Abstract
In diabetes, a loss of pancreatic β-cells causes insulin dependency. When insulin dependency is caused by type 1 diabetes or pancreatic diabetes, for example, pancreatic β-cells need to be regenerated for definitive treatment. The methods for generating pancreatic β-cells include a method of creating pancreatic β-cells in vitro and implanting them into the body and a method of regenerating pancreatic β-cells in the body via gene introduction or the administration of differential proliferation factors to the body. Moreover, the number of pancreatic β-cells is also low in type 2 diabetes, caused by the compounding factors of insulin secretory failure and insulin resistance; therefore, if pancreatic β-cells can be regenerated in a living body, then a further amelioration of the pathology can be expected. The development of pancreatic β-cell-targeting regenerative medicine can lead to the next generation of diabetes treatment.
Introduction
For diseases caused by the failure of vital organs, the definitive treatment is to restore the organs' functions. This can be done by transplanting an organ provided by a third party, but this method relies on sufficient donors. On the other hand, regenerative medicine, which aims to restore functionality that has once failed by applying knowledge from fields such as developmental biology, molecular biology, and stem cell biology to the medical field, is not dependent on the supply of donor organs, and therefore the clinical application of such treatments offers great hope for the future. On the other hand, diabetes characterized by high blood glucose concentrations is caused by a decreased insulin activity in the body, a known background factor of which is the loss or significant decrease of pancreatic β-cells that excrete insulin. Therefore, the regeneration of pancreatic β-cells is necessary for the definitive treatment of diabetes. This review article describes the regenerative medical strategies that aim to treat diabetes by targeting pancreatic β-cells, as well as other related issues.
The Significance of Regenerative Medicine for Diabetes
The status of diabetes is divided into three disease stages, according to the level of insulin action deficiency and the pathology: the normal zone, the boundary zone, and the diabetes zone. The diabetes zone is divided into a period of insulin dependency in which the administration of insulin preparations is necessary for survival and a period of insulin nondependency. In type 1 diabetes, pancreatic β-cells are specifically destroyed by autoimmune abnormalities, etc., resulting in insulin dependency. Moreover, a significant decrease or loss of pancreatic β-cells due to chronic pancreatitis or pancreatic resection can lead to the same state. On the other hand, in type 2 diabetes, which develops through the compounding factors of insulin secretory failure and insulin resistance, insulin dependency is rarely caused, but it is known that the decrease of pancreatic β-cells is caused by glucose toxicity, exhaustion, and such. Insulin excreted from pancreatic β-cells is the only hormone that stimulates distal cells and tissues and allows for the absorption of glucose that can be used as source of energy. Therefore, the depletion of endogenous insulin secretion due to the loss of pancreatic β-cells causes metabolic abnormalities such as hyperglycemia, thus leading to a poor prognosis for survival.
Currently, simple blood glucose measurements and various types of insulin analogues have been developed, and methods of controlling blood glucose levels have advanced at a remarkable rate. However, even with the full use of insulin secretion intensification therapy with multiple daily blood glucose measurements and the administration of corresponding amounts of insulin, many patients still experience unstable blood glucose levels. This reflects the difficulty faced by current blood glucose measurement systems and insulin preparations in fully regulating blood glucose, which is naturally performed by the living body. In a healthy body, pancreatic β-cells continuously detect blood glucose levels and immediately release appropriate amounts of insulin secretion into the blood properly to maintain the blood glucose level in the living body within a narrow range. It is not difficult to understand why it is not easy to control blood glucose levels thoroughly with insulin administrations only a few times a day based on blood glucose levels measured only a few times a day. Insulin-dependent diabetes that exhibits unstable blood glucose with insulin therapy is associated with various complications. More specifically, excessive dosages of insulin can lead to low blood glucose levels, so-called hypoglycemia, thereby causing insulin shock and thus posing a risk of endangering a patient's life. The maintenance of higher blood glucose levels in order to avoid such a life-threatening condition results in the loss of vision caused by retinopathy, the need for dialysis due to chronic renal failure, and necrosis and amputation of the lower limbs due to severe nerve and/or microvascular damage.
The purpose of regenerating pancreatic β-cells in insulin-dependent diabetes is not to eliminate the daily problems of insulin therapy. Rather, the purpose is to reinitiate the glycemia control that was originally performed by the body itself by treating the root cause of insulin dependency as well as to resolve the issues related to the patient's survival that cannot be treated by insulin secretion therapy. Moreover, in type 2 diabetes, there are indications that insulin secretion failure is caused by decreased amounts of pancreatic β-cells (16). Therefore, it is necessary to restore the number of pancreatic β-cells to regain the body's original ability to regulate blood glucose levels, and the necessity of developing regenerative medicine is being seriously considered.
Regenerative Medical Approaches after the Depletion of Endogenous Insulin Secretion
There are two major methods of regenerating pancreatic β-cells that have been lost. One is an ex vivo approach in which pancreatic β-cells or insulin-producing cells are created extracorporeally and transplanting them into the body (Fig. 1), and the other is an in vivo approach that aims to regenerate pancreatic β-cells inside the body via gene introduction or the administration of differentiation-inducing factors (Fig. 2).
Ex vivo approach for pancreatic β-cell-targeted regenerative medicine. With the ex vivo approach, cells are eventually transplanted back into the body. Various manipulations can be performed on freely selected cells, which include embryonic stem cells, induced pluripotent stem cells, adult tissue stem/progenitor cells, and terminally differentiated pancreatic islet cells (Tx; transplantation).
In vivo approach for pancreatic β-cell-targeted regenerative medicine. The in vivo approach is to create pancreatic β-cells or glucose-sensitive insulin secretory cells by performing manipulation on cells existing inside the body by means of gene transfer and/or administration of differentiation-inducing factors.
Ex Vivo Approach (Fig. 1)
With the ex vivo approach, cells are eventually returned to the body by transplantation, but because such transplantable cells are already functional, the beneficial effects can be expected within a short period of time. Because an extracorporeal manipulation is applied, various operations can be performed on freely selected cells that will become the source of pancreatic β-cells. Moreover, the issue of creating a supply of cells for transplantation can also be addressed by creating them on a sufficiently large scale. The conditions for sources of pancreatic β-cells used in the ex vivo approach include controllable proliferation and capabilities of the efficient expression of function and the maintenance of function. Embryonic stem cells and tissue stem/progenitor cells are the most common candidates.
It has been reported that, in a study using mice, glucose-sensitive insulin secretory cells were differentiated from stem cells and that diabetic status was ameliorated by transplantation of the cells (8,12), but in general, it is recognized that it is still difficult to intentionally create pancreatic β-cells in mass quantities from embryonic stem cells and tissue stem/progenitor cells using the currently available differentiation methods (7,17). In contrast, we established a cell line that secreted insulin in response to glucose concentrations with terminally differentiated human adult pancreatic islet β-cells as a cell source by applying a reversible immortalizing system (9,13). After performing a monolayer culture of human pancreatic islets, it was immortalized using retroviral vectors SSR#69 that simultaneously encode SV40T and fusion genes of hygromycin resistance (HygroR) and herpes simplex virus-thymidine kinase (HSV-TK) between loxP sequences that are targeted by a Cre-recombinant enzyme, as well as recombinant retroviral vectors SSR#197 that express both hTERT, which prevents the shortening of telomere length, and an enhanced green fluorescent protein (EGFP). The immortalized cells were selected with hygromycin and then screened with single-cell cloning to establish a human pancreatic β-cell line (NAKT-15). After infection of NAKT-15 cells with a recombinant adenovirus vector AxCANCre that expresses Cre recombinase, transplantation of the reverted form of NAKT-15 cells with immortalized genes removed in diabetic mice resulted in regulation of blood glucose levels.
Furthermore, Yamanaka, and coworkers have developed induced pluripotent stem cells (iPS cells) that have abilities equivalent to embryonic stem cells generated from adult human fibroblasts (21). The problems faced with embryonic stem cells are not all solved with iPS cells, due to the fact that a differentiation induction method for pancreatic β-cells is still needed. However, it circumvents the ethical issues regarding the use of human embryonic stem cells, and because the development of a method of differentiation induction for pancreatic β-cells from stem cells is expected to accelerate, future progress is also expected.
In Vivo Approach (Fig. 2)
The in vivo approach is to create pancreatic β-cells or glucose-sensitive insulin secretory cells by performing manipulation on cells existing inside the body. It has been reported that in an experiment of gene therapy using rodents, diabetic symptoms such as the depletion of endogenous insulin secretion were alleviated. Lee et al. cured a case of diabetes by inserting short-chain insulin genes downstream of glucose sensitive promoters, targeting hepatocytes, into an individual suffering from a loss of pancreatic β-cells (11). Moreover, there are reports claiming that the introduction of differentiation factor genes that target progenitor cells of pancreatic β-cells existing in the liver have restored the body's ability to regulate blood glucose levels (6,10). The in vivo approach uses stem/progenitor cells that exist inside the body and can therefore be considered a more physiological approach. It does not require implantation techniques as in the ex vivo approach, and there is no need to consider ethical issues regarding cell sources. There are still many issues to be resolved regarding the aspects of efficacy and safety, such as confirmation of differentiation induction and control of proliferation, as well as assessments of systemic adverse effects caused by factors used for the operations. However, because it is physiologically more appealing and less invasive, it has been recognized as a useful approach and vigorous studies are currently under way.
Regenerative Medical Approaches before the Depletion of Endogenous Insulin Secretion
In type 2 diabetes, the amount of pancreatic β-cells decreases, but is not completely lost in most patients. Because it is not yet insulin dependent, medical treatment should therefore be minimally invasive. More specifically, the in vivo approach that involves the administration of differential proliferation factors and such targeting stem/progenitor cells in the pancreas is an appropriate means. For example, the application of GLP-1 excreted from gut endocrine cells (L cells), which has an insulin tropic effect, can be considered. Recent studies have reported that GLP-1 promotes differentiation into pancreatic β-cells and has a proliferative effect on pancreatic β-cells (2,22). Moreover, Melton and coworkers have reported that adult pancreatic β-cells, which were assumed to not differentiate or proliferate, retain self-propagating abilities (5), thus indicating that they may be sensitive to differential proliferation factors. If the causal factors of decreases in the amount of pancreatic β-cells can be eliminated through an intervention in the early stages of diabetes, it is possible for the amount of pancreatic β-cells to recover to normal levels (14).
Related Issues Regarding the Regeneration of Pancreatic β-Cells for Completely Curing Diabetes
It is known that insulin secretory cells can be created ex vivo using stem cells as a cell source, but the capability of that of pancreatic β-cells is inferior in comparison to those generated in the original pancreatic islet (18). It has been clarified that a developed capillary net exists in the pancreatic islet (1) and that β-cells communicate with other β-cells or non-β-cells to excrete hormones for blood glucose regulation (3,19). To create highly functional pancreatic β-cells such as those existing in the body, it may be necessary to reproduce the tissue structure of the pancreatic islet. We believe that this would require more than one β-cell, analyses of the intercellular communication between β-cells and non-β-cells, the development of a restructuring technique, and the techniques for tissue engineering and revascularization for extracellular environments.
The regeneration of pancreatic β-cells is essential for curing diabetes but it cannot act as a cure by itself. Unless the root cause of the loss of pancreatic β-cells is eliminated, they will continue to be destroyed via the same mechanisms, even if the pancreatic β-cells can be regenerated. Such causes include self-reactive immune abnormalities seen especially in type 1 diabetes (15). However, there is no definitive solution for this at present. High expectations are now being placed on the development of such innovations, such as new immunosuppressive agents and immunological modification through the induction of regulatory T cells. In in vivo regeneration therapy performed before the depletion of endogenous insulin secretion, a better prognosis can be expected when the therapy is performed earlier. Currently, preclinical diagnoses of diabetes are done using glucose intolerance, but if the amount of pancreatic β-cells can be measured, diabetes can be diagnosed even earlier. Techniques of imaging pancreatic islets in which pancreatic β-cells exist are being developed for the purpose of such super-early diagnoses (4,20).
Conclusion
Owing to recent developments in basic science, regenerative medicine that will be effective on diabetes is now approaching fruition. In addition to the regeneration of pancreatic β-cells, important factors for completely curing diabetes include technological developments for tissue engineering of pancreatic islets, the construction of a bioartificial pancreas (23) and the establishment of treatments for the etiology of pancreatic β-cell destruction, and the development of diagnostic techniques for the early detection of the onset of diabetes. In order for regenerative medical approaches to achieve their purpose of completely curing diabetes, it is necessary to accumulate knowledge and promote integrative studies across various research fields such as immunology and the fields of basic science and clinical medicine, which are already closely involved with diabetes.