Murine ESC-IPC differentiation protocols
Diabetes mellitus is a chronic metabolic disease that results in high levels of glucose in the blood. Normally, glucose is transported into cells from the blood for energy, and this transport is initiated in response to the hormone, insulin. In diabetic patients, cellular glucose uptake is defective, in part due to the inability of cells to respond to insulin, or the inability of the body to produce the insulin itself. According to the International Diabetes Foundation, 366 million people worldwide had been diagnosed with diabetes in 2011, and this number is continuing to increase in every country (http://www.idf.org/). It is an especially challenging health problem, as treatments to both control hyperglycemia as well as the debilitating side effects of diabetes, such as injury to the blood vessels and nerves, must be addressed. Amongst the three types of diabetes (Type I - autoimmune, gestational and Type II – adult onset), Type II diabetes is the most prevalent, in which hyperglycemia is uncontrolled due to the body’s inability to produce enough insulin or due to the body’s inability to respond appropriately to lower the blood glucose level. In contrast, in patients with Type I diabetes (TID), insulin-producing beta (β) cells are destroyed by the immune system. Studies of TID by many groups have provided extensive insight to the fields of immunological tolerance and pancreatic developmental biology. In this chapter, we will briefly describe TID, provide a synopsis of pancreatic β cell development in mice as compared to humans, review of some of the medical treatments currently available for TID, and discuss current studies that have explored the use of stem cells as alternative possible therapies for TID.
2. Type I diabetes in humans and mice
2.1. TID in humans
A fully comprehensive comparison of TID in humans and mice has recently been published, so we will only highlight certain areas (van Belle, Coppieters et al. 2011). TID is a multigenic disease, and several candidates have been discovered that implicate disruptions in the normal process of negative selection of autoreactive T cells during thymic development, genetic association with specific major histocompatibility complex (MHC) genes in the human leukocyte antigen (HLA) locus, as well as dysregulation of mature T lymphocyte responses. For example, patients with mutations in the
2.2. TID in mice
The identification of the mechanisms underlying the autoimmunity of TID in humans has been facilitated by the use of rodent models of the disease (Van Belle, Taylor et al. 2009; von Herrath and Nepom 2009). Despite the fact that TID is a multigenic disease with heterogeneous etiologies, there is overall consensus that TID is caused by the destruction of pancreatic β cells by sets of immune cells that recognize many potential autoantigens (such as insulin, glutamate decarboxylase (GAD), islet antigens, and heat shock proteins-60 and -70) and that the disease can be transferred by the transplantation of autoreactive T lymphocytes that are specific for these antigens (Mallone, Brezar et al. 2011). Several spontaneous, induced and transgenic mouse models currently exist, which have provided important insights into the genetics, cellular mechanisms and immunological aspects of TID. However, the non-obese diabetic (NOD) mouse strain remains the primary animal strain for studies of TID. The utility of the NOD mouse and comparison of the TID in the NOD mouse with human TID have been extensively reviewed and debated, and we direct the reader to these excellent reviews for further reference (Anderson and Bluestone 2005; Roep and Peakman 2011). The NOD mouse strain, when housed in specific-pathogen free conditions, displays the onset of diabetes after 12 weeks of age in both sexes. Insulitis is observed as mononuclear infiltration in the islets before full blown diabetes is evident, and this cellular infiltrate is comprised of primary T lymphocytes, but other lymphoid and myeloid cells are also present. The insulitis results in the destruction of the pancreatic islets and the insulin-producing β cells. The NOD mouse also displays defects in functional macrophages and natural killer cells, NKT cells and regulatory T cells and complement. The NOD mouse, therefore, has allowed for the investigation of numerous immune mechanisms that contribute to the development of TID. The peripheral neuropathy that accompanies TID in humans is also evident in the NOD mouse, which has allowed for analysis and management of the side effects of TID (Anderson and Bluestone 2005; Roep and Peakman 2011). Similar to human diabetes, the MHC of the NOD mouse contains several loci that are linked to increased susceptibility to TID. The NOD mouse background has also been used to derive the NOD/
Many studies in mice have focused on mechanisms to control the aberrant autoimmune response in TID, as well as strategies to replace dysfunctional β cells, using simple insulin replacement or rescue/regeneration of the β cells. Despite the similarities between TID in humans and in the NOD mouse, clear differences have been noted. TID in the NOD mouse appears to be a more aggressive disease compared than that in humans, so the NOD mouse may be more beneficial for studies of therapies for long-term TID than for discovery of pre-susceptibility markers of TID. β cells in the NOD mouse may also be more proliferative and could possibly regenerate better than other strains (Sherry, Kushner et al. 2006), and evidence that pancreatic development is altered in the NOD mouse also exists (Homo-Delarche 2001). Therefore, in addition to its clear autoimmune etiology, TID may also result from defects in pancreatic development.
2.3. Pancreatic development in the mouse and human
The adult pancreas organ is comprised of many cell types, including exocrine and endocrine cells, and is highly vascularized. Although TID is a disease that occurs in the fully formed pancreas, strategies to treat or cure TID via β cell regeneration or β cell differentiation from embryonic stem cells (ESCs) or induced pluripotent cells (iPSCs) have relied on the current knowledge of pancreatic development during the fetal period. Here, we briefly review the development of pancreas in the mouse and human, and then move to a description of development of β cells only.
In the mouse, pancreas formation occurs in three phases (Jorgenson, Ahnfelt-Ronne et al. 2007). In the first phase, pancreatic formation from the endodermal gut tube begins at embryonic day (e) 8.5. Patterning of the gut tube in the pancreatic region is reliant on retinoic acid (RA) signaling from the mesoderm. The dorsal and ventral pancreas buds form from the pancreatic epithelium in the primitive gut tube. Recently, the critical role of the Wnt signaling in the pancreatic mesenchyme in the differentiation of pancreatic epithelial cells was shown
Islet development in humans occurs at a slower rate compared to what is observed in mice, but the reasons for this are not understood. The size of the human islet is comparable to that of the mouse, but in the mouse, the mean area ratio of β cells is higher than observed in the human (Kim, Miller et al. 2009). Fetal antigen-1 is a protein expressed exclusively in human endodermal tissue and pancreatic epithelial cells before islet formation (Tornehave, Jansen et al. 1993). Islets can be located in the human fetus as early as 12-13 weeks post coitus (Piper, Brickwood et al. 2004), although functional β cells that are insulin-positive can be detected as early as 8 weeks.
An overview of pancreatic endocrine cell development is shown in Figure 1. Definitive endodermal progenitor cells can differentiate into pancreatic progenitor cells, which have been identified by the expression of two critical transcriptional factors:
For β cell development, the essential role of three key transcription factors, Pax4, Nkx6.1, Nkx2.2, in the mouse and their relationships to the human β cell development is clear. Pax4 (paired box 4) is required for the commitment to the pancreatic lineages (Wang, Elghazi et al. 2004), as well as for the regulation of β cell mass size, proliferation and survival in mice (Brun and Gauthier 2008), and mutations in the
2.4. Current therapies for TID
A fully mature, functional β cell produces and appropriately secretes the mature form of insulin (i.e. proteolytic processing of pro-insulin to form C-peptide is evident), in response to glucose to maintain normal glycemic levels in the blood. The current treatment for TID is long-term insulin replacement therapy that is delivered via injection, insulin pens or pumps, and has been quite successful for control of hyperglycemia. However, insulin replacement therapy is not considered a cure for TID, as patients receiving insulin long-term still manifest abnormalities in metabolism, as measured by above-normal levels of glycated hemoglobin (HbA1c). Furthermore, for children, the daily requirement for insulin replacement can limit their day-to-day activities, and TID-related complications, such as vascular disease, retinopathy and neuropathy, still persist even with insulin therapy. Pancreatic transplantation has been performed for TID patients with success, but the main limitations of this strategy are the low number of suitable donors required for transplant and the decision if the risks of surgery and transplantation outweigh the benefits (Jahansouz, Kumer et al. 2011). Furthermore, even with insulin replacement therapy or pancreatic transplantation, autoreactive immune cells that can attack and destroy any residual β cells in the patient can still remain. For this reason, therapies that modulate the immune response to β cells and β cell antigens also exist, and their safety and efficacy have been or are currently being tested in clinical trials (Bluestone, Herold et al. 2010; Waldron-Lynch and Herold 2011). GAD65 specific-antigen-based immunotherapy with alum adjuvant demonstrated preservation of C-peptide fasting levels in younger new-onset patients four years after treatment (Ludvigsson, Hjorth et al. 2011), but this was not supported in the larger Phase III trial (Ludvigsson, Krisky et al. 2012). Humanized anti-CD3 antibody (in which human Fc immunoglobulin domains are engineered to mouse CD3-binding portions of the antibody) therapy given in a single dose reduced the decline of insulin production and the amount of exogenous insulin required in the patients (Herold, Gitelman et al. 2009). Interestingly, the anti-CD3 antibodies reduced the numbers of circulating T lymphocytes, but whether or not autoreactive T cells are specifically depleted is unclear. There is evidence that the anti-CD3 antibody acts as a weak TCR agonist and stimulates the production of regulatory T cells (Tregs) in humans. B lymphocytes in TID have been postulated to act as antigen-presenting cells and also to produce autoantibodies that can result in β cell destruction (Bluestone, Herold et al. 2010). Anti-CD20 antibodies target and deplete B lymphocytes, and lead to a preservation of β cell function in patients, as measured by C-peptide production and reduction of HbA1c levels compared to controls. However, the level of autoantibodies in these TID patients was not determined, so the mechanism by which anti-CD20 therapy works in TID patients is still uncertain. As we will explain further below, these immune modulation therapies may also be important for the future success of β cell regeneration and transplantation.
3. Experimental alternative treatments for TID: Stem cells
Pancreas transplantation has succeeded as a replacement for insulin-secreting β cells for TID patients, but this relies on a limited source of cadaveric pancreatic tissue (Danovitch, Cohen et al. 2005). Early studies demonstrated that isolated human islets did not proliferate in suspension culture, and that adherent islet cells showed limited replication of β cells (Nielsen, Brunstedt et al. 1979; Nielsen, Galsgaard et al. 2001). Therefore, alternative sources of IPCs are needed, and stem cells have been suggested as this source. Stem cells are undifferentiated cells that are capable of self-renewal and differentiation into any cell type. They can be classified depending on their origin. Embryonic stem cells (ESCs) are derived from the inner cell mass of implanted embryos (Evans and Kaufman 1981), induced pluripotent stem cells (iPSCs) are adult or fetal cells that have been “reprogrammed” to an ESC-like state (Takahashi and Yamanaka 2006), adult stem cells are isolated from adult tissues (Becker, Mc et al. 1963) and germline-derived stem cells are generated from embryonic or adult gonads (Shamblott, Axelman et al. 1998). In this section, we will discuss the state-of-the-art technologies in which the different types of stem cells are generated and differentiated and their possible clinical applications as cellular replacement therapies for TID.
3.1. Tissue-specific pancreatic stem cells
The existence of pancreatic stem cells was proposed in the last decade (Ramiya, Maraist et al. 2000), and is still a topic of debate (Dor, Brown et al. 2004; Smukler, Arntfield et al. 2011) (Figure 1). When defining a “stem cell” by function, the standard tests are to examine the ability of a putative stem cell to self-renew, often using proliferation assays; and differentiate, measured by changes in function, cell surface marker and/or gene expression.
Evidence from fetal and adult pancreas biology in physiological as well as pathological conditions support the hypothesis of the existence of pancreatic adult stem cells based on proliferation assays. For example, pancreas from pregnant rats and mice displayed an uptake of bromodeoxyuridine (BrdU) during pregnancy, suggesting the proliferation of islets (Parsons, Brelje et al. 1992; Karnik, Chen et al. 2007). This proliferation was stimulated by prolactin (Nielsen, Svensson et al. 1999; Rieck and Kaestner) that reduced expression of
As mentioned previously,
To test whether β cells can self-replicate in the adult, transgenic mice in which pre-existing β cells were specifically labeled were utilized (Dor, Brown et al. 2004). In the absence of pancreatic injury, these labeled cells were not replaced by new IPCs, arguing against the existence of an adult pancreatic stem cell. However, the labeled IPCs were able to uptake BrdU after pancreatectomy, indicating that adult β cells contain regenerative ability (Dor, Brown et al. 2004). A different model in which apoptosis was induced specifically in β cells using a diphtheria toxin receptor (DTR) transgenic mouse model showed β cell recovery, restoration to euglycemia, and that the surviving β cells were involved in their own regeneration (Nir, Melton et al. 2007). These studies assumed that insulin expression was limited exclusively to cells, but it has been shown that in certain circumstances, other cells and tissues can express insulin (Kojima, Fujimiya et al. 2004). In support of this, rare multipotent stem cells which express insulin have been described in both embryonic and adult mouse and human pancreas that are distinct from mature β cells (Smukler, Arntfield et al. 2011). Importantly, these cells were shown to self-renew, expressed
Questions regarding the existence and location of pancreatic stem cells and progenitors outside of the pancreas are still an area of investigation. Hepatic and pancreatic cells are derived from a common endodermal progenitor during embryogenesis, and this has led to the hypothesis that cells from fetal liver tissue could be used as an alternative β cell source. Pancreatic duct cells and liver cells can be induced to express certain β cell gene products in culture (Heremans, Van De Casteele et al. 2002; Sapir, Shternhall et al. 2005). In support of this, CD45- Ter119- c-kit- hepatic stem cells were isolated from mouse fetal liver and differentiated into IPCs in vitro. These differentiated cells were then transplanted into drug-induced diabetic mice, which showed a significant reduction of their blood glucose levels (Feng, Du et al. 2005). Bone marrow has also been proposed as another possible source of IPCs. For example, human fetal bone marrow-derived CD45- Glycophorin A- mesodermal progenitor cells generated insulin secreting islet-like clusters when injected into human fetal pancreatic tissues that were implanted in STZ-treated NOD/severe combined immunodeficiency (SCID) mice (Ai, Todorov et al. 2007). In the mouse, progeny of Mac-1- Sca+ bone marrow cells cultured with cytokines such as IL-3, IL-6, IL-11 and stem cell factor (SCF) migrate into the pancreas islets and have been reported to convert into CD45- CD34+ insulin-expressing cells (Luo, Luo et al. 2009). The mechanisms involved in the conversion of these bone marrow cells into IPCs were not investigated.
3.2. Transdifferentiated cells as heterologous sources of β cellsTransdifferentiation is defined as a process by which a “terminally” differentiated cell changes into another type of differentiated cell. Transdifferentiation of pancreatic α cells into β cells has been suggested as a source of β cell replacement for TID. Transdifferentiation of α cells into β cells has been observed
One important question that has not yet been answered pertains to the location of the cells that can undergo transdifferentiation into β cells
Taken together, the studies cited in this section demonstrate that there are many possible pluripotent and even terminally differentiated cells
3.3. Differentiation of embryonic stem cells into insulin producing cells
The use of ESCs for cell replacement therapies has great promise, due to the potentially unlimited self-renewal of ESC in their undifferentiated state, and the potential of ESC to differentiate into any type of cellular derivatives. Many groups have used diverse methodologies to differentiate human or murine ESCs into IPCs for β cell replacement in diabetes (Figure 3). The most common obstacles amongst the different protocols are the inability of ESC-derived IPCs (ESC-IPCs) to secrete insulin after glucose-stimulation, the risk of teratoma formation due to undifferentiated ESC that remain in the ESC-IPC culture, and immune rejection of the ESC-IPC after transplantation
The efficiency of ESC-IPC differentiation
|ES cell lines used||Insulin mRNA||c-peptide protein expression||Rescue of diabetic mice||Differentiation protocol reference|
|R1||Not done||Not done||Yes||Yes||(Soria, Roche et al. 2000)|
|B5, E14.1 and ESF122||Yes||Yes|
|Yes||Failed (Fujikawa, Oh et al. 2005)||(Lumelsky, Blondel et al. 2001)|
|JM1, ROSA, and ESF122||Yes||Yes||Yes||Yes||(Hori, Rulifson et al. 2002)|
|R1 and ESF122||Yes||Yes||Yes||Yes with ||(Blyszczuk, Asbrand et al. 2004)|
|D3||Yes||Not done||Not done||Not done||(McKiernan, O'Driscoll et al. 2007)|
|D3||Yes||Not done||Yes||Not done||(Wang and Ye 2009)|
|B6||Yes||Yes||Yes||Not done||(Kim, Hoffman et al. 2010)|
|EB3||Yes||Yes||Yes||Not done||(Miyazaki, Yamato et al. 2004)|
|R1||Yes||Yes||Yes||Not done||(Banerjee, Sharma et al.)|
|ES cell lines|
|Insulin mRNA||c-peptide protein expression||Rescue of diabetic mice||Differentiation protocol reference|
|CyT25, CyT49 CyT203||Yes||Yes||Yes||Yes||(D'Amour, Bang et al. 2006; Kroon, Martinson et al. 2008)|
|H1and H9||Yes||Yes||Yes||Yes||(Jiang, Shi et al. 2007)|
|H1, H7 and H9||Yes||Yes||Yes||Not done||(Jiang, Au et al. 2007)|
|Hes3||Yes||Yes||Yes||No||(Phillips, Hentze et al. 2007)|
The first successful approach differentiating ESCs into functional IPCs utilized murine ESCs that were transfected with a neomycin selection gene under the control of the human
To settle this controversy, the detection of C-peptide, a by-product of endogenous insulin synthesis, will be needed in order to prove that ESC-IPCs produce insulin
Blyszczuk et al. described a third ESC-IPC differentiation protocol (Blyszczuk, Asbrand et al. 2004). This protocol consisted of three steps: 1) formation of EBs, 2) spontaneous differentiation of EBs into cells of ectodermal, mesodermal and endodermal lineages by transferring EBs into gelatin-coated plates, and 3) induction of differentiation into IPCs by culturing the gelatin-plated EB cell suspension with culture media containing progesterone, laminin, putrescine, selenium, nicotinamide, insulin, transferrin and B27 (Blyszczuk, Asbrand et al. 2004; Schroeder, Kania et al. 2006; Schroeder, Rolletschek et al. 2006). ESC-IPCs generated by the Blyszczuk protocol released insulin
Solving the ESC-IPC differentiation problem is complicated, as individual ESC lines do not behave similarly before and after differentiation, which also makes direct cross-comparison of the published ESC-IPC differentiation protocols difficult (Osafune, Caron et al. 2008). The three ESC-IPC differentiation protocols described above were compared using three different ESC cell lines, with the ES122 ESC line providing the best results (Boyd, Wu et al. 2008). When the ES122 ESC line was differentiated using the Lumelsky, Hori or Blyszczuk protocols, the Blyszczuk protocol proved to be superior: higher insulin-1 and insulin-2 gene and protein expression, greater increase in insulin secretion after glucose challenge
More differentiation protocols to generate ESC-IPCs from murine ESCs have been described, but all of the known assays to test function of ESC-IPC have not been performed (Table 1). A comparative analysis in which the murine ESC D3 line was differentiated through the formation of EBs in the presence of retinoic acid showed how the subsequent exposure to sodium butyrate produced IPCs that expressed insulin-1 and insulin-2. However, C-peptide was not expressed and
For human ESCs, differentiation into IPCs through monolayers, without EB formation, is the methodology most commonly followed. The protocol by D’Amour
Further modifications to the D’Amour protocol have shown some improvements in IPC properties. For example, human ESC-IPC that secrete insulin and respond to glucose were generated by combining activin A and ATRA in a chemically defined medium, and other maturation factors, such as bFGF and nicotinamide in DMEM/F12. ESC-IPCs differentiated by this system were able to rescue 30% of diabetic
Human ESC-IPCs have been also generated through the formation of EBs. Human EBs in a 3D matrix were induced to differentiate into definitive endoderm by the presence of activin A and Bmp4, and then into IPCs via the addition of growth factors such as FGF18, EGF, TGF-α, IGF-1, IGF-1 and VEGF. In contrast with other reports, these ESC-IPCs expressed only insulin and no other pancreatic hormones; however, they did not rescue diabetic mice and formed teratomas instead. When
Therefore, for human and murine ESC-IPC differentiation, no “standard” protocol currently exists, as the results appear to be dependent on the ESC line utilized. In addition, there is no consensus on the composition of the differentiation culture mediums to guide cells from definitive endoderm to the IPC fate. However, some compounds appear to be key factors for the success of ESC-IPC differentiation. Activin A, a member of the transforming growth factor-beta (TGF-β) family, is involved in cellular proliferation and differentiation, and seems to be essential at the first steps of differentiation of human ESC into definitive endodermal cells (D'Amour, Bang et al. 2006; Kroon, Martinson et al. 2008). Nicotinamide, the amide of vitamin B3, is a well-known inducer of endocrine differentiation (Otonkoski, Beattie et al. 1993), and has been used in the differentiation of human and murine ESCs into IPCs (Jiang, Shi et al. 2007; Boyd, Wu et al. 2008). Epidermal Growth Factor (EGF) and Basic Fibroblast Growth Factor (bFGF) have been also applied in human and murine ESC-IPC differentiation, as both factors induce cell proliferation and differentiation (Lumelsky, Blondel et al. 2001; Jiang, Au et al. 2007). However, EGF, but not bFGF, needs to be removed from the cultures during the last stages of the differentiation protocols in order to allow the formation of islet-like cell aggregates (Cras-Meneur, Elghazi et al. 2001; Hardikar, Marcus-Samuels et al. 2003). Hepatocyte Growth Factor (HGF) is an inducer of cell proliferation that is secreted by mesenchymal cells, and has been used for human ESC-IPC differentiation (Otonkoski, Cirulli et al. 1996; D'Amour, Bang et al. 2006; Phillips, Hentze et al. 2007) as well as murine fetal liver differentiation into IPCs (Feng, Du et al. 2005). Exendin-4 stimulates β cell proliferation (Xu, Stoffers et al. 1999) and it has been used for murine and human ESC-IPC differentiation (Tang, Cao et al. 2004; Wu, Liu et al. 2007; Gabr, Sobh et al. 2008; Gao, Wu et al. 2008; Li, Lam et al.). A combination of insulin, transferrin, selenium and fibronectin (ITSFn) has been a common cocktail in murine ESC-IPC differentiation, although the presence of insulin in the medium brought some controversy about the endogenous insulin production of IPCs (Lumelsky, Blondel et al. 2001; Hori, Rulifson et al. 2002; Blyszczuk, Asbrand et al. 2004; Hansson, Tonning et al. 2004). The concentration of glucose in the culture media can also influence ESC-IPC differentiation and their properties. Low glucose concentrations (5mM) increase the content of insulin in islet-like clusters, whereas higher concentrations (20-30 mM) induce their replication (Bonner-Weir, Deery et al. 1989; Guillemain, Filhoulaud et al. 2007). Importantly, the presence or absence of serum has a clear effect on ESC-IPC differentiation. It is widely accepted by all cell culture professionals that lot-to-lot variation of serum sources can affect the success of
In summary, despite the progress made to date, investigation must continue to optimize ESC-IPC differentiation before their clinical use is possible. ESC-IPC cultures contain heterogeneous cell populations, and have not been well-identified. Moreover, the ESC-IPCs do not always respond to glucose challenge and do not release insulin, limiting their use as a pancreatic β cell replacement in TID. Identification of novel factors to induce maturation of ESC-IPC phenotypes may help to achieve full functional status. However, perhaps a better strategy would be to identify and transplant the immature pancreatic progenitor cells within the ESC-IPC cultures, as they may mature and respond more appropriately
3.4. Identifying markers of endoderm and pancreatic progenitor cells in ESC-IPC cultures
ESC-IPCs to date have not recapitulated pancreatic mature β cell phenotypes. Pancreatic progenitors that are generated within the heterogeneous ESC-IPCs may be suitable for cellular replacement therapy in TID. The pancreatic progenitors may not self-renew as stem cells do, but could differentiate into endocrine cells after transplantation, including β cells. The identification of specific cell surface markers on pancreatic progenitors would allow for their isolation and for the transplantation of a homogenous population of ESC-derived pancreatic progenitors. We will discuss some current advances in the identification of pancreatic progenitors
3.4.1. Definitive endoderm progenitors
In mice, putative definitive endodermal cells that co-express E-cadherin and Decay Accelerating Factor (DAF1/CD55) cell surface markers also express
3.4.2. Pancreatic progenitors
First isolated using
The establishment of intermediate lineage-restricted progenitor cell lines (similar to the pancreatic progenitor cells) that generate homogeneous IPCs could be useful for scalable IPC production. Several clones were isolated from differentiation protocols into IPCs with the E-RoSH cell line (an endothelial lineage-restricted clonal murine ES cell derived line). The eight clones that were isolated could be frozen and thawed for months, and when cultured, they displayed properties of pancreatic β cells, such as expression of
In summary, isolation and characterization of pancreatic progenitors from embryonic development and ESC-IPCs could provide another possible β cell replacement source for the treatment of diabetes. These multipotent cells are not teratoma-forming, and can differentiate into IPCs and other hormone expressing cells, and have been shown to reverse hyperglycemic blood levels after transplantation. However, the isolation of pure or enriched pancreatic progenitors using cell-specific surface markers from ESC-IPCs cell differentiation cultures that are able to cure diabetic mice still has not been demonstrated. More studies to assess the efficiency of human ESC-IPC differentiation and optimization of sorting methods to remove any undifferentiated teratoma-forming cells are necessary before clinical translation. Nevertheless, even if fully functional, teratoma-free ESC-IPCs are derived, they must circumvent another possible barrier after transplantation: the host immune system (see Section 3.6). For this reason, induced pluripotent stem cells have been proposed as an autologous alternative to ESCs.
3.5. Differentiation of induced pluripotent stem cells into insulin-producing cellsInduced pluripotent stem cells (iPSCs) are reprogrammed somatic cells that behave like ESCs and can be re-differentiated into all three germ layers and potentially, any terminally differentiated cell type (Takahashi and Yamanaka 2006). The generation of iPSCs was firstly achieved by the viral delivery of reprogramming factors (Takahashi and Yamanaka 2006) in murine cells, and later by non-viral methods (Yu, Hu et al. 2009). Human iPSCs have also been generated (Yu, Hu et al. 2009). iPSC are envisioned as the future of “personalized” medicine since they could be used to generate autologous therapies that will not require pharmacologic immune suppression after transplantation (Okita, Ichisaka et al. 2007). However, the immunogenicity of syngeneic iPSC, which was unexpected by the stem cell biology field, demonstrated that this is not the case (Zhao, Zhang et al. 2011). Moreover, autologous iPSC generated from patients with genetic diseases likely will retain the propensity to develop disease following re-differentiation, unless they are modified (Raya, Rodriguez-Piza et al. 2009). The use of allogeneic iPSC could alleviate this possibility, but would have the disadvantage of possible immune rejection. iPSCs differ from ESCs in gene expression profiles (Chin, Mason et al. 2009), persistence of donor-cell gene expression (Marchetto, Yeo et al. 2009; Ghosh, Wilson et al. 2010), differentiation abilities (Feng, Lu et al. 2010; Hu, Weick et al. 2010) and genetic stability (Mayshar, Ben-David et al. 2010; Laurent, Ulitsky et al. 2011).
Currently, there is no standard iPSC to IPC differentiation protocol, but scientists have followed strategies similar to ESC to IPC differentiation (Table 3). Murine skin fibroblasts reprogrammed into iPSCs were differentiated into IPCs that were glucose responsive
|Source||Insulin mRNA||c-peptide protein expression||Rescue of diabetic mice||Differentiation protocol reference|
|Murine skin fibroblast||Yes||Not done||Yes||Yes||(Alipio, Liao et al. 2010)|
|Human fibroblast||Yes||Yes||Not done||Not done||(Zhang, Jiang et al. 2009)|
|Skin human TID adult and healthy neonatal fibroblast||Yes||Yes||Yes||Not done||(Maehr, Chen et al. 2009; Thatava, Nelson et al. 2010)|
3.6. Addressing the immunogenicity of ESC-IPCs and iPSC-IPCs
Inflammation, due to local tissue damage, is a regular occurrence during transplantation of cells or organs, even in a syngeneic setting (Turvey, Gonzalez-Nicolini et al. 2000; Carvalho-Gaspar, Billing et al. 2005). For instance, cellular infiltrates are evident in rat islet β cell allografts within one week post-transplant, and are completely destroyed within 2 weeks. Depletion of major histocompatibility complex class II (MHC II)-positive islet cells (which presumably are antigen-presenting cells) delays β cell rejection up to 5 weeks, while re-aggregation of islet β cells after islet dissociation and β cell purification promotes allograft survival up to 20 weeks (Pipeleers, Pipeleers-Marichal et al. 1991). Embryonic tissue has not been considered to be highly immunogenic, mainly based on the “immune privileged” status of the fetus during gestation, which protects it from the maternal immune system. However, this immune privileged status is not generally observed outside the womb. For example, transplanted human β cells from fetal pancreas, which have a greater proliferative capacity than adult tissue (Hayek and Beattie 1997; Castaing, Peault et al. 2001), normalize blood glucose levels in diabetic immunoincompetent rodents (Hullett, Falany et al. 1987; Tuch, Osgerby et al. 1988; Tuch and Monk 1991; Castaing, Peault et al. 2001), but are rejected when transplanted into humanized SCID mice (immune reconstituted with human fetal liver and thymus tissue) (Rouleau, Namikawa et al. 1996). Human pancreas obtained from first trimester fetus are less immunogenic that pancreas from the second trimester of gestation, as indicated by cellular infiltrates that contain high levels of host CD45+ cells. This is consistent with an upregulation of T cell activating molecules, such as MHC II, the chemokine ligand 19 (CCL19), complement component 3 (C3) and tumour necrosis factor superfamily 10 (TNFSF10, also known as TNF-related apoptosis-inducing ligand, TRAIL), in second trimester human fetal pancreas compared to first trimester tissue (Brands, Colvin et al. 2008).
ESCs and iPSCs were also considered to be non-immunogenic, until recently. Murine ESCs do not express MHC I or MHC II, but increase MHC I expression when they differentiate spontaneously (Wu, Boyd et al. 2008). Despite of the lack of MHC I expression, ESCs are not deleted by natural killer (NK) cells (Koch, Geraldes et al. 2008). Interestingly, MHC I-positive murine ESCs are not recognized by cytotoxic CD8+ T cells, even when antigen presenting cells (APCs) are used to prime T cells, suggesting that ES cells have some immune privilege. Nevertheless, this immune privilege is fragile and the addition of CD4+ helper T cells can induce ESC rejection
To date, there have been few descriptions of the immunogenicity of ESC-IPCs, but the existing data indicate that both innate and adaptive arms of the host immune system could be activated after ESC-IPC transplantation. ESC-IPCs can normalize blood glucose levels of diabetic immune compromised mice short-term, but when transplanted into immunocompetent hosts, their functionality is impaired, even under syngeneic conditions (Wu, Boyd et al. 2008). One explanation for the immune rejection of ESC-IPCs is that they express higher levels of MHC I than ES cells, and express higher levels of both MHC I and MHC II after exposure with IFNγ
Strategies to circumvent the immune system barrier to ESC-IPCs will be needed if they will be used for the treatment of diabetic patients. For instance, a short term immunosuppression approach blocking cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4), CD40 ligand (CD40L) or lymphocyte function-associated antigen 1(LFA-1) for 6 days, has shown to allow for the engraftment of murine and human ESCs (Pearl, Lee et al. 2011). Since CTLA4Ig treatment has previously induced long-term survival of xenogeneic pancreatic islet grafts (Lenschow, Zeng et al. 1992; Eventov-Friedman, Tchorsh et al. 2006), it is possible that similar pre-conditioning treatments in recipients will allow for the acceptance of ESC-IPC grafts. An alternative strategy is the co-culture of ESC-IPC with bone marrow-derived mesenchymal stem cells, since co-culture of β cells with bone marrow-derived mesenchymal stem cells induces the expression of protective molecules such as IL6 (in a paradoxically anti-inflammatory role) and TGFβ1, an immunosuppressive cytokine, and anti-apoptotic genes like
3.7. Hematopoietic stem cell transplantation to induce immune tolerance for type I diabetes
The autoimmune nature of TID provides two main targets in which hematopoietic stem cell transplantation (HSCT) could be used to ameliorate disease: the replacement of autoimmune T cells to prevent the onset of TID, and the induction of immunological tolerance to islet replacements via a previous HSCT or HSC co-transplantation. In both cases, induction of immunological tolerance to donor antigens is required for long-term survival of a transplanted organ. This is more easily achieved with autologous bone marrow transplantation, but can also occur when allogeneic bone marrow donors are used. In allogeneic bone marrow transplantation, induction of central and peripheral tolerance to donor antigens has been accomplished by creating mixed hematopoietic chimerism using non-myeloablative conditioning regimens (Sykes 2007; Sykes 2009). An important component to the induction of this tolerance is the education of donor and host T cells to recognize each other as “self” to avoid graft-versus-host disease (GVHD) and well as host-versus-graft reactions.
HSCT has been used to induce immune tolerance to TID antigens. Gene therapy approaches in which transplantation of syngeneic HSCs that were engineered to express pro-insulin under the MHC-II promoter transferred protection against insulitis and the development of spontaneous autoimmune diabetes (Steptoe, Ritchie et al. 2003; Chan, Clements et al. 2006). Transplant of fully allogeneic purified adult hematopoietic stem cells into lethally irradiated NOD mice (Beilhack, Scheffold et al. 2003) and the induction of mixed allogeneic hematopoietic chimerism using a non-myeloablative conditioning regimen cured diabetes in NOD mice and established clear immunological tolerance to both host and donor MHC as well as to insulin (Nikolic, Takeuchi et al. 2004). Recently, it has been shown that mixed chimerism across MHC mismatches is superior than mixed chimerism amongst MHC matched hematopoietic cells for prevention of insulitis in TID (Racine, Wang et al. 2011). Moreover, mesenchymal stem cells (MSC) co-transplanted with hematopoietic stem cells without islets prevented the onset of TID in allogeneic recipients, and co-transplantation of MSC with allogeneic hematopoietic stem cells and islets have been used to prevent the onset of TID in rodent models (Itakura, Asari et al. 2007; Asari, Itakura et al. 2011).
The results of the first human clinical trial of the use of autologous HSC transplant to treat early-stage TID patients were excellent (Voltarelli, Couri et al. 2007). Importantly, in these studies TID was alleviated using hematopoietic grafts alone (i.e. without co-transplantation of islets). However, patients with late-stage TID can only currently be cured by islet transplantation protocols which require islets from multiple matched donors, exceeding donor availability (Shapiro, Ricordi et al. 2006), or other insulin producing cell sources (such as ESC-IPC and iPS-IPC), which will also require induction of immune tolerance. Combined islet and HSC allotransplantation using an 'Edmonton-like' immunosuppression, (i.e. without ablative conditioning) in which high doses of donor HSCs (4.3 + 1.9 x 106 HSCs/kg) were transplanted after 5 and 11days of islet transplantation, has not resulted in stable hematopoietic chimerism or graft tolerance after 1 year post-transplantation (Mineo, Ricordi et al. 2008). Whether prior transplantation of HSCs could improve the engraftment of later islet transplantation is not clear, and is not easily tested in the clinic.
The induction of immunological tolerance to ESC-derived tissues is an active and emerging area of investigation. Recent studies have provided proof-of-principle that the induction of immunological tolerance to ESC-derived tissues can be achieved, but the mechanisms of tolerance induction to ESC-derived hematopoietic-progenitors (ES-HP), as well as the ability of ES-HP themselves to induce immunological tolerance across allogeneic barriers, are still not well understood (reviewed in (Thompson and Manilay 2011)). Transplantation of HoxB4-transduced ES-HP resulted in mixed hematopoietic chimersm and the induction of specific transplantation tolerance to allogeneic cardiac grafts (Bonde and Zavazava 2006; Bonde, Chan et al. 2008). It appears that HoxB4-ES-HP-derived T cells are actively “tolerized” to donor antigens in the thymus, and that if not, they can cause lethal GVHD (Kim, Stultz et al. 2011). In line with this, recipients of allogeneic ES-HP lacked evidence of GVHD (Burt, Verda et al. 2004; Bonde and Zavazava 2006; Bonde, Chan et al. 2008). Finally, another study has shown that non-HoxB4 transduced ES-HP can prevent onset of TID in mice (Verda, Kim et al. 2008), but the method of ES-HP production in this study is quite different from the other more recent protocols. Therefore, it appears that ES-HP are similar to bone-marrow derived hematopoietic progenitors, and that they could be used to induce tolerance of allogeneic tissues that are derived from the same ESC line, or that share MHC antigens. The same theory could apply to hematopoietic progenitors that are derived from iPSC (Niwa, Umeda et al. 2009).
The replacement of β cells is a promising therapy for diabetic patients, but the process depends on the discovery and detailed study of sources other than cadaveric pancreas. Pancreatic stem cells would be an ideal source for β cell replacement, since they are committed to differentiate into pancreatic cells. However, their cellular marker phenotype, as well as their anatomic location, has not yet been identified. If these questions are resolved, the conditions for their expansion and differentiation will require further optimization, such as emulating the properties of the pancreatic stem cell niche, improving their expansion, and perfecting the composition of culture media to induce their differentiation into endocrine cells. In addition, as pancreatic stem cells will likely be isolated from healthy patients, the immune system barrier to allogeneic transplants will also need to be addressed.
Another alternative to pancreatic stem cells is the differentiation of IPCs from ESC or iPSC, but this first requires the development and acceptance of a standard methodology to generate them. IPCs differentiated by this standard methodology will have to be completely mature and functional (i.e. respond appropriately when blood glucose level are altered, and have absolutely no teratoma-forming potential). The generation of a well-defined homogenous population of transplantable pancreatic progenitors that can be identified with extracellular markers would be an alternative to IPCs. Although they are not completely mature, pancreatic progenitors could perhaps differentiate more readily into mature endocrine cells in response to the signals in their
This manuscript is supported by funding from the University of California and the California Institute for Regenerative Medicine (RN1-00554-1).