WO2008153299A1

WO2008153299A1 - Method for rejuvenating isolated pancreatic islets for transplantation

Info

Publication number: WO2008153299A1
Application number: PCT/KR2008/003228
Authority: WO
Inventors: Jun-Seop Shin
Original assignee: Jun-Seop Shin
Priority date: 2007-06-10
Filing date: 2008-06-10
Publication date: 2008-12-18

Abstract

The present invention relates to a novel method for rejuvenating isolated pancreatic islets for transplantation, more particularly to a method to increase or enhance insulin content, glucose-stimulated insulin secretion, viability, and engraft capacity upon transplantation into diabetic recipients of isolated pancreatic islets. According the present invention, the rejuvenated islets have 1.7-fold and 1.9-fold greater intracellular insulin content and glucose-stimulated insulin secretion (GSIS), respectively than freshly isolated islets. Moreover, many genes important for β-cell survival and function such as insulin, GLUT2, Bcl-2, and DNA repair enzymes were significantly increased by rejuvenation. These enhanced islet capacity endow them greater resistance against oxidative stresses, reduced immunogenicity, and at least 2-fold increase of engraft efficacy in islet xeno- and allogeneic transplantation model. In conclusion, according to the present invention, it is possible that islet transplantation with smaller amount of islets should cure type 1 diabetes.

Description

Technical Field

The present invention relates to a novel method for rejuvenating isolated pancreatic islets for transplantation, more particularly to a method to increase or enhance insulin content, glucose-stimulated insulin secretion, viability, and engraft capacity upon transplantation into diabetic recipients of isolated pancreatic islets.

Background Art

Diabetes is one of most serious health problems worldwide, affecting nearly 200 million people with its number growing to 400 million at 2030. Type 1 diabetes consists of about 5% of all diabetic patients and is characterized by β-cells destruction by autoimmune attacks. Type 2 diabetes is common form of diabetes and the peripheral insulin resistance and gradual dysfunction of β-cells are main features of this disease. Although two forms of diabetes are different regarding the etiology, β-cells loss whether it is absolute or relative is common pathophysiological outcome, and thus the replacement or regeneration of β-cells has been extensively studied over the past decades.

Islet transplantation is a preferred option for type 1 diabetes treatment, particularly when patients are easily succumbed to severe hypoglycemic episodes (Shapiro, A.M. et al. Islet transplantation in seven patients with type 1 diabetes mellitus using a glucocorticoid-free immunosuppressive regimen. N Engl J Med 343, 230-238 (2000)). However, donor shortage remains a serious limitation of this therapeutic regimen to broad usage. Moreover, islet isolation techniques are so imperfect as to fail as many as half the isolation attempts from cadaveric donor pancreata, thereby worsening shortage of the transplantable islet mass. Therefore, various methods have been studied over the years to circumvent this problem; First, reagents required for islet isolation process such as collagenase have continuously improved with respect to purity and potency (Kin, T. et al. Factors influencing the collagenase digestion phase of human islet isolation. Transplantation 83, 7-12 (2007)). Various parameters affecting islet quality and quantity such as cold ischemia time, organ storage solution, collagenase incubation time, etc have been tested to optimize islet yield (Lakey, J. R., Burridge, P.W. & Shapiro, A.M. Technical aspects of islet preparation and transplantation. Transpl lnt 16, 613-632 (2003)). Second, several surrogate β-cells have been developed (Ferber, S. et al. Surrogate beta cells. Diabetologia 40 Suppl 3, B39-43 (1997)). Third, pancreatic progenitor cells have been directed toward islet-like clusters (Ramiya, V.K. et al. Reversal of insulin-dependent diabetes using islets generated in vitro from pancreatic stem cells. Nat Med 6, 278-282 (2000)). Fourth, differentiation procedure of adult and embryonic stem cells into insulin-producing cells has been extensively studied (Lumelsky, N. et al. Differentiation of embryonic stem cells to insulin-secreting structures similar to pancreatic islets. Science 292, 1389-1394 (2001)). Finally, many researchers have tried to increase viability of isolated islets through beneficial gene delivery (Rao, P. et al. Hepatocyte growth factor gene therapy for islet transplantation. Expert Opin Biol Ther 4, 507-518 (2004)) or with treatment of anti- apoptotic agents (Emamaullee, J.A. et al. Caspase inhibitor therapy enhances marginal mass islet graft survival and preserves long-term function in islet transplantation. Diabetes 56, 1289-1298 (2007)) or pro-survival agent (Yang, Z. et al. Inflammatory blockade improves human pancreatic islet function and viability. Am J Transplant 5, 475-483 (2005)).

Given the hot debates on β-cell source in adult pancreatic islets whether it is from proliferation of pre-existing β-cells or from neogenesis from the precursor or progenitor cells, the clinically relevant β-cells from either proliferation of adult islets or differentiation of progenitor cells await further research. Likewise, differentiation of islet- like cells from stem cells has put serious ethical problem in the case of embryonic stem cells or very low efficiency even when they are transplanted into diabetic animals in the case of islet-like cells derived from adult stem cells. Therefore, it is not surprising that among various strategies, only improved collagenase brand has been applied to clinic, whilst others remain animal experiment stages. In the present invention, we have developed a new culture technique, which is comprised of the following three steps; degranulation, chromatin remodeling, and regranulation. Using this novel culture technique, we were able to increase islet function with regard to glucose-stimulated insulin secretion and resistance to oxidative stresses such as hydrogen peroxide or pro-inflammatory cytokine mixtures. Moreover, in islet transplantation model, we were able to show that rejuvenated islets are at least 2-fold superior to freshly isolated islets regarding the correction of high blood glucose level induced by streptotozocin (STZ) and have significantly reduced immunogenicity.

Disclosure of Invention

The object of the present invention is to provide a method for rejuvenating isolated pancreatic islets, which increases isolated islet's viability, function, and the engraft efficacy upon transplantation into diabetic patients. Further, another object of the present invention is to provide a composition for transplantation into diabetic recipients, comprising the pancreatic islets rejuvenated according to the present invention.

Further, another object of the present invention is to provide a method for treating Type I diabetes in a patient in need thereof, comprising administering to said patient an effective amount of the pancreatic islets rejuvenated according to the present invention.

According to one aspect of the present invention, there is provided a method for rejuvenating isolated pancreatic islets for transplantation, comprising following steps: a) degranulating step, in which the isolated pancreatic islets are treated with insulin secretagogue ; b) chromatin remodeling step, in which the degranulated islets are treated with chromatin modifier; and c) regranulating step, in which the chromatin remodeled islets are treated with serum-supplemented culture medium. In the rejuvenating method of the present invention, the isolated pancreatic islets may be obtained from rodents, such as mouse and rats, or higher mammals including human beings, preferably cadaver donors.

As used herein the term "rejuvenating" means to increase or refresh the isolated islet's function as similar to in vivo counterpart or even better than them, for example, to increase or enhance insulin content, insulin secretion activity, viability, and/or engraft capacity of the isolated islets.

As used herein the term "degranulating step" means to make the isolated pancreatic islets release intracellular insulin as much as possible, leading to decrease of the number of insulin secretory granules. The decrease of insulin secretory granules can be demonstrated from that intracellular insulin content was decreased after step 1 in Fig. 5a. In the present invention, insulin secretagogue was used for the degranulating step.

In the degranulating step of the invention, the insulin secretagogue may be any substance which can cause the intracellular insulin of the pancreatic β-cells to be secreted. Preferably, the insulin secretagogue includes, but not limited to, glucose, KCI, arginine, 2 (alpha)-ketoisocaproate, sulfonylureas, and so on. (Liang, Y. and Matschinsky, F. M. Mechanisms of action of nonglucose insulin secretagogues. Annu Rev Nutr. 14, 59-81 (1994)) In the degranulating step of the invention, the insulin secretagogue may be treated in a stimulatory or depolarizing concentration, for example 15-50 mM of glucose or KCI. Preferably, the isolated pancreatic islets may be maintained or cultured for 12- 72 hrs in serum-supplemented (7-15%) culture medium.

As used herein the term "chromatin remodeling step" means to relaxe(s)£§ ^,^fc the compact chromatin into more open structure, which promotes transcription by facilitating accessibility. In the present invention, chromatin modifier was used for the chromatin remodeling step.

In the chromatin remodeling step of the invention, the chromatin modifier may be any substance which can relax the compact chromatin into more open structure. Preferably, the chromatin modifier includes, but not limited to, histone deacetylase (HDAC) inhibitor, arginine methyltransferase inhibitor, lysine methyltransferase inhibitor, chromatin remodeling factors, and so on. (Kouzarides, T. Chromatin modifications and their function. Cell 128, 693-705 (2007), Cairns, B. R. Chromatin remodeling complexes: strength in diversity, precision through specialization. Curr. Opin. Genet. Dev. 15, 185-190 (2005))

In the chromatin remodeling step of the invention, the histone deacetylase (HDAC) inhibitor may be any substance which can inhibit the activity of histone deacetylase (HDAC). Preferably, the histone deacetylase (HDAC) inhibitor includes, but not limited to, trichostatin A, sodium butyrate, SAHA (suberoylanilide hydroxamic acid), and so on. (Marks, P. A. and Dokmanovic, M. Histone deacetylase inhibitors: discovery and development as anticancer agents. Expert Opin Investig Drugs. 14, 1497-1511 (2005))

In the chromatin remodeling step of the invention, the chromatin modifier may be treated in a chromatin relaxing concentration, for example 1 nM-10 mM of histone deacetylase (HDAC) inhibitor. Preferably, the degranulated islets may be maintained or cultured for 12-72 hrs in serum-reduced (0.5-3%) culture medium. The term "serum- reduced" means that the concentration of serum is reduced from that of the degranulating step, for example from 7-15% to 0.5-3%. That is because high concentration of serum may interfere with the activity of the chromatin modifier. As used herein the term "regranulating step" means to make the chromatin remodeled islets restore insulin gene transcription, insulin biosynthesis, and insulin storage in the secretory granules, leading to increase of the number of insulin secretory granules. The increase of insulin secretory granules can be demonstrated from that intracellular insulin content was increased after step 3 in Fig. 5a. In the regranulating step of the invention, the chromatin remodeled islets may be maintained or cultured for 12-72 hrs in serum-supplemented (7-15%) culture medium. The serum-supplemented culture medium may contain 5-10 mM of glucose as an energy source.

In a preferred embodiment of the present invention, the rejuvenated pancreatic islets are characterized to have increased insulin content, enhanced insulin secretion activity, higher viability, and enhanced engraft capacity upon transplantation, compared with non-rejuvenated isolated pancreatic islets. The non-rejuvenated isolated pancreatic islets means freshly isolated islets which are not treated with the present rejuvenating method. According to another aspect of the present invention, there is provided a composition for transplantation into diabetic recipients, comprising the pancreatic islets rejuvenated according to the method of the present invention.

According to another aspect of the present invention, there is provided a method for treating type 1 diabetes in a patient in need thereof, comprising the step of administering to said patient an effective amount of the pancreatic islets rejuvenated according to the method of the present invention.

Administration can involve transferring or transplanting the rejuvenated pancreatic islets into a patient by injection, surgical implantation or perfusion of the islets. The route of administration will be determined by the need for the islets to reside in a particular tissue or organ and by the ability of the islets to find and be retained by the desired target tissue or organ.

A therapeutically effective amount of the rejuvenated pancreatic islets rejuvenated is defined primarily by clinical response in a patient, and ranges from about 5,000 IEQ/kg body weight to about 20,000 IEQ /kg body weight of islets.

It is proposed in accordance with the present invention that the rejuvenated pancreatic islets are transplanted to subjects with type 1 diabetes or a related condition. Preferably, the transplantation process may further include appropriate treatment to prevent recurrent autoimmunity and/or immune-mediated rejection. For example, an immunosuppressive agent can be treated by administering to a patient in need thereof.

An "immunosuppressive agent" is any agent that prevents, delays the occurrence of or reduces the intensity of an immune reaction against a foreign cell in a host, particularly a transplanted cell. Preferred are immunosuppressive agents which suppress cell-mediated immune responses against cells identified by the immune system as non-self. Examples of immunosuppressive agents include but are not limited to cyclosporin, cyclophosphamide, prednisone, dexamethasone, methotrexate, azathioprine, mycophenolate, thalidomide, FK-506, systemic steroids, as well as a broad range of antibodies, receptor agonists, receptor antagonists, and other such agents as known to one skilled in the art.

Hereinafter, the present invention will be described more clearly as follows. The present invention aims to increase isolated islet's viability, function, and the engraft efficacy upon transplantation into diabetic patients by culturing isolated islets up to 7 days in vitro under a defined sequence of treatment. This procedure is composed of three key steps with each step focusing a specific aim (Fig. 1).

First, the degranulating step is performed to release intracellular insulin as much as possible, and thus reduce the intracellular insulin content and increase extracellular insulin concentration in culture medium. This is done by treating islets with well-known insulin secretagogues such as glucose, KCI, arginine, 2- ketoisocaproate, and so on. For example, in this invention, the degranulating step is made by treating isolated islets with the stimulatory concentration of glucose (25 mM) and depolarizing concentration of KCI (30 mM) for 12-72 hrs in suspension culture maintained in 10% fetal bovine serum (FBS)-supplemented CMRL1066 medium.

Second, the chromatin remodeling step is done to make compact chromatin structure into more relaxed state, which might increase expression of subset of genes essential for β-cell function and survival. This is done by treating the above cultured islets with histone deacetylase (HDAC) inhibitor such as trichostatin A, sodium butyrate, SAHA (suberoylanilide hydroxamic acid), and so on. For example, in this invention, the chromatin remodeling step is done by treating the above cultured islets with trichostatin A (TSA, 100 nM) for 12-72 hrs in suspension culture maintained in serum-reduced (1 %) CMRL1066 medium.

Third, the regranulating step is performed to restore insulin gene transcription, insulin biosynthesis, and insulin storage in the secretory granules. This is done by treating the above cultured islets for 12-72 hrs with serum-supplemented regular culture medium. For example, in this invention, this is made by treating the above cultured islets for 12-72 hrs with 10% FBS-supplemented CMRL1066 medium in suspension culture.

Islet transplantation is a promising therapeutic regimen for type 1 diabetes, but donor shortage is the main obstacle for wider application. To overcome this problem, the inventor developed a novel culture technique of isolated islets, which is composed of three discrete steps such as degranulation, chromatin remodeling, and regranulation. Using this rejuvenation procedure, the inventor demonstrated that the rejuvenated islets have 1.7-fold and 1.9-fold greater intracellular insulin content and glucose-stimulated insulin secretion (GSIS), respectively than freshly isolated islets. Moreover, many genes important for β-cell survival and function such as insulin, GLUT2, Bcl-2, and DNA repair enzymes were significantly increased by rejuvenation. These enhanced islet capacity endow them greater resistance against oxidative stresses, reduced immunogenicity, and at least 2-fold increase of engraft efficacy in islet xeno- and allogeneic transplantation model. In conclusion, the inventor develops a novel culture technique of isolated islets and rejuvenates them, making it possible that islet transplantation with smaller amount of islets should cure type 1 diabetes.

Hereinafter, the results and effects of the present invention will be described more clearly as follows.

Development of islet rejuvenation method Islet β-cells have been shown to have insulin receptor, insulin receptor substrate, and various downstream signaling components such as phosphoinositide 3- OH kinase, AKT/PKB, and so on and insulin itself is deemed as autocrine and/or paracrine survival factor for β-cells. Therefore, in the degranulation step (step 1), we wanted to increase islet viability by releasing intracellular insulin using well-known insulin secretagogues, glucose and high concentration of K⁺ ion. Then, histone deacetylase inhibitor (HDACi) was treated to induce chromatin remodeling (step 2), during which the relative lysine acetylation levels at the histone 3 and 4 are increased, thereby inducing expression of the repressed subset of genes. Among these gene sets, several important genes for β-cell survival and function such as insulin, glucose transporter-2 (GLUT2), Bcl-2 might be expected to increase. Finally, after removing residual HDACi, islets were incubated in regular serum-containing media to promote insulin biosynthesis and storage in the secretory vesicles (step 3). With respect to the gross morphology during the culture steps, yellow-brownish islets became paler after step 1 , and then the color was restored at the end of culture. Because islets were suspension cultured during the entire period, they became more and more rounded with time and very often fused with each other, giving rise to very large multi-lobed giant islets (Fig. 2). As seen by scanning electron microscope, their surfaces are very smooth and numerous microvilli are observed (Fig. 3A). Interestingly, even cells in the innermost layer appears to be healthy without any sign of necrosis, which is frequently observed in islets cultured for the same period without rejuvenation procedure (Fig. 3B). As seen in transmission electron microscope, the typical insulin granules are evenly distributed throughout the whole cytoplasm and cellular organelles such as mitochondria, endoplasmic reticulum, and Golgi complex appear intact (Fig. 4).

Rejuvenated islets have enhanced GSIS capacity and intracellular insulin content

As we measured intracellular insulin content and GSIS from the batch of size- matched islets, freshly isolated islets (FIs) had 70.8 ng/IEQ of intracellular insulin and showed 2.0-fold insulin secretion at 16.7 mM glucose over 5.6 mM glucose concentration (1.76 ng/IEQ at 16.7 mM glucose vs 0.87 ng/IEQ at 5.6 mM glucose). However, rejuvenated islets (RIs) had 136.3 ng/IEQ of intracellular insulin and 3.4-fold insulin response to glucose stimuli (3.72 ng/IEQ at 16.7 mM glucose vs 1.09 ng/IEQ at 5.6 mM glucose). This represents 1.9-fold more insulin in intracellular spaces and 1.7- fold better insulin response to glucose stimuli of RIs compared with FIs. In the control experiments, when islets were cultured for the same period to rejuvenation procedure with regular media changes, intracellular insulin increased to 104.8 ng/IEQ, but their insulin response to glucose stimuli is completely blunted (0.5 ng/IEQ at 16.7 mM glucose vs 0.43 ng/IEQ at 5.6 mM glucose). Importantly, valproic acid, another HDACi had similar effect on intracellular insulin content and GSIS. When the intracellular insulin content was measured during the courses of rejuvenation procedure, it decreased to 60% of the level from FIs after the step 1 , but then gradually increased, reaching to 1.9-fold after the step 3 (Fig. 5A). These results indicate that our culture method should rejuvenate isolated islets as evidenced by increased intracellular insulin content and enhanced GSIS capacity. Moreover, through detailed protocols such as omitting the first step or second step, we could dissect out the effect of each step of rejuvenation procedure on intracellular insulin and GSIS; Increase of intracellular insulin content was due to the degranulation step and the enhanced insulin response to high glucose was due to HDACi treatment (Fig. 5B).

Rejuvenated islets exhibit increased expression of important penes in β-cell survival and function

Treatment of HDACi and observation of the increased biochemical capacity of RIs prompted us to hypothesize that rejuvenation procedure might affect gene expression profiles of isolated islets leading to enhancement of islet function. When RIs and FIs were subjected to semi-quantitative RT-PCR analyses, insulin 1 and 2, GLUT2, Bcl-2, and several genes for DNA repair were significantly increased up to 2- 4-fold in RIs compared with FIs. By contrast, β-actin and was unaffected (Fig. 6A). Interestingly, all of HDACs including class I (HDAC1 , -2, -3, and -4) and class Il (HDAC6, -7, -8, and -10) were expressed in isolated islets and the expression of HDAC 4 and 7 was selectively decreased by TSA (Fig. 6B). These results indicate that HDACi treatment during the rejuvenation method led to chromatin remodeling at the various loci throughout the whole genome with essential genes for β-cell survival and function being increased. Furthermore, wide expression of HDACs and selective regulation of HDAC 4 and 7 by TSA strongly suggest that HDACs are important for maintenance and/or regulation of islet function.

Rejuvenated islets are more resistant against oxidative stresses than freshly isolated islets

Islet β-cells are more vulnerable to oxidative stress than other cell types, because levels of anti-oxidative enzymes handling this insult are relatively low. Therefore, prior to confirming the enhanced islet function of RIs in vivo by transplantation, we set out to examine islet viability and apoptosis rate under various oxidative stresses in vitro. When FIs and RIs were treated with H₂O₂ or cytokine mixtures composed of IL-1 β, TNF-α, and IFN-γ, viability and apoptosis rate of RIs were consistently 10-20% higher and lower than those of FIs, respectively (Fig. 7). Moreover, under the same condition, RIs had 10-15% higher intracellular insulin content than FIs despite GSlS was completely impaired by oxidative insults (Fig. 8).

Rejuvenated islets are at least 2-fold superior to freshly isolated islets with respect to correction of hyperglycemia induced by STZ in xenotransplantation model

To examine the enhanced islet function of RIs in vivo, we used islet xenotransplantation model by transplanting islets from SD rats into STZ-induced diabetic nude mice. As shown in Fig. 9, all mice receiving 150 IEQ or 200 IEQ SD FIs (4 out of 4 mice in both groups) restored prompt normoglycemia after 2-5 days lagging period depending on the severity of diabetes. As we reduced islet mass to 100 IEQ FIs, half of mice (7 out of 14 mice) receiving islet graft became normoglycemic, but the other showed chronic hyperglycemia or became eventually hyperglycemic with a short period of graft functioning. By contrast, when 100 IEQ RIs were transplanted into diabetic nude mice, all mice (5 out of 5 mice) restored normoglycemia within 1 week after transplantation. Even when 50 IEQ RIs were transplanted, half of mice (5 out of 10 mice) became normoglycemic, but the other did not. These results indicate that the marginal islet mass sufficient for correction of hyperglycemia of nude mice is about 100 IEQ SD FIs and RIs are at least 2-fold superior to FIs with respect to curing capacity of STZ-induced diabetes in nude mice.

Rejuvenated islets are at least 2-fold superior to freshly isolated islets with respect to correction of hyperglycemia induced by STZ in inbred rat transplantation model

To further extend the enhanced islet capacity of RIs in other transplantation model, we employed inbred rats (Wistar-Kyoto strain) as both islet donor and recipient. In this experiment, we isolated islets from one donor and transplanted them into STZ- induced another diabetic WK rat with (Rl group) or without rejuvenation procedure (Fl group). As shown in Fig. 10, only one animal out of five recipients in both groups became normoglycemic, but transplanted islet mass of Rl group was 46% less than that of Fl group (Table 1). These results are fully consistent with data from xenotransplantation model, and suggest that rejuvenation method should be equally effective for islets from different strain of animals.

Rejuvenated islets exhibit less immunogenicity than freshly isolated islets in outbred rat transplantation model

Finally, to examine the effect of rejuvenation on islet immunogenicity, we performed islet transplantation using outbred rats as both islet donor and recipient in a similar scheme to inbred islet transplantation. When islets from Fl or RI group were transplanted into STZ-induced diabetic SD rat, blood glucose levels of some animals were transiently decreased to normal value, but it elevated quickly to over 300 mg/dl, suggesting that transplanted islets were rejected by immune reaction (Fig. 11). Very importantly, when the histological samples obtained at 10 days after transplantation from both groups were examined by insulin immunostaining, nearly normal islet architectures with strong insulin immunoreactivity were observed in only Rl group, but not in Fl group. In this latter group, islets were destroyed by numerous immune cells and consequently only islet vestiges without insulin immunoreactivity were observed (Fig. 12). Brief Description of the Drawings

The above and other objects, features and other advantages of the present invention will be more clearly understood from the following detailed description taken in conjunction with the accompanying drawings, in which;

Figure 1 depicts a general scheme of rejuvenating method of the present invention

Figure 2 depicts the gross morphology of islets during culture period. Isolated islets from SD rats were pre-incubated in regular media overnight, and suspension cultured according to rejuvenating method. The islets were incubated in regular media supplemented with high concentration of glucose and KCI for 48 hrs (step 1), treated with HDACi (100 nM TSA) for 24 hrs (step 2), and then incubated with regular media for

48 hrs (step 3). Islets were gathered into the dish center by gentle swirling and photomicrographs were taken at 4Ox magnification under inverted microscope. Arrow indicated multi-lobed islets, which are formed by fusion of a few islets during culture period.

Figure 3 shows scanning electron microscopic pictures of rejuvenated islets.

Isolated islets were cultured according to rejuvenating method, and observed by scanning electron microscope. Islet has very smooth surfaces with well-defined cellular junctions and numerous microvilli (A). Cells in the innermost layer are well organized and show no sign of necrosis (B).

Figure 4 shows a transmission electron micrograph of rejuvenated islets.

Figure 5 shows intracellular insulin content and GSlS of FIs and RIs. The isolated islets were divided into multiple portions, and intracellular insulin content and GSIS were measured at indicated time points during rejuvenation procedure. In all experiments, TSA was used as HDACi except for valproic acid (VA). Suspension control, which cultured for the same period with regular media changes were used as control. Data are the mean ± SD of at least three independent experiments, and the statistical significance (p<0.05) between control and test groups of intracellular insulin content or insulin secretion between at 0 and 16.7 mM glucose was made by ANOVA or Student t- test, respectively.

Figure 6 shows gene expression pattern between FIs and RIs. The isolated islets were divided into two groups (FIs and RIs) and gene expression pattern was examined by semi-quantitative RT-PCR analyses. Essential genes for β-cell function (lnsulin-1 and -2 and GLUT2) or DNA repair (Ogg1 , APEX, and NtM) were significantly increased in Rl group compared with Fl group, whereas β-actin was similar between two groups (A). Gene expression pattern of class I and class Il HDACs was also measured (B). Figure 7 shows viability of FIs or RIs treated with H₂O₂ or cytokine mixtures.

Isolated islets were divided into two portion (Fl vs Rl group), and they were treated with 500 μM H₂O₂ for 30 min, or cytokines mixtures (100 ng/ml of rat IL-1 β, 50 ng/ml of TNF- α, and 100 unit/ml of IFN-γ).for 12 hrs. Viability was measured by MTT assay.

Figure 8 shows intracelluar insulin content and GSIS of FIs or RIs treated with H₂O₂ or cytokine mixtures. Isolated islets were divided into two portion (Fl vs Rl group), and they were treated with 500 μM H₂O₂ for 30 min, or cytokines mixtures (100 ng/ml of rat IL-1 β, 50 ng/ml of TNF-α, and 100 unit/ml of IFN-γ).for 12 hrs. Intracellular insulin content and GSIS from size-matched batch of islets (10 islets) were measured by 1-hr static incubation and ELISA. Figure 9 shows blood glucose levels after transplantation of FIs or RIs in islet xenotransplantation. All nude mice were rendered diabetic by STZ injection, and the indicated amounts of FIs or RIs were transplanted under kidney subcapsule at day 0. Blood glucose levels were measured every other day until 1 month and weekly thereafter. Recipients showing chronic hyperglycemia or unstable blood glucose pattern were marked with arrowhead.

Figure 10 shows blood glucose levels after transplantation of FIs or RIs in inbred rat islet transplantation. WK rats were rendered diabetic by STZ injection, and the indicated amounts of islets isolated from separate donor WK rat were transplanted into portal vein at day 0. Blood glucose levels were measured every other day until 3 months. Figure 11 shows blood glucose levels after transplantation of FIs or RIs in outbred rat islet transplantation. SD rats were rendered diabetic by STZ injection, and the indicated amounts of islets isolated from separate donor SD rat were transplanted into portal vein at day 0. Blood glucose levels were measured every other day until 2 months. Transient normoglycemia and time point, during which liver samples bearing islet graft were retrieved were indicated by circle and arrow, respectively.

Figure 12 shows histochemical insulin immunostaining of liver samples retrieved at 17 days after islet transplantation in outbred rat transplantation. Liver samples were obtained at 17 days after islet transplantation and were subjected to insulin immunostaining. Only islet vestiges with numerous immune cell infiltrations were observed in Fl group, whereas nearly normal islet architectures with strong insulin immunoreactivity were seen in Rl group.

Best Mode for Carrying Out The Invention

Practical and presently preferred embodiments of the present invention are illustrated as shown in the following Examples. However, it will be appreciated that those skilled in the art, on consideration of this disclosure, may make modifications and improvements within the spirit and scope of the present invention.

Animals and Reagents

Nude mice (Balb/c sic nu-nu), inbred Wistar-Kyoto (WK), and outbred Sprague- Dawley (SD) rats were purchased from Central Laboratory Animal Inc. (Seoul, Korea). STZ, trichostatin A (TSA), 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) and all other chemicals were obtained from Sigma-Aldrich Korea Co. (Seoul, Korea) unless otherwise indicated. Hank's balanced salt solution (HBSS), CMRL1066 media, fetal bovine serum (FBS), antibiotics (penicillin and streptomycin), and other cell culture wares were purchased from Invitrogen (Calsbad, USA). Recombinant rat interleukin 1β (IL-1β), tumor necrosis factor-α (TNF-α), and interferon^ (IFN-γ) were purchased from R&D systems, Inc. (Minneapolis, MN). Fibrin gel kit (Greenplast^®) was purchased from Green Cross Corp. (Kyunggi-do, Korea). All animal experiments were performed in accordance with approved protocols by the Korea University Institutional Animal Care and Experimentation Committee.

Example 1 : Islet Isolation

SD rats weighing 250-280 g were anesthetized with sodium pentobarbital (50 mg/kg, i.p.). After abdomen cavity was opened, common bile duct was cannulated with 10 ml syringe and 7 ml of collagenase solution (type Xl, 0.7 mg/ml and DNase I, 0.05 mg/ml in HBSS) was injected. Inflated pancreata were excised and incubated for 18 min at 37⁰C. The tissue was broken down by gentle shaking and the filtrates through 500 μm mesh were obtained. Islets were purified by Ficoll density gradient centrifugation using Ficoll solution with the density of 1.041g/ml, 1.075g/ml, and 1.085g/ml. For inbred rat islet transplantation, islets from WK rats weighing 200-250 g were obtained as above.

Example 2: Islet culture

The isolated islets were pre-incubated overnight in 10% FBS-supplemented CMRL 1640 medium (regular media) in bacterial dish to prevent attachment. These overnight incubated islets were considered as freshly isolated islets and we refer them to FIs throughout this specification. By contrast, rejuvenated islets (RIs) were obtained as described below. During the degranulation step, the islets were treated with the stimulatory concentration of glucose (25 mM) and depolarizing concentration of KCl (30 mM) for 48 hrs in suspension culture maintained in 10% fetal bovine serum (FBS)- supplemented CMRL1066 medium to promote intracellular insulin release. Then, during the chromatin remodeling step, the media was switched to 1 % FBS-supplemented CMRL 1640 media and the islets were treated with 100 nM trichostatin A (TSA) for 24 hrs. During the regranulation step, the islets were washed with regular media twice and then incubated in regular media, 10% FBS-supplemented CMRL1066 media, for further 48 hrs. To minimize islet batch variations, in all experiments, the isolated islets were divided into two portions (Fl group vs Rl group) and used for measurement of various parameters.

Example 3: Morphological changes of the isolated islets during the culture period

During the culture period, Islet morphology was examined by the inverted light microscope and photomicrographed by the digital camera (Oympus CK-2, Japan) under a 4Ox magnification (Fig. 2). Freshly isolated islets (FIs) appear bright brown, and shows various size distribution ranging from 50 to 400 μm in diameter with rather irregular surface. During the degranulating step, islets become pale yellowish with smooth surface. After chromatin remodeling step and regranulaing step, islets show brown color and high cell density with smooth and well-defined surface. By examining the diameter of each islet, the mean islet equivalent (IEQ) was calculated according to Ricordi method.

Example 4: Ultrastructural features of the islets

For scanning electron microscopy (Fig. 3), FIs or RIs were fixed in 1% paraformaldehyde-1 % glutaraldehyde in 0.1 M phosphate buffer, and post-fixed in 2%

OsO₄ in 0.1 M phosphate buffer. Samples were dehydrated, and dried using Hitachi

HCP-2 critical point dryer. They were then mounted and coated by RMC-Eiko Ion

Coater followed by observation. For transmission electron microscopy (Fig. 4), islets or tissue samples (kidney and liver bearing islet graft) were fixed in 1 % paraformaldehyde- 1% glutaraldehyde in 0.1 M phosphate buffer, and post-fixed in 2% OsO₄ in 0.1M phosphate buffer. They were dehydrated and embedded in Lowicryl resin. Then, the ultra-thin section of samples were stained by uranyl acetate and lead citrate and observed in JEOL electron microscope. Consistent with light microscope observations, surface of the cultured islets is very smooth and every single cell is well-defined between neighboring cells. Compact and viable cells are observed inside of the islets and numerous microvilli are seen on the surface. As seen in TEM, the typical insulin granules are evenly distributed throughout the whole cytoplasm and cellular organelles such as mitochondria, endoplasmic reticulum, and Golgi complex appear intact.

Example 5: Insulin content and glucose-stimulated insulin secretion (GSIS) of the islets

Isolated islets were either overnight incubated in regular media or cultured as above. Batch of islets with the similar size distribution ranging from 150-200 μm in diameter (10 islets) were dispensed in Eppendorf tube. GSIS was measured by 1-hr static incubation in Kreb's Ringer bicarbonate (KRB) buffer containing 0, 5.6, and 16.7 mM glucose sequentially. Finally, islets were sonicated in acidic alcohol and intracellular insulin was extracted overnight at 4^°C. Insulin was measured in duplicate by enzyme- linked immunosorbent assay (ELISA) according to the manufacturer's protocol

(Mercodia AB, Uppsala, Sweden) (Fig. 5). Intracellular insulin content and GSIS are increased by 1.9- and 1.7-fold, respectively in cultured islets compared with those of freshly isolated islets.

Example 6: Gene expression profiles of the islets

Total RNAs were isolated from FIs or RIs using Trizol reagent and cDNAs were synthesized using oligo d(T) and superscript Il reverse transcriptase (Invitrogen) according to manufacturer's protocols. Semi-quantitative RT-PCR was performed as described in 5 (Fig. 6). Important genes for maintaining β-cell function such as insulin and glucose transporter 2 (GLUT2) in cultured islets are increased up to 2.5-fold compared with those in freshly isolated islets. DNA repair enzymes (Ogg1 , Apexi , and Nth1) and cell cycle regulator (p16) are also increased in cultured islets. Expression levels of several other genes (β-actin, Smcx, MeCP2) are unchanged, suggesting the subset of genes, in particular, for β-cell survival and function may be activated by procedure of this invention.

Example 7: Resistance against oxidative stresses

Isolated islets were either incubated in regular media as control or cultured as above. Batch of islets (30 islets) were dispensed in new culture dish and treated with 500 μM H₂O₂ for 30 min or with cytokine mixtures (100 ng/ml of rat IL-1β, 50 ng/ml of TNF-α, and 100 unit/ml of IFN-γ) for 12 hrs. GSIS was measured as above. Viability and apoptosis were measured by MTT assay and ApopPercentage™ apoptosis assay (Biocolor Ltd., UK), respectively.

Example 8: Hyperglycemia correction by islet transplantation

Nude mice weighing 20—25 g were rendered diabetic by injecting STZ (200 mg/kg, i.p.) after 12 hr-fasting period. STZ was freshly made in 0.04M citric acid buffer (pH 4.0). For WK and SD rats, STZ (60 mg/kg, i.p.) was injected after 12 hr-fasting period. Diabetic induction was confirmed by monitoring tail blood glucose level >300 mg/dl for at least two consecutive days using automatic glucometer (CareSens II, I Sense Co., Seoul, Korea).

Diabetic nude mice were anesthetized with sodium pentobarbital (50 mg/kg, i.p.) and left kidney was exposed by local laparotomy. Varying amounts of islets (200, 150, 100, and 50 IEQ) were embedded in fibrin gels by mixing 0.75 μl of fibrinogen and 0.75 μl of thrombin solution with brief centrifugation, and transplanted under the kidney subcapsule. For rat islet transplantation, WK or SD rats were anesthetized with sodium pentobarbital (40 mg/kg, i.p.), and abdomen was opened by midline surgery. After the exposure of portal vein, the indicated amounts of islets in the figure legend resuspended in serum-free CMRL 1640 media containing 1 unit of heparin were slowly injected. Gelfoam was immediately put onto a puncture site to prevent bleeding. In our hands, survival rates of nude mice transplantation and rat transplantation were >95% and >90%, respectively. Abnormal blood glucose levels of diabetic nude mice are corrected by transplanted islets within 2-3 weeks and animals remain healthy up to further 1 month follow-up period without any sign of adverse effect.

Tablei . Transplanted islet mass in inbred WK rat transplantation

Group Islet number Total IEQ Body weight Transplanted islets IEQ/g

FI 434.20±67.20 559.04±68.64 210.00±13.70 2.67±0.32

RI 315.80±29.24 368.54±25.81 259.00±15.57 1.43±0.13

Isolated islets from each donor rat were counted and photomicrographed at 4Ox magnification under inverted microscope. Four areas were randomly chosen and the diameter of about 150 islets were counted and converted to IEQ. FIs were transplanted into portal vein after one day preculture of isolated islets, but RIs were transplanted after further 5 day-long rejuvenation procedure. Body weight of each recipient rat at transplantation time was measured.

Example 9: Histology and lmmunohistochemistry

After 2-month follow-up period, nude mice exhibiting normal glycemia were anesthetized with sodium pentobarbital (50 mg/kg, i.p.) and left kidney bearing transplanted islets was resected. Kidney was fixed in 10% neutral formalin and embedded in paraffin according to standard protocol. Kidney section (3 μm-thick) was deparaffinized and then immunostained using anti-insulin antibody (DAKO North America, Inc., Carpinteria, CA, USA) and peroxidase-conjugated secondary antibody with diamino benzimidine (DAB) as chromogen. Liver samples bearing islet graft from WK and SD rats were also processed and immunostained as above. Statistics

All data are expressed as means+standard deviation (SD). A difference was considered significant if the p value was less than 0.05, using Student's t-iesϊ or oneway analysis of the variance (ANOVA) when multiple comparisons were made between groups.

Those skilled in the art will appreciate that the conceptions and specific embodiments disclosed in the foregoing description may be readily utilized as a basis for modifying or designing other embodiments for carrying out the same purposes of the present invention. Those skilled in the art will also appreciate that such equivalent embodiments do not depart from the spirit and scope of the invention as set forth in the appended claims.

Claims

1. A method for rejuvenating isolated pancreatic islets for transplantation, comprising following steps: a) degranulating step, in which the isolated pancreatic islets are treated with insulin secretagogue ; b) chromatin remodeling step, in which the degranulated islets are treated with chromatin modifier; and c) regranulating step, in which the chromatin remodeled islets are treated with serum-supplemented culture medium.

2. The method according to claim 1 , in which, in the degranulating step, the insulin secretagogue is any one selected from a group consisting of glucose, KCI, arginine, 2 (alpha)-ketoisocaproate, and sulfonylureas.

3. The method according to claim 1 , in which, in the degranulating step, the isolated pancreatic islets are maintained for 12-72 hrs in serum-supplemented culture medium.

4. The method according to claim 1 , in which, in the chromatin remodeling step, the chromatin modifier is any one selected from a group consisting of histone deacetylase (HDAC) inhibitor, arginine methyltransferase inhibitor, lysine methyltransferase inhibitor, and chromatin remodeling factors.

5. The method according to claim 4, in which the histone deacetylase (HDAC) inhibitor is any one selected from a group consisting of trichostatin A, sodium butyrate, and SAHA (suberoylanilide hydroxamic acid).

6. The method according to claim 1 , in which, in the chromatin remodeling step, the degranulated islets are maintained for 12-72 hrs in serum-reduced culture medium.

7. The method according to claim 1 , in which, in the regranulating step, the chromatin remodeled islets are maintained for 12-72 hrs in serum-supplemented culture medium.

8. The method according to claim 1 , in which the rejuvenated pancreatic islets have increased insulin content, enhanced insulin secretion activity, higher viability, and enhanced engraft capacity upon transplantation, compared with non-rejuvenated isolated pancreatic islets.

9. A composition for transplantation into diabetic recipients, comprising the pancreatic islets rejuvenated according to any one claim of claim 1 to 8.

10. A method for treating type 1 diabetes in a patient in need thereof, comprising the step of administering to said patient an effective amount of the pancreatic islets rejuvenated according to any one claim, of claim 1 to 8.