WO2022149649A1 - Procédé de différenciation de cellules productrices d'insuline à l'aide d'atélocollagène ionisé, et pancréas artificiel fabriqué à l'aide de celui-ci - Google Patents

Procédé de différenciation de cellules productrices d'insuline à l'aide d'atélocollagène ionisé, et pancréas artificiel fabriqué à l'aide de celui-ci Download PDF

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WO2022149649A1
WO2022149649A1 PCT/KR2021/001527 KR2021001527W WO2022149649A1 WO 2022149649 A1 WO2022149649 A1 WO 2022149649A1 KR 2021001527 W KR2021001527 W KR 2021001527W WO 2022149649 A1 WO2022149649 A1 WO 2022149649A1
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insulin
cells
atelocollagen
ionized
medium
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김송철
심인경
이유나
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재단법인 아산사회복지재단
울산대학교 산학협력단
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    • C12M21/00—Bioreactors or fermenters specially adapted for specific uses
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    • C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
    • C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
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    • C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
    • C12N2506/45—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from artificially induced pluripotent stem cells
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    • C12N2533/00—Supports or coatings for cell culture, characterised by material
    • C12N2533/50—Proteins
    • C12N2533/54—Collagen; Gelatin

Definitions

  • the present invention relates to a method for differentiation of insulin-producing cells using ionized atelocollagen and an artificial pancreas manufactured using the same.
  • Diabetes mellitus is a disease characterized by chronic high blood sugar as the most important feature, and if prolonged, it causes complications such as delayed wound healing due to microvascular damage, neurological disease, renal failure, heart disease, retinal disease, etc., thereby increasing social and economic burden.
  • insulin enhancement therapy which repeatedly administers insufficient insulin in diabetic patients, has been used in clinical practice for a long time. The reality is that diabetes complications cannot be prevented.
  • Type 1 diabetes Diabetes is largely divided into two types (type 1 and type 2) depending on the cause.
  • type 1 diabetes diabetes occurs when one's own immune cells destroy insulin-producing cells.
  • type 2 diabetes organs/tissues/cells in the body become resistant to insulin due to various causes. As a result, diabetes symptoms showing high blood sugar and accompanying complications are induced.
  • Islet cell transplantation has been recognized as a successful strategy for the treatment of diabetes.
  • islet transplantation for the treatment of diabetes is limiting its use.
  • IPC insulin-producing cells
  • PSCs pluripotent stem cells
  • Recent studies have shown that PSCs can differentiate into IPCs in a manner similar to the differentiation stage during embryonic formation.
  • differentiated IPCs have many problems due to low insulin secretion, differentiated cell diversity, and teratoma-forming ability. Therefore, studies on methods for inducing differentiation into efficient insulin-producing cells are being actively conducted.
  • ECM extra-cellular matrix
  • collagen has been used as a very important biomaterial among extracellular matrix-related biomaterials.
  • Collagen is known to be distributed in almost all tissues in the body and accounts for about 1/3 of the proteins in the body. It is known as an essential protein for construction.
  • collagen in the extracellular matrix can change its intrinsic properties through various chemical treatments.
  • collagen is generally insoluble in neutral water, methanol, ethanol, succinic anhydride, acetic anhydride, etc.
  • the collagen transformed with There are many tissues that contain collagen, such as skin, ligaments, bones, blood vessels, amniotic membrane, pericardium, heart valve, placenta, and cornea, but the type of collagen is different for each tissue.
  • type 1 collagen is most widely used in tissue engineering because it is contained in a large amount in almost all tissues such as skin, ligaments, and bone. At both ends of the type 1 collagen molecule, there is a portion called telopeptide that does not form a helix, which is the main cause of the immune response. Collagen (atelocollagen) is used.
  • biomaterials using collagen still have limitations in using them directly in human tissues due to their low tensile strength and biodegradable properties.
  • the present invention relates to a method for differentiating insulin-producing cells using ionized atelocollagen and an artificial pancreas prepared using the same, and to human induced pluripotent stem cells (iPSCs) endoderm (definitive endoderm), pancreatic progenitor cells (pancreatic progenitor cell) , and a method of sequentially inducing differentiation into pancreatic endocrine cells, as a result of using hydrogel containing ionized atelocollagen, insulin producing cells with a more mature degree of differentiation compared to two-dimensional culture were produced.
  • iPSCs human induced pluripotent stem cells
  • endoderm definitive endoderm
  • pancreatic progenitor cells pancreatic progenitor cell
  • the present invention provides a method for inducing differentiation of insulin-producing cells using an ionized atelocollagen-containing hydrogel, comprising (1) human induced pluripotent stem cells (iPSCs) containing activin A, CHIR99021 and Y-27632.
  • iPSCs human induced pluripotent stem cells
  • step (1) Inducing differentiation into endoderm (definitive endoderm) by culturing in a medium; (2) The endoderm differentiation induced in step (1) is cultured in a medium containing B27-insulin, Dorsomorphin, Retinoic acid, SB431542, and SANT1 to pancreatic progenitor cells ) to induce differentiation; and (3) containing the ionized atelocollagen containing B27-insulin, dexamethasone, nicotinamide, forskolin and Exendin-4 in the pancreatic progenitor cells induced in step (2). Inducing differentiation into pancreatic endocrine cells by encapsulating in hydrogel and culturing; provides a method for inducing differentiation of insulin-producing cells, including the.
  • the present invention also provides insulin-producing cells prepared according to the method for inducing differentiation of insulin-producing cells.
  • the present invention provides an artificial pancreas comprising insulin-producing cells prepared according to the method for inducing differentiation of insulin-producing cells.
  • iPSCs human induced pluripotent stem cells
  • endoderm definitive endoderm
  • pancreatic progenitor cells pancreatic progenitor cells
  • pancreatic endocrine cells pancreatic endocrine cells
  • FIG. 3 is a result of observing the structure of the hydrogel containing ionized atelocollagen prepared according to the present invention with a scanning electron microscope.
  • FIG. 4 is a schematic diagram showing a method for differentiating insulin-producing cells using the hydrogel containing ionized atelocollagen prepared according to the present invention.
  • FIG. 5 is a microscopic observation of the cell morphology according to the step of differentiating insulin-producing cells using the hydrogel containing ionized atelocollagen prepared according to the present invention.
  • FIG. 6 is a result of confirming the expression level of a pancreatic-related gene in order to evaluate the degree of differentiation of insulin-producing cells using the hydrogel containing ionized atelocollagen prepared according to the present invention.
  • glucose secretion reactivity (glucose stimulated insulin secretion:GSIS) according to the glucose concentration in order to evaluate the degree of differentiation of insulin-producing cells using the hydrogel containing ionized atelocollagen prepared according to the present invention.
  • FIG. 9 is a result of observing insulin-producing cells using immunochemical staining to evaluate the degree of differentiation of insulin-producing cells using the hydrogel containing ionized atelocollagen prepared according to the present invention.
  • FIG. 10 is a tissue photograph 3 months after subcutaneous transplantation of insulin-producing cells encapsulated in an ionized atelocollagen-containing hydrogel prepared according to the present invention into a diabetic NSG mouse (NOD scid gamma mouse).
  • FIG. 11 is a tissue photograph 3 months after subcutaneous transplantation of insulin-producing cells encapsulated in BME (Basal Medium Eagle) hydrogel into diabetic NSG mice (NOD scid gamma mouse).
  • BME Basal Medium Eagle
  • the present invention provides a method for inducing differentiation of insulin-producing cells using an ionized atelocollagen-containing hydrogel, comprising (1) human induced pluripotent stem cells (iPSCs) containing activin A, CHIR99021 and Y-27632.
  • iPSCs human induced pluripotent stem cells
  • step (1) Inducing differentiation into endoderm (definitive endoderm) by culturing in a medium; (2) The endoderm differentiation induced in step (1) is cultured in a medium containing B27-insulin, Dorsomorphin, Retinoic acid, SB431542, and SANT1 to pancreatic progenitor cells ) to induce differentiation; and (3) containing the ionized atelocollagen containing B27-insulin, dexamethasone, nicotinamide, forskolin and Exendin-4 in the pancreatic progenitor cells induced in step (2). Inducing differentiation into pancreatic endocrine cells by encapsulating in hydrogel and culturing; provides a method for inducing differentiation of insulin-producing cells, including the.
  • the medium is DMEM (Dulbecco's Modified Eagle's Medium), MEM (Minimal Essential Medium), improved MEM (improved MEM), BME (Basal Medium Eagle), RPMI 1640 (Roswell Park Memorial Institute medium 1640), Advanced RPMI 1640 (Advanced) RPMI1640), F-10, F-12, DMEM-F12, ⁇ -MEM ( ⁇ -Minimal Essential Medium), G-MEM (Glasgow's Minimal Essential Medium) and IMDM (Iscove's Modified Dulbecco's Medium) any one selected from the group consisting of .
  • DMEM Disbecco's Modified Eagle's Medium
  • MEM Minimum Essential Medium
  • improved MEM improved MEM
  • BME Base Medium Eagle
  • RPMI 1640 Roswell Park Memorial Institute medium 1640
  • Advanced RPMI 1640 Advanced RPMI1640
  • step (1) human induced pluripotent stem cells (iPSCs) were cultured in a medium containing 10 to 400 ng/mL activin A, 1 to 5 ⁇ M CHIR99021, and 5 to 50 ⁇ M Y-27632.
  • iPSCs human induced pluripotent stem cells
  • step (3) (3-1) B27-insulin, dexamethasone, nicotinamide, forskolin in a concave micro-well of (3-1) differentiation-induced pancreatic progenitor cells and preparing cell spheroids of pancreatic progenitor cells by culturing them in a medium containing Exendin-4; and (3-2) containing ionized atelocollagen containing B27-insulin, dexamethasone, nicotinamide, and Exendin-4 in the cell spheroid prepared in step (3-1). It further includes; encapsulating in hydrogel and culturing.
  • the ionized atelocollagen-containing hydrogel contains cationized atelocollagen in an amount of 0.5 to 3% by weight and has a viscosity in the range of 1 Pa.s to 100 Pa.s at a shear rate of 1s -1 and , to form a nanofibrous network.
  • the present invention also provides insulin-producing cells prepared according to the method for inducing differentiation of insulin-producing cells.
  • the present invention provides an artificial pancreas comprising insulin-producing cells prepared according to the method for inducing differentiation of insulin-producing cells.
  • Example 1 Differentiation of insulin producing cells from induced pluripotent stem cells (iPSCs)
  • iPSCs Human induced pluripotent stem cells used in the present invention were provided by the Asan Research Center for Stem Cell Research, and iPSCs were 8TM containing essential 8TM supplements (Gibco, USA) and antibiotics (Gibco, USA). It was cultured in a culture dish coated with vitronectin using a basal medium (Gibco, USA). Inducible pluripotent stem cells were subcultured at 1:5 to 1:6 using StemPro Accutase medium (Gibco, USA), and cultured at 37°C and 5% CO 2 conditions.
  • induced pluripotent stem cells In order to differentiate induced pluripotent stem cells (iPSCs) into insulin-producing cells, induced pluripotent stem cells (iPSCs) are sequentially transformed into definitive endoderm, pancreatic progenitor cells, and pancreatic endocrine cells. to induce differentiation to prepare insulin-producing cells.
  • iPSCs induced pluripotent stem cells
  • RPMI Roswell Park Memorial Institute medium
  • FBS Fetal Bovine Serum, Invitrogen, USA
  • iPSCs induced pluripotent stem cells
  • activin A activin A, PeproTech, USA
  • 3 ⁇ M CHIR99021 Sigma-Aldrich, USA
  • MEM medium Improved MEM Zinc Option culture medium, Invitrogen, USA
  • B27 Invitrogen, USA
  • vitronectin vitronectin
  • endoderm definitive endoderm
  • pancreatic progenitor cells were in a concave micro-well using MEM medium containing 1% B27, 0.1 ⁇ M dexamethasone (SigmaAldrich, USA), 10 mM Spheroids containing 1000 cells treated with nicotinamide (Sigma-Aldrich, USA), 10 ⁇ M forskolin (Sigma-Aldrich, USA), and 10 nM Exendin-4 (Sigma-Aldrich, USA) (spheroid) was prepared in the form. Single cells of pancreatic progenitor cells or the prepared cell spheroids were transferred to hydrogel containing ionized atelocollagen and cultured for a long time under the same medium conditions as above to induce differentiation into insulin-producing cells. .
  • iPSCs induced pluripotent stem cells
  • Non-ionized atelocollagen was prepared through pretreatment of animal tissue, removal of telopeptide, and extraction of atelocollagen, which are well known in the art. It was prepared according to known information.
  • atelocollagen in order to prepare ionized atelocollagen, 1-5% by weight of atelocollagen is added to 70-90% ethanol (or methanol) and 0.5-1M acetic acid or 0.1-0.5M HCl is added. Put in, adjusted to pH 2-4, stirred and mixed at 4 °C for 4-10 days to prepare an atelocollagen dispersion.
  • the atelocollagen dispersion was adjusted to pH 7.4 with 0.1-0.5 M NaOH, and centrifuged to obtain a precipitate.
  • the obtained precipitate was diluted in purified water at a ratio of about 10 to 100 mL per 1 g, and then put into a dialysis membrane and dialyzed in purified water (dialysis buffer).
  • the purified water (dialysis buffer) was replaced, and after that, the purified water (dialysis buffer) was replaced 3 to 12 times every 3 to 5 hours.
  • the cationized atelocollagen precipitate dialyzed by the above process was freeze-dried at -70°C for 30 hours or more.
  • the dried cationic atelocollagen was dissolved in distilled water at 0.5 to 3%, and a cell culture solution was added to prepare a hydrogel.
  • the viscosity was measured, the shape of the hydrogel was observed with a scanning electron microscope, and the protein content was analyzed.
  • BME2 Cultrex Basement Membrane Extract
  • Viscosity was measured with Advanced Rheometric Expansion System (TA Instruments, USA), and as a result of the analysis, as shown in FIG. 1 , the hydrogel containing ionized atelocollagen at 0.5%, 1%, and 1.5% was a cancer cell-derived hydrogel. Similar to BME (Matrigel), it exhibited a viscosity in the range of 1 Pa.s to 100 Pa.s at a shear rate of 1s -1 , and a tendency to decrease as the shear rate increases.
  • the hydrogel containing ionized atelocollagen prepared according to the present invention has a viscosity similar to that of the bio-ink mixed with the cells used in the wet 3D cell printing method, and can be used for culturing various cells. It proves that various types of three-dimensional structures can be manufactured and used by a three-dimensional printing method, etc. because not only has a suitable viscosity, but also can be smoothly discharged using a syringe.
  • the modulus was rapidly phased and increased at 37 ° C. It was confirmed that it was changed to the form of a gel.
  • the above results prove that the hydrogel containing ionized atelocollagen prepared according to the present invention can be cultured by maintaining it at a constant temperature at the cell culture temperature because it has suitable temperature sensitivity.
  • iPSCs induced pluripotent stem cells
  • endoderm definitive endoderm
  • pancreatic progenitor cells pancreatic progenitor cells
  • pancreatic endocrine cells pancreatic endocrine cells
  • insulin Production cells were prepared.
  • FIG. 4 at the stage of differentiation into pancreatic endocrine cells expressing PDX1, a major transcription factor of the pancreas, the hydrogel containing ionized atelocollagen prepared according to the present invention or other hydrogel After that, the cells were cultured for a long time to induce differentiation into insulin-producing cells.
  • Pancreatic endocrine cells cultured in a two-dimensional culture dish were isolated from single cells using enzymes such as Trypsin-EDTA, Accutase, and TrypLE, and cultured in a concave micro-well to 1000 cells. It was prepared in the form of a spheroid containing Pancreatic endocrine cells (pancreatic endocrine cells) single cells or spheroids were encapsulated in a hydrogel containing ionized atelocollagen and cultured.
  • enzymes such as Trypsin-EDTA, Accutase, and TrypLE
  • IPCs Insulin producing cells differentiated from pancreatic endocrine cells were prepared in a two-dimensional culture dish, concave micro-well, spheroid shape confirmation, and hydrogel encapsulation. cultured, and the morphology of each cell was observed.
  • single cells or spheroid forms were respectively encapsulated in ionized atelocollagen-containing hydrogel and BME hydrogel, and the culture morphology was observed under a microscope on the 1st and 10th days of culture. observed. As shown in FIG. 5 , it was confirmed that the cells encapsulated in the hydrogel maintained their shape well and differentiated.
  • IPCs insulin producing cells
  • pancreatic-related gene expression insulin secretion
  • C-peptide secretion insulin secretion reactivity
  • GSIS insulin secretion reactivity
  • the insulin gene is a gene related to pancreatic constituent cells such as pancreatic endocrine cells (Insulin, glucagon, somatostatin), exocrine cells (amylase), and duct cells (CK19) after mRNA is extracted from differentiated cells on the 0, 5, and 10 days. , the expression of pancreatic-related transcription factors (PDX1, NGN3, NKX2.2, NKX6.1) was confirmed. Total RNA was isolated using triazole (TRIzol reagent; Invitrogen, USA) according to the product manual, and cDNA was synthesized using Reverse transcription master premix (ELPiS, Korea).
  • pancreatic constituent cells such as pancreatic endocrine cells (Insulin, glucagon, somatostatin), exocrine cells (amylase), and duct cells (CK19) after mRNA is extracted from differentiated cells on the 0, 5, and 10 days. , the expression of pancreatic-related transcription factors (PDX1, NGN3, NKX2.2
  • PCR Real-time PCR was performed using Cyber Green Supermix (SsoAdvancedTM Universal SYBR Green Supermix; BIO-RAD, USA), and each mRNA was amplified using the primer sets in Table 2. PCR was performed at 95°C for 15 seconds. , 30 seconds at 58 °C, and 30 seconds at 72 °C as 1 cycle, 45 cycles were repeated.
  • the secretion levels of insulin and C-peptide were measured using each ELISA (Enzyme-Linked Immunosorbent Assay) by collecting cell cultures. As shown in FIG. 7 , the secretion levels of insulin and C-peptide were found to be evidenced when encapsulated in hydrogel compared to two-dimensional culture, and, in particular, when cultured in hydrogel containing atelocollagen in the form of spheroids. , the secretion levels of insulin and C-peptide were the highest.
  • the insulin secretion reactivity (GSIS) according to the glucose concentration was evaluated.
  • Insulin secretion reactivity according to glucose concentration (GSIS) The amount of insulin secreted for one hour at a low glucose concentration (2mM) or a high glucose concentration (20mM) was measured.
  • the insulin secretion level was measured by insulin ELISA (Enzyme-Linked Immunosorbent Assay), and the reactivity (glucose stimulation index) was calculated as the amount of insulin secreted from low glucose / amount of insulin secreted from high glucose. .
  • the insulin secretion reactivity according to the glucose concentration increased compared to the two-dimensional culture. In particular, when cultured in a hydrogel containing atelocollagen in the form of single cells and spheroids, the BME culture In comparison, it was found that the insulin secretion reactivity was excellent.
  • intracellularly differentiated insulin-producing cells were identified through immunochemical staining.
  • the cells were fixed with formalin.
  • the hydrogel was cut to a thickness of 4 ⁇ m by making a paraffin block to prepare a tissue slide.
  • the slides were deparaffinized and dehydrated, followed by antigen retrieval. After blocking for 1 hour, Guinea pig anti-insulin (1:200; Abcam) and mouse anti-glucagon (1:1000; Abcam, MA, USA) rabbit anti-PDX1 (1:200; Abcam, MA, USA) at 4°C overnight.
  • pancreatic endocrine cell spheroids were introduced into hydrogel containing ionized atelocollagen, and the resulting cells were subcutaneously transplanted into diabetic NSG mice (NOD scid gamma mice).
  • pancreatic endocrine cell spheroids were introduced and transplanted into BME hydrogel, which is a cancer cell-derived extracellular matrix.
  • the mouse model was NSG with regulated immune response, and the implantation was subcutaneously in the form of hydrogel.

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Abstract

La présente invention concerne un procédé de différenciation de cellules productrices d'insuline à l'aide d'atélocollagène ionisé, et un pancréas artificiel fabriqué à l'aide de celui-ci. Dans la mesure où il a été constaté que l'utilisation d'un hydrogel contenant de l'atélocollagène ionisé dans un procédé d'induction de différenciations consécutives de cellules souches pluripotentes induites humaines (iPSC) en endoderme définitif, en cellules progénitrices pancréatiques et en cellules endocrines pancréatiques, aboutit à la production de cellules productrices d'insuline avec un niveau de différenciation plus mature par rapport à une culture bidimensionnelle, la présente invention fournit un procédé d'induction de la différenciation de cellules productrices d'insuline en utilisant un hydrogel contenant de l'atélocollagène ionisé, et des cellules productrices d'insuline et un pancréas artificiel ainsi produits.
PCT/KR2021/001527 2021-01-05 2021-02-05 Procédé de différenciation de cellules productrices d'insuline à l'aide d'atélocollagène ionisé, et pancréas artificiel fabriqué à l'aide de celui-ci WO2022149649A1 (fr)

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KR1020210000801A KR20220098914A (ko) 2021-01-05 2021-01-05 이온화 아텔로콜라겐을 이용한 인슐린 생성세포의 분화 방법 및 이를 이용하여 제조된 인공 췌장

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