In vitro reconstitution of pancreatic islets

Abstract

The lack of transplantable pancreatic islets is a serious problem that affects the treatment of patients with type 1 diabetes mellitus. Beta cells can be induced from various sources of stem or progenitor cells, including induced pluripotent stem cells in the near future; however, the reconstitution of islets from β cells in culture dishes is challenging. The generation of highly functional islets may require three-dimensional spherical cultures that resemble intact islets. This review discusses recent advances in the reconstitution of islets. Several factors affect the reconstitution of pseudoislets with higher functions, such as architectural similarity, cell-to-cell contact, and the production method. The actual transplantation of naked or encapsulated pseudoislets and islet-like cell clusters from various stem cell sources is also discussed. Advancing our understanding of the methods used to reconstitute pseudoislets should expand the range of potential strategies available for developing de novo islets for therapeutic applications.

Keywords: :

• pancreatic islets
• pseudoislets
• islet-like cell clusters
• diabetes mellitus
• tissue engineering

Introduction

Transplantation of the pancreas or pancreatic islets is the most efficient means of treating type 1 diabetes mellitus.^1,2 Whole pancreas transplantation allows the patients to remain insulin-independent for more than 10 y.³ Islet transplantation does not need major surgery and the function of the islet grafts maintained over five years.^4,5 However, a shortage of donors prevents this therapy from being implemented, although the methodology is well established. The generation of artificial pancreas is an alternative means of providing transplantable islets; islets can be isolated from pigs or other animals with the hydrogel capsule and separation membrane attached to prevent the access of immunoglobulins, which would lead to immunoreactions.^6-8

Another approach is to reconstitute pancreatic islets in vitro. At present, it is not possible to induce fully differentiated β cells from induced pluripotent stem (iPS) cells. However, after these methods have been established, it will be possible to obtain a cell source to form islets, which would be adapted to the immune systems of patients. Various tissue engineering methods are also needed to handle the cells so that they form islets with an appropriate architecture. Thus, it would be useful to understand the process of reconstituting islet structures using freshly isolated primary islet endocrine cells, established cell lines, and β cells, such as cells induced from iPS or embryonic stem (ES) cells. In this review, we focus on in vitro organogenesis to form islet-like tissues using primary endocrine cells, cell lines, and various stem cells.

Pseudoislets from Single-Cell Preparations

The first reports of pseudoislet formation appeared around 1980. Scharp et al. reported the digestion of dog pancreas using trypsin to obtain single endocrine cells, where subsequent rotational culture yielded selective aggregation in 4–8 d. Pseudoislets contained all islet cell types and were stable for 4 wk, releasing hormones in response to appropriate stimuli.⁹ They also succeeded in forming pseudoislets from neonatal pig islets.¹⁰ Pseudoislet formation was also confirmed using adult rats,^11-14 neonatal rats,^11,15-17 cadaveric pancreata from children,¹⁸ and neonatal humans.¹⁹ These studies clearly demonstrate that pancreatic endocrine cells, such as α, β, and delta, and pancreatic polypeptide-producing cells have the potential to reconstitute their original architecture and form islets. Halban et al. also demonstrated that their pseudoislets were remarkably similar to adult rat islets in terms of the cellular composition and organization, although the pseudoislets were approximately half the size of the native islets.¹⁴ This encouraged the generation of islets in vitro using islet cells derived from various sources as well as primary cells.

Mechanisms of Pseudoislet Formation with an Appropriate Architecture

Aggregates can form with similar cell positioning to intact islets. Thus, determining the mechanisms that underlie this process would improve our understanding of islet regenerative medicine. Halban et al. compared pseudoislets formed from native pancreatic β cells and transformed (RINm5F) cells.²⁰ On mixing native β cells, non-β cells, and RINm5F cells, they observed that the native β cells were centrally located and were surrounded by zones of non-β cells, as reported previously, whereas the RINm5F cells were found only in the peripheral region. They concluded that a cell surface antigen recognized by a monoclonal antibody was important for the behavior of native β cells in pseudoislets. In 1991, a related study observed that E-cadherin was important for the aggregation of islet cells and that calcium-independent cell adhesion molecules distinguished the islet cell types.²¹ The cell surface calcium-independent adhesion molecule expressed by β cells that regulates β cell aggregation was shown to be controlled by tumor necrosis factor-α signaling.²² They found that neural cell adhesion molecule was expressed by non-β cells and showed that it had a role in islet cell type segregation.²³ These studies demonstrate the role of cell surface molecules in selective aggregation and the formation of the islet-like architecture of pseudoislets. However, the relationships between cell positioning and the functions of pseudoislets remain to be elucidated and may help in designing a better method for the production of more functional pseudoislets for use in regenerative medicine.

Relationship between Cell-to-Cell Contact and Function

It has been reported that the architecture of islets (the arrangement of endocrine cells) is important for their functions such as insulin secretion, thereby indicating that simple aggregates may exhibit relatively weak functions.^24-28 Pseudoislets are suitable for studying the importance of structure and the relationships between the configurations of cells in islets, rather than β cells alone. It has been shown that cell-to-cell contact is indispensable for the initiation of insulin secretion when stimulating intact islets with glucose. Pseudoislet formation is sufficient for reversing the loss of structure–function relationships in single-cell preparations of islets cells from adult rat islets.²⁹ Pseudoislets were also formed using MIN6, an established pancreatic β cell line, to show that cell-to-cell interactions are essential for integrated responses to nutrient stimuli.³⁰ The β cell line-based pseudoislet system is different from pseudoislets formed using whole endocrine cells, although it is a powerful tool for studying various aspects of islets in vitro, as shown by Jones et al. in several studies.^31-38

The reconstitution of islets using two types of cells can help to elucidate the relationships between them. For example, Josefsen et al. separated islet cells obtained from rat islets into β cells and non-β cells using fluorescence-activated cell sorting. They reconstituted the separated cells and found that α cells are necessary for insulin secretion but not for glucose sensing.³⁹ On the other hand, Hamaguchi et al. reported pseudoislets formed from α and β cell lines and showed that cell-to-cell contact reduced the level of insulin secretion by β cells.⁴⁰ Brereton et al. concluded that α cells did not affect insulin secretion by β cells via cell-to-cell contact,⁴¹ although these results are controversial. Importantly, insufficient studies have used pseudoislets to test this hypothesis. Thus, the roles of interactions between endocrine cells must be intensively analyzed to facilitate the effective reconstitution of islets for use in regenerative medicine. A possible explanation for previous contrasting results is the ratio of α and β cells. Thus, we tested broader ranges of cell type ratios compared with previous studies, i.e., Hamaguchi et al. used α:β ratios of 1:2, 1:1, or 2:1, and Brereton et al. used an α:β ratio of 1:3. We found that the level of insulin secretion was higher when the α:β ratio was 1:8 than when only β cells were used. When the percentage of α cells was 20% or more, insulin secretion was not induced, remained the same or reduced.⁴² These results may be reflecting the importance of endocrine cell ratio in islet. For example Brissova et al. reported that mouse islets were composed of 14–19% α cells, 75–80% β cells, and 6% delta cells.⁴³ They also reported the population of human islets, 34% α cells, 54% β cells, and 10% delta cell.⁴³ Kim et al. compared islet architecture including β cell ratio between different species, human (64%), monkey (79%), pig (89%), rabbit (79%), bird (46%), and mouse (90%).⁴⁴ It is an issue to be addressed whether the optimal cell ratio and arrangement in pseudoislets is different when we use endocrine cells from other species.

Effective Methods for Pseudoislet Production and Maintenance of Their Functions

The formation of pseudoislets is not difficult, which is an advantage for pancreas studies using pseudoislets. Early studies of pseudoislets reported culture in an untreated culture dish with or without shaking (Table 1). However, analyses of the effective factors that facilitate the formation of pseudoislets with higher functions are indispensable. Matta et al. studied the conditions of islet dissociation and reaggregation of islated endcrine cells for effective pseudoislet formation, and found that dissociation with dispase and reaggregation in rotation culture with a high glucose medium (11 mM) was better than the condition using trypsin, static culture, and low glucose medium (4 mM).⁴⁵ Supplementation with all-trans retinoic acid or a conditioned medium obtained from exocrine cell culture also helped in effective aggregation of dissociated islet cells, which exhibited normal insulin secretion after glucose stimulation.⁴⁶ The addition of nicotinamide to the culture medium also maintained or enhanced the functions of pseudoislets obtained from pig neonatal or human fetal islets.^47-49

Table 1. Methods used to form pseudoislets

Using single-cell suspensions of pancreatic cells
Non-adherent dish under static condition	Refs. 12, 13, 17, 20, 22, 23, 29, 38, 39, 45, 46, 47, 48, 50, 54, 55, 56
Non-adherent dish with shaking	Refs. 9, 10, 21, 39, 40, 45, 49
Adherent dish	Refs. 18, 36
On a gelatin-coated dish	Refs. 30, 31, 32, 33, 34, 35, 37, 41
Round-bottom 96-well plate	Ref. 57
Droplets on an untreated culture dish	Ref. 14
Centrifugation in tubes	Ref. 11
In collagen gel or synthetic polymer gel	Refs. 15, 59, 62, 63, 64
In methylcellulose sol	Ref. 42
Microencapsulation	Ref. 53
Specific surfaces	Ref. 51
Dielectrophoresis	Ref. 52
Using small pieces of pancreatic tissue
Non-adherent dish	Ref. 19

Genetically engineered islets may have various potential applications. Compared with cells in native islets, dissociated endocrine cells on plates are easier to transfect with exogenous genes using viral or chemical methods. Caton et al. reported the lentivirus-mediated transduction of endocrine cells on plates and the formation of pseudoislets with gene-transferred cells,⁵⁰ which were used to elucidate the functions of connexin during insulin secretion. The expression or knockdown of specific genes would be indispensable for enhancing or regulating pseudoislets in regenerative medicine.

Recent trials have investigated the formation of pseudoislets using various biomaterials, including different surfaces. Yang et al. reported that poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) is a good surface for the aggregation of pseudoislets that exhibit higher insulin production.⁵¹ Pethig et al. demonstrated the dielectrophoretic assembly of a β cell line to form pseudoislets at approximately 1000 cells/aggregate in a 10 × 10 array.⁵² The β cell aggregates included nanosensors to detect the pH and cellular oxygen levels. Tsang et al. aggregated adult human pancreatic islet cells in the intracapsular spaces of alginate-poly-l-lysine microcapsules.⁵³ These capsules had potent effects on reducing hyperglycemia and produced human C-peptide after transplantation into mice.

Transplantation of Pseudoislets

Better results are obtained in transplantation using pseudoislets that form successfully. Tze and Tai reported the allotransplantation and xenotransplantation of rat-derived pseudoislets intraportally into diabetic rats and mice.^12,54 Rejection is one of the major problems of transplantation. Wolf-Jochim et al. compared the survival time after transplantation of fresh or cultured pancreatic islets, pseudoislets, and single cells beneath the kidney capsule in non-immunosuppressed diabetic rats. They concluded that transplantation of pseudoislets resulted in the prolonged acceptance of allografts, without immunosuppression of the host.⁵⁵ Vascularization is another problem that affects pseudoislet transplantation. Beger et al. reported the vascularization of pseudoislets in the dorsal skin folds using Syrian golden hamsters.⁵⁶ The vascularization of pseudoislet after transplantation was slower than that of native islets containing the original endothelial cells. However, they argued that pseudoislets did not disable the formation of a microvasculature. To improve the vascularization in pseudoislets, Penko et al. formed mosaic pseudoislets, which comprised β cells with interspersed vasculogenic endothelial progenitor cells.⁵⁷ They did not report the results of transplantation, although these mosaic pseudoislets exhibited higher insulin secretion after glucose stimulation than normal pseudoislets that comprised only β cells.

Production of Bioartificial Islets Using Reconstituted Islets

The use of immunophagophores is effective for protecting islet tissues from the immune system, which is generally achieved by producing bioartificial islets using intact islets.⁵⁸ This method is also adaptable to pseudoislets. Ohgawara et al. formed pseudoislets using a mouse β cell line and transferred them into diffusion chambers to create a bioartificial endocrine pancreas (Bio-AEP).⁵⁹ The implantation of Bio-AEP into stereptozotocin-induced diabetic rats resulted in normoglycemia for ≥12 wk without any immunosuppressants. As mentioned previously, the capsulation of pseudoislets is another technique that facilitates immunoprotection and helps control the size of islets.⁵³ Sakai et al. reported advanced methods for forming cell aggregates with outer cell layers as a model of islets.⁶⁰ Onoe et al. established islet tissues by reconstituting them in hydrogel fibers.⁶¹ One of the advantages of producing islets in fibers is their ease of handling during transplantation or the removal of fibers. In contrast to particles beneath the kidney capsule, the fibers remain fixed; thus, it is easy to locate them after transplantation.

Islet-Like Cell Clusters (ICCs) from Various Stem Cells

The term ICCs refers to islet-like cellular aggregates derived from stem cell culture. They differ from pseudoislets because ICCs are “grown” by culturing cells on tissue culture plates, which includes no step to dissociate cells into single-cell suspensions. ICCs have been produced from pancreatic tissue-derived stem cells such as rat embryonic pancreatic precursor cells,^62-64 chick islet-derived stellate-like cells,⁶⁵ human non-islet pancreatic cells,⁶⁶ human mesenchymal stem cells (MSCs) derived from pancreatic islets,⁶⁷ and human adult pancreatic endocrine progenitors.⁶⁸ MSCs derived from the bone marrow of mice,^69,70 rats,^71,72 and humans⁷³ have also been used to produce ICCs. Adipose tissue-derived stem cells from mice⁷⁴ or humans^75,76 are sources of pseudoislets. Additional stem cell sources include human peripheral blood monocytes,⁷⁷ human umbilical cord blood-derived stem cells,⁷⁸ human skin fibroblasts,⁷⁹ human umbilical cord matrix-derived MSCs,⁸⁰ human placenta-derived MSCs,⁸¹ human amnion-derived MSCs,⁸² human multipotent dermal fibroblasts,⁸³ human placenta-derived multipotent stem cells,⁸⁴ and human dental pulp stem cells.⁸⁵ Previous studies have induced ICCs from mouse ES cells,^86-89 monkey ES cells,⁹⁰ and human ES cells.^91-94 Mouse and human iPS cells can also be adapted to induce ICCs.^95,96 In particular, the islet-like tissues induced by Saito et al. exhibited distinct three-dimensional structures, which were similar to adult pancreatic islets, and they secreted insulin in response to glucose concentrations. If the induction efficiency of islets can be improved, these systems for inducing ICCs from stem cells will be very important methods to compensate for the lack of transplantable islets. In addition, although the structure is not similar to that of native islets, it is possible that single cells from trypsinized ICCs can be used to reconstitute pseudoislets.

Blastocyst complementation allows pancreatic replacement by donor cells in pigs,⁹⁷ thereby indicating that human ES or iPS cells may contribute to pancreatic organogenesis. This approach may be used to generate pancreatic islets with an intact architecture via capsulation with immunoisolation materials, because blood vessels would not be able to replace the donor cells. Human endocrine cells isolated from humanized pancreas may also be used to form pseudoislets.

Conclusions

Pancreatic endocrine cells have the potential to reorganize islet-like structures even when they are dissociated into single cells. This characteristic is highly beneficial for the in vitro formation of islets (pseudoislets or ICCs), which could compensate for the lack of transplantable pancreas because the structure of the islets is important for their function. In addition, the best structures for specific functions may differ from those of native islets. Thus, the in vitro reconstitution of islets should be tested depending on factors such as the ratio of α:β cells. Expansion of the concept of islets is also important for enhancing the effectiveness of islets for transplantation. “Fiber-formed islets” represent a breakthrough in this area. Many studies have induced ICCs using various types of stem cells, some of which were able to induce ICCs that resembled native islets. However, although the structures of ICCs may be very different from intact islets, it may be possible to form pseudoislets using single cells obtained from dissociated ICCs. Thus, it is essential to integrate the concepts of pseudoislets, ICCs, and artificial islets to facilitate their practical use.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

10.4161/org.28351