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WO2008153299A1 - Procédé pour rajeunir des îlots pancréatiques isolés en vue d'une greffe - Google Patents

Procédé pour rajeunir des îlots pancréatiques isolés en vue d'une greffe Download PDF

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WO2008153299A1
WO2008153299A1 PCT/KR2008/003228 KR2008003228W WO2008153299A1 WO 2008153299 A1 WO2008153299 A1 WO 2008153299A1 KR 2008003228 W KR2008003228 W KR 2008003228W WO 2008153299 A1 WO2008153299 A1 WO 2008153299A1
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islets
transplantation
insulin
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pancreatic islets
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Jun-Seop Shin
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0676—Pancreatic cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/16—Amides, e.g. hydroxamic acids
    • A61K31/165—Amides, e.g. hydroxamic acids having aromatic rings, e.g. colchicine, atenolol, progabide
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A61K31/19—Carboxylic acids, e.g. valproic acid
    • A61K31/195—Carboxylic acids, e.g. valproic acid having an amino group
    • A61K31/197—Carboxylic acids, e.g. valproic acid having an amino group the amino and the carboxyl groups being attached to the same acyclic carbon chain, e.g. gamma-aminobutyric acid [GABA], beta-alanine, epsilon-aminocaproic acid or pantothenic acid
    • A61K31/198—Alpha-amino acids, e.g. alanine or edetic acid [EDTA]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/64—Sulfonylureas, e.g. glibenclamide, tolbutamide, chlorpropamide
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    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
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    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/05—Inorganic components
    • C12N2500/10—Metals; Metal chelators
    • C12N2500/12—Light metals, i.e. alkali, alkaline earth, Be, Al, Mg
    • C—CHEMISTRY; METALLURGY
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    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2500/00—Specific components of cell culture medium
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    • C—CHEMISTRY; METALLURGY
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/065—Modulators of histone acetylation

Definitions

  • 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.
  • Type 1 diabetes consists of about 5% of all diabetic patients and is characterized by ⁇ -cells destruction by autoimmune attacks.
  • Type 2 diabetes is common form of diabetes and the peripheral insulin resistance and gradual dysfunction of ⁇ -cells are main features of this disease. Although two forms of diabetes are different regarding the etiology, ⁇ -cells loss whether it is absolute or relative is common pathophysiological outcome, and thus the replacement or regeneration of ⁇ -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)).
  • donor shortage remains a serious limitation of this therapeutic regimen to broad usage.
  • 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.
  • 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.
  • 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.
  • 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.
  • the isolated pancreatic islets may be obtained from rodents, such as mouse and rats, or higher mammals including human beings, preferably cadaver donors.
  • 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.
  • 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.
  • insulin secretagogue was used for the degranulating step.
  • the insulin secretagogue may be any substance which can cause the intracellular insulin of the pancreatic ⁇ -cells to be secreted.
  • the insulin secretagogue includes, but not limited to, glucose, KCI, arginine, 2 (alpha)-ketoisocaproate, sulfonylureas, and so on.
  • the insulin secretagogue may be treated in a stimulatory or depolarizing concentration, for example 15-50 mM of glucose or KCI.
  • a stimulatory or depolarizing concentration for example 15-50 mM of glucose or KCI.
  • the isolated pancreatic islets may be maintained or cultured for 12- 72 hrs in serum-supplemented (7-15%) culture medium.
  • chromatin remodeling step means to relaxe(s)£ ⁇ ⁇ , fc the compact chromatin into more open structure, which promotes transcription by facilitating accessibility.
  • chromatin modifier was used for the chromatin remodeling step.
  • the chromatin modifier may be any substance which can relax the compact chromatin into more open structure.
  • the chromatin modifier includes, but not limited to, histone deacetylase (HDAC) inhibitor, arginine methyltransferase inhibitor, lysine methyltransferase inhibitor, chromatin remodeling factors, and so on.
  • HDAC histone deacetylase
  • arginine methyltransferase inhibitor arginine methyltransferase inhibitor
  • lysine methyltransferase inhibitor chromatin remodeling factors, and so on.
  • the histone deacetylase (HDAC) inhibitor may be any substance which can inhibit the activity of histone deacetylase (HDAC).
  • HDAC histone deacetylase
  • the histone deacetylase (HDAC) inhibitor includes, but not limited to, trichostatin A, sodium butyrate, SAHA (suberoylanilide hydroxamic acid), and so on.
  • the chromatin modifier may be treated in a chromatin relaxing concentration, for example 1 nM-10 mM of histone deacetylase (HDAC) inhibitor.
  • HDAC histone deacetylase
  • the degranulated islets may be maintained or cultured for 12-72 hrs in serum-reduced (0.5-3%) culture medium.
  • 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.
  • 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.
  • 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.
  • 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.
  • a composition for transplantation into diabetic recipients comprising the pancreatic islets rejuvenated according to the method of the present invention.
  • 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.
  • the rejuvenated pancreatic islets are transplanted to subjects with type 1 diabetes or a related condition.
  • the transplantation process may further include appropriate treatment to prevent recurrent autoimmunity and/or immune-mediated rejection.
  • an immunosuppressive agent can be treated by administering to a patient in need thereof.
  • 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.
  • immunosuppressive agents which suppress cell-mediated immune responses against cells identified by the immune system as non-self.
  • 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.
  • 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).
  • 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.
  • 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.
  • FBS fetal bovine serum
  • 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 ⁇ -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.
  • HDAC histone deacetylase
  • 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.
  • 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.
  • 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.
  • GSIS glucose-stimulated insulin secretion
  • Islet ⁇ -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 ⁇ -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.
  • HDACi histone deacetylase inhibitor
  • 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).
  • Fig. 3A As seen by scanning electron microscope, their surfaces are very smooth and numerous microvilli are observed (Fig. 3A).
  • 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
  • Rejuvenated islets exhibit increased expression of important penes in ⁇ -cell survival and function
  • 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).
  • Rejuvenated islets are more resistant against oxidative stresses than freshly isolated islets
  • Islet ⁇ -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 H 2 O 2 or cytokine mixtures composed of IL-1 ⁇ , TNF- ⁇ , and IFN- ⁇ , 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
  • islet xenotransplantation model by transplanting islets from SD rats into STZ-induced diabetic nude mice.
  • 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.
  • islet mass 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.
  • mice 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.
  • 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
  • Rejuvenated islets exhibit less immunogenicity than freshly isolated islets in outbred rat transplantation model
  • 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
  • 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.
  • 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.
  • FIG. 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.
  • TSA was used as HDACi except for valproic acid (VA).
  • VA valproic acid
  • 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 ⁇ -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 ⁇ -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 H 2 O 2 or cytokine mixtures.
  • Isolated islets were divided into two portion (Fl vs Rl group), and they were treated with 500 ⁇ M H 2 O 2 for 30 min, or cytokines mixtures (100 ng/ml of rat IL-1 ⁇ , 50 ng/ml of TNF- ⁇ , and 100 unit/ml of IFN- ⁇ ).for 12 hrs. Viability was measured by MTT assay.
  • Figure 8 shows intracelluar insulin content and GSIS of FIs or RIs treated with H 2 O 2 or cytokine mixtures. Isolated islets were divided into two portion (Fl vs Rl group), and they were treated with 500 ⁇ M H 2 O 2 for 30 min, or cytokines mixtures (100 ng/ml of rat IL-1 ⁇ , 50 ng/ml of TNF- ⁇ , and 100 unit/ml of IFN- ⁇ ).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.
  • mice 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.
  • FIG 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.
  • 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.
  • TSA trichostatin A
  • MTT 5-diphenyltetrazolium bromide
  • HBSS Hank's balanced salt solution
  • FBS fetal bovine serum
  • antibiotics penicillin and streptomycin
  • other cell culture wares were purchased from Invitrogen (Calsbad, USA).
  • Recombinant rat interleukin 1 ⁇ (IL-1 ⁇ ), tumor necrosis factor- ⁇ (TNF- ⁇ ), and interferon ⁇ (IFN- ⁇ ) 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.
  • 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 37 0 C. The tissue was broken down by gentle shaking and the filtrates through 500 ⁇ m mesh were obtained.
  • collagenase solution type Xl, 0.7 mg/ml and DNase I, 0.05 mg/ml in HBSS
  • 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.
  • islets from WK rats weighing 200-250 g were obtained as above.
  • 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.
  • 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.
  • FBS fetal bovine serum
  • 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.
  • TSA trichostatin A
  • the islets were washed with regular media twice and then incubated in regular media, 10% FBS-supplemented CMRL1066 media, for further 48 hrs.
  • 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
  • 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 ⁇ m in diameter with rather irregular surface.
  • islets become pale yellowish with smooth surface.
  • islets show brown color and high cell density with smooth and well-defined surface.
  • IEQ mean islet equivalent
  • FIs or RIs were fixed in 1% paraformaldehyde-1 % glutaraldehyde in 0.1 M phosphate buffer, and post-fixed in 2%
  • islets or tissue samples were fixed in 1 % paraformaldehyde- 1% glutaraldehyde in 0.1 M phosphate buffer, and post-fixed in 2% OsO 4 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 ⁇ m 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
  • 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.
  • 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 ⁇ -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 ( ⁇ -actin, Smcx, MeCP2) are unchanged, suggesting the subset of genes, in particular, for ⁇ -cell survival and function may be activated by procedure of this invention.
  • Important genes for maintaining ⁇ -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.
  • 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 ⁇ M H 2 O 2 for 30 min or with cytokine mixtures (100 ng/ml of rat IL-1 ⁇ , 50 ng/ml of TNF- ⁇ , and 100 unit/ml of IFN- ⁇ ) for 12 hrs. GSIS was measured as above. Viability and apoptosis were measured by MTT assay and ApopPercentageTM 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).
  • STZ 60 mg/kg, i.p.
  • 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 ⁇ l of fibrinogen and 0.75 ⁇ l 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.
  • 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.
  • kidneys 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 ⁇ m-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.
  • DAB diamino benzimidine

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Abstract

La présente invention concerne un nouveau procédé pour rajeunir des îlots pancréatiques isolés en vue d'une greffe, plus particulièrement un procédé pour améliorer ou augmenter la teneur en insuline, la sécrétion d'insuline stimulée par le glucose, la viabilité, et la capacité de prise de greffe après une greffe d'îlots pancréatiques isolés chez des sujets diabétiques. Selon la présente invention, les îlots rajeunis présentent une teneur en insuline intracellulaire et une sécrétion d'insuline stimulée par le glucose (GSIS) 1,7 et 1,9 fois supérieure, respectivement, par rapport à des îlots fraîchement isolés. De plus, le rajeunissement a permis d'augmenter significativement la quantité de nombreux gènes importants pour la survie et la fonction des cellules β comme l'insuline, GLUT2, Bcl-2, et les enzymes de réparation de l'ADN. La capacité augmentée des îlots confère à ces derniers une plus grande résistance au stress oxydatif, une immunogénicité réduite, et une augmentation au moins 2 fois supérieure de l'efficacité de prise de greffe dans un modèle de xénogreffe et d'allogreffe d'îlots. En conclusion, selon la présente invention, la greffe d'îlots avec un nombre d'îlots réduit peut guérir le diabète de type 1.
PCT/KR2008/003228 2007-06-10 2008-06-10 Procédé pour rajeunir des îlots pancréatiques isolés en vue d'une greffe Ceased WO2008153299A1 (fr)

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EP2896688A1 (fr) * 2014-01-20 2015-07-22 Centre National de la Recherche Scientifique (CNRS) Procédé de production de cellules pancréatiques bêta à partir de cellules progénitrices par l'utilisation de peroxyde d'hydrogène

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2896688A1 (fr) * 2014-01-20 2015-07-22 Centre National de la Recherche Scientifique (CNRS) Procédé de production de cellules pancréatiques bêta à partir de cellules progénitrices par l'utilisation de peroxyde d'hydrogène

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