimization of function while reducing negative effects of waste product accumulation. Re-engineering additional components of a pancreatic islet with the optimal composition of endocrine and non-endocrine niche cells, including vascular niche cells and extracellular matrix (ECM) components, may lead to an optimally functional beta cell population for transplantation into patients.
Applying Lessons Learned from Whole-Organ Pancreas and Islet Transplantation to Stem Cell-Derived Beta Cells
There is growing appreciation for the fact that the transplant site itself influences the functionality and survival of islets. Unlike the insulin independence that always occurs following a technically successful pancreas transplant, the marginal mass of engrafted islets associated with infusion through the portal vein often requires multiple infusions from different deceased donors (Hering et al., 2016). Furthermore, it is more challenging to create states of insulin independence in patients with higher BMIs and higher insulin requirements (Balamurugan et al., 2014). Portal infusion limits the amount of tissue that can be infused secondary to the development of portal hypertension. In addition, isolated islets engraft poorly secondary to IBMIR and ischemia injuries (Eich et al., 2007). Of equal significance, islets infused into the portal vein are not retrievable, and this remains a concern for the clinical translation of any trials using hESC-derived beta cells until it has been unequivocally proven that such cells are equivalent to fully mature human islet cells and lack any possibility of neoplastic transformation. However, while the risk of teratoma formation from undifferentiated stem cells cannot be completely ignored at present, further optimizing the generation of fully differentiated islet and beta cells from stem cells should alleviate this concern.
Considering the information gained from solid organ and islet transplantation, it is clear that a more optimal site for islet tissue is necessary. Multiple investigators are optimizing subcutaneous or intramuscular sites for islet transplantation to enhance engraftment and permit removal of the graft in the event of tissue dysfunction or transformation (Bertuzzi et al., 2018, Gamble et al., 2018). Similarly, exploring optimal sites that permit retrieval of hESC-derived beta cells will be important for clinical translation. Successful pancreas transplantation in T2D patients suggests that given an unlimited source of beta cells, cell replacement may provide a benefit for the vast majority of people suffering from T2D (Kandaswamy et al., 2018). Of note, transplantation of any allogeneic cell source requires mitigation of the recipient immune system.
Immune Modulation in Beta Cell Replacement Therapy
The recent success in the use of beta cell replacement to treat T1D, combined with advances in the generation of hESC- or iPSC-derived beta cells, provides a roadmap for effective treatment of the disease. However, both the alloimmune and autoimmune systems remain major barriers to the widespread adoption of cell replacement therapies in T1D and, potentially, T2D. Current immunosuppressive drugs are effective but require lifelong treatment, and in many cases lead to side effects and toxicities that make the adoption of this treatment strategy limited to only the most severe cases of disease. Here, we focus on new efforts to understand and control immunity, with the goal of creating a combinatorial therapy that takes advantage of drugs, bioengineering, and gene editing to bring beta cell replacement to the larger diabetes community.
Control of Alloimmune Responses against Beta Cells
In T1D patients, transplanted allogeneic beta cells face alloimmune-mediated rejection that can eliminate the graft within days if not suppressed. T cells are the principal drivers of alloimmunity; their activation results from T cell receptor (TCR) recognition of antigenic peptides presented by the major histocompatibility complexes (MHCs). The highly polymorphic proteins of the MHC, referred to as human leukocyte antigens (HLAs) in humans, are the major targets of alloimmune attacks. Minor histocompatibility antigens, such as male antigens in female recipients or mitochondrial antigens, can also trigger rejection responses, although at a slower pace. MHC class I is expressed on all cell types, including beta cells, and beta cells overexpress MHC I in inflamed conditions (Hamilton-Williams et al., 2003). MHC class II is expressed on antigen presenting cells (APCs) such as B cells, dendritic cells, and macrophages and can be induced on non-APCs, including beta cells (Foulis and Farquharson, 1986, Pujol-Borrell et al., 1987). After transplantation, MHCs released by donor cells are taken up by recipient APCs, broken down into antigenic fragments, and presented by recipient MHC on the surface of recipient APC. This normal process of T cell activation is referred to as the “indirect” pathway in the context of transplantation. This is in contrast to the “direct” pathway that is unique to transplantation immunology. In the direct pathway, TCRs on the recipient’s T cells bind to allogeneic MHC expressed on donor cells, leading to T cell activation and direct attack of the graft cells. Both direct and indirect T cell pathways contribute to graft rejection and often act cooperatively to carry out the attacks (Brennan et al., 2009, Jiang et al., 2004, Makhlouf et al., 2003).
The current approach to control transplant rejection is to globally suppress T cell activation by inhibiting TCR signaling with CNIs, prevent T cell proliferation with mycophenolate, and dampen inflammation with steroids. In addition to this triple-drug regimen, transplant patients often receive “induction” therapies, defined as short-term treatments in the peri-transplant period. Clinical islet transplant experiences have shown that stronger induction therapies that deplete T cells, combined with blockade of inflammatory cytokines TNFα and IL-1, lead to better long-term islet graft function (Barton et al., 2012).
Although immunosuppression protects grafts from rejection, it globally compromises the patients’ immune systems, leaving them vulnerable to infections and malignancies. An ideal strategy would be to train the immune system to accept the transplanted graft as self and avoid chronic global immunosuppression. Immune tolerance to transplanted tissue has been achieved in many preclinical animal models, and potential tolerance-promoting therapies are currently under development for humans.
Most commonly used immunosuppressive drugs inhibit both effector cells and tolerance-promoting immune regulatory mechanisms (Table 1). However, different drugs have varying selectivity in inhibiting anti-graft effector responses versus tolerance-promoting regulatory T cells (Tregs) (Furukawa et al., 2016). For example, the use of CNIs decreases the percentages of Tregs, whereas rapamycin use is associated with an increase in percentages of Tregs. CTLA-4Ig (Belatacept) inhibits effector T cell activation and clonal expansion but can also impair Treg homeostasis and function at high doses (Bour-Jordan et al., 2004, Salomon and Bluestone, 2001). Thus, by selecting immunosuppressive drugs and their dosing, it may be possible to preferentially inhibit effector cells and to leverage the immune system’s self-control mechanisms to reduce the burden of immunosuppression. Achieving immune tolerance requires the immune system to be aware of the antigen and to actively avoid activation toward that antigen. Tolerance to self-antigens is achieved through a combination of deletion, cell-intrinsic checkpoints, and suppression by regulatory mechanisms such as Tregs, IL-10, and transforming growth factor ß (TGFß) signaling. By blocking immune activation, currently available immunosuppressive drugs also inhibit these tolerance-inducing mechanisms. Thus, attaining tolerance is very inefficient using currently available immunosuppressive drugs.
