WO2022149649A1 - Method of differentiating insulin-producing cells using ionized atelocollagen, and artificial pancreas manufactured using same - Google Patents
Method of differentiating insulin-producing cells using ionized atelocollagen, and artificial pancreas manufactured using same Download PDFInfo
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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
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- atelocollagen
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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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- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
Abstract
The present invention relates to a method of differentiating insulin-producing cells by using ionized atelocollagen, and to an artificial pancreas manufactured using same. As it was found that the use of a hydrogel containing ionized atelocollagen in a method of inducing sequential differentiations of human induced pluripotent stem cells (iPSC) to definitive endoderm, pancreatic progenitor cells, and pancreatic endocrine cells, results in the production of insulin-producing cells with a more mature differentiation level compared to a two-dimensional culture, the present invention provides a method of inducing differentiation of insulin-producing cells by using a hydrogel containing ionized atelocollagen, and insulin-producing cells and an artificial pancreas produced thereby.
Description
본 발명은 이온화 아텔로콜라겐을 이용한 인슐린 생성세포의 분화 방법 및 이를 이용하여 제조된 인공 췌장에 관한 것이다.The present invention relates to a method for differentiation of insulin-producing cells using ionized atelocollagen and an artificial pancreas manufactured using the same.
당뇨병은 만성적인 고혈당을 가장 중요한 특징으로 하는 질환으로 장기화되면 미세혈관 손상에 기인한 창상치유 지연, 신경질환, 신부전증, 심장질환, 망막질환 등의 합병증을 유발하여 사회적 경제적 부담을 가중시키는 질환이다. 현재까지 당뇨병을 완치시킬 수 있는 치료법은 없으며 당뇨환자에서 부족한 인슐린을 반복적으로 투여하는 인슐린 강화요법이 오랜 기간 임상에서 활용되어 왔지만 하루에도 수 차례 반복해서 투여해야 하는 불편함이 존재하며, 그럼에도 불구하고 당뇨 합병증을 막을 수 없는 것이 현실이다.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. To date, there is no treatment that can cure diabetes, and 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.
당뇨병은 원인에 따라 크게 두 가지 형태(제1형, 제2형)로 구분된다. 제1형 당뇨병의 경우 자신의 면역세포가 인슐린 생성세포를 파괴하여 발생하는 당뇨병으로 인슐린 생성세포가 부족하여 혈당을 조절하지 못하기 때문에 발생한다. 제2형 당뇨병의 경우 다양한 원인에 의하여 신체 내 장기/조직/세포 등이 인슐린에 대한 저항성을 가지게 되는 것에 기인하는 것으로, 고혈당에 기인한 인슐린 생성세포의 기능저하 및 소실로 혈당의 조절이 원활하지 않게 되며 결과적으로 고혈당을 보이는 당뇨병 증상과 함께 그에 따른 합병증이 유발된다.Diabetes is largely divided into two types (type 1 and type 2) depending on the cause. In the case of type 1 diabetes, diabetes occurs when one's own immune cells destroy insulin-producing cells. In the case of 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.
췌도세포 이식은 당뇨병 치료를 위한 성공적인 전략으로 인식되어왔다. 그러나 췌장 공유자의 부족으로 당뇨병 치료를 위한 췌도이식은 그 이용에 한계를 드러내고 있다. 이러한 점을 극복하기 위해서, 인슐린 생성세포(insulin-producing cells, IPC)로 분화 가능한 후보자를 찾기 위한 연구가 진행 중이다. 만능줄기세포(pluripotent stem cells, PSC)는 자기증식능과 다양한 세포로의 분화능을 가지고 있기 때문에 IPC로의 분화 가능한 세포치료제가 될 수 있다. 최근 연구에 따르면, PSC는 배아 형성 시기의 분화 단계와 유사한 방법으로 IPC로 분화가 가능함을 보여주었다. 그러나, 분화된 IPC는 낮은 인슐린 분비, 분화된 세포의 다양성, 그리고 테라토마 형성능으로 인해 많은 문제점을 가지고 있다. 따라서 효율적인 인슐린 생성세포로 분화를 유도하기 위한 방법에 대한 연구가 활발히 이루어지고 있다.Islet cell transplantation has been recognized as a successful strategy for the treatment of diabetes. However, due to the lack of shared pancreas, islet transplantation for the treatment of diabetes is limiting its use. In order to overcome this point, research is underway to find candidates capable of differentiating into insulin-producing cells (IPC). Because pluripotent stem cells (PSCs) have the ability to self-renew and differentiate into various cells, they can be a cell therapy capable of differentiating into IPCs. Recent studies have shown that PSCs can differentiate into IPCs in a manner similar to the differentiation stage during embryonic formation. However, 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.
한편, 췌도세포의 이식 후에 췌도세포 기능의 일부 또는 완전한 손실이 일어나게 되는데, 이와 같은 췌도세포 기능의 손실은 췌도세포를 췌장에서 분리 및 정제할 때 필연적으로 발생하는 세포외기질(Extra-cellular matrix, ECM)의 파괴가 가장 큰 원인으로 알려져 있다. 특히, 세포외기질은 세포의 부착과 이동에 필수적인 역할은 물론, 세포자극을 위한 신호전달에도 중요한 역할을 한다고 알려져 있으며, 이로 인해 췌도세포를 비롯한 많은 종류의 세포의 부착성과 생존율 그리고 증식을 크게 향상시킨다는 많은 보고가 있다. 따라서, 췌도세포 배양 및 이식과 인공췌도 제조방법과 관련한 기술분야에서 세포외기질은 큰 주목을 받고 있다.On the other hand, some or complete loss of islet cell function occurs after transplantation of islet cells. This loss of islet cell function is an extra-cellular matrix (extra-cellular matrix, ECM) is known to be the biggest cause. In particular, it is known that the extracellular matrix plays an essential role in cell adhesion and movement as well as in signal transduction for cell stimulation, thereby greatly improving the adhesion, viability, and proliferation of many types of cells including islet cells. There are many reports that Therefore, the extracellular matrix is receiving great attention in the technical field related to islet cell culture and transplantation and artificial pancreatic islet production methods.
세포외기질을 생체재료로 이용할 때 세포외기질 관련 생체재료들 중에서도 콜라겐(collagen)은 매우 중요한 생체 소재로서 사용되어 왔음을 알 수 있다. 콜라겐은 생체 내 거의 모든 조직에 분포하여 체내에 존재하는 단백질 중 1/3 가량을 차지하는 것으로 알려져 있으며, 세포의 지지 및 증식을 위한 구조체로서 세포와 결합하여 장기와 조직의 형태를 유지함으로써 생체 구조를 구축하는데 필수 불가결한 단백질로 알려져 있다. When using the extracellular matrix as a biomaterial, it can be seen that 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.
특히, 세포외기질의 콜라겐은 여러 가지 화학적 처리를 통하여 콜라겐 고유의 성질을 변화시키는 것이 가능한데, 예를 들어 일반적으로 콜라겐은 중성의 물에서는 잘 녹지 않는 반면에, 메탄올, 에탄올, 무수숙신산, 무수아세트산 등으로 변형시킨 콜라겐은 양이온화 혹은 음이온화되어 중성의 물에서도 녹게 된다. 생체에는 피부, 인대, 골, 혈관, 양막, 심막, 심장판막, 태반, 각막 등 콜라겐이 함유되어 있는 조직이 많지만 콜라겐의 종류는 각 조직마다 다르다. 특히 제1형 콜라겐은 피부, 인대, 골 등 거의 모든 조직에 다량 포함되어 있기 때문에 조직공학에서 가장 널리 이용되고 있다. 제1형 콜라겐 분자의 양쪽 말단에는 나선을 형성하지 않은 텔로펩타이드(telopeptide)라고 불리는 부분이 있는데, 이는 면역반응을 일으키는 주된 원인이므로, 의약품이나 화장품 등의 원료로 사용 시에는 이 부분을 제거한 아텔로콜라겐(atelocollagen)을 이용한다. 콜라겐 사용에 의한 창상 치료 효과가 우수함에도 불구하고, 콜라겐을 이용한 생체재료 물질은 아직도 낮은 인장강도와 생분해적인 특성 때문에 사람 조직에서 바로 사용하기에 한계를 가지고 있다.In particular, collagen in the extracellular matrix can change its intrinsic properties through various chemical treatments. For example, while 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. In particular, 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. In spite of the excellent wound healing effect by using collagen, biomaterials using collagen still have limitations in using them directly in human tissues due to their low tensile strength and biodegradable properties.
따라서 최근에는 이러한 콜라겐의 특성을 활용하여 치료용 세포를 배양하는 방법 및 인공조직 제조 방법에 대한 연구가 활발하게 진행되고 있다.Therefore, recently, studies on a method of culturing cells for treatment and a method of manufacturing artificial tissues by utilizing the characteristics of collagen are being actively conducted.
[선행기술문헌][Prior art literature]
[특허문헌][Patent Literature]
한국공개특허공보 제10-2003-0033638호 (2003.05.01)Korean Patent Publication No. 10-2003-0033638 (2003.05.01)
본 발명은 이온화 아텔로콜라겐을 이용한 인슐린 생성세포의 분화 방법 및 이를 이용하여 제조된 인공 췌장에 관한 것으로, 인간 유도만능줄기세포(iPSC)를 내배엽(definitive endoderm), 췌장 전구세포(pancreatic progenitor cell), 및 췌장 내분비 세포(pancreatic endocrine cell)로 순차적으로 분화를 유도하는 방법에서 이온화 아텔로콜라겐 함유 수화젤을 이용한 결과 2차원 배양에 비해 분화 정도가 더 성숙된 인슐린 생성세포 (Insulin producing cell)가 제조되는 것을 확인하고, in vivo에 이식한 경우 BME에 비해 인슐린 생성세포를 성숙시키는 효과가 우수함을 확인함으로써, 이온화 아텔로콜라겐 함유 수화젤을 이용하여 인슐린 생성세포의 분화 유도 방법 및 이를 통해 제조된 인슐린 생성세포 및 인공 췌장을 제공하는 것이다.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 A method for inducing differentiation of insulin-producing cells using ionized atelocollagen-containing hydrogel and insulin produced therefrom To provide a production cell and an artificial pancreas.
본 발명은 이온화 아텔로콜라겐 함유 수화젤을 이용하여 인슐린 생성세포의 분화 유도 방법으로서, (1) 인간 유도만능줄기세포(iPSC)를 액티빈 A(activin A), CHIR99021 및 Y-27632를 포함하는 배지에 배양하여 내배엽(definitive endoderm)으로 분화를 유도하는 단계; (2) 상기 (1) 단계에서 분화가 유도된 내배엽을 B27-insulin, 돌소몰핀(Dorsomorphin), 레티논산(Retinoic acid), SB431542, 및 SANT1을 포함하는 배지에 배양하여 췌장 전구세포(pancreatic progenitor cell)로 분화를 유도하는 단계; 및 (3) 상기 (2) 단계에서 분화가 유도된 췌장 전구세포를 B27-insulin, 덱사메타손(dexamethasone), 니코틴아미드(nicotinamide), 포스콜린(forskolin) 및 Exendin-4를 포함하는 이온화 아텔로콜라겐 함유 수화젤에 봉입하여 배양하여 췌장 내분비 세포(pancreatic endocrine cell)로 분화를 유도하는 단계;를 포함하는 인슐린 생성세포의 분화 유도 방법을 제공한다.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. 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.
또한, 본 발명은 상기 인슐린 생성세포의 분화 유도 방법에 따라 제조된 인슐린 생성세포를 제공한다.The present invention also provides insulin-producing cells prepared according to the method for inducing differentiation of insulin-producing cells.
또한, 본 발명은 상기 인슐린 생성세포의 분화 유도 방법에 따라 제조된 인슐린 생성세포를 포함하는 인공 췌장을 제공한다.In addition, the present invention provides an artificial pancreas comprising insulin-producing cells prepared according to the method for inducing differentiation of insulin-producing cells.
본 발명에 따르면, 인간 유도만능줄기세포(iPSC)를 내배엽(definitive endoderm), 췌장 전구세포(pancreatic progenitor cell), 및 췌장 내분비 세포(pancreatic endocrine cell)로 순차적으로 분화를 유도하는 방법에서 이온화 아텔로콜라겐 함유 수화젤을 이용한 결과 2차원 배양에 비해 분화 정도가 더 성숙된 인슐린 생성세포 (Insulin producing cell)가 제조되는 것을 확인하고, in vivo에 이식한 경우 BME에 비해 인슐린 생성세포를 성숙시키는 효과가 우수함을 확인함으로써, 이온화 아텔로콜라겐 함유 수화젤을 이용하여 인슐린 생성세포의 분화 유도 방법 및 이를 통해 제조된 인슐린 생성세포 및 인공 췌장을 제공을 제공할 수 있다.According to the present invention, in a method for sequentially inducing differentiation of human induced pluripotent stem cells (iPSCs) into endoderm (definitive endoderm), pancreatic progenitor cells, and pancreatic endocrine cells (pancreatic endocrine cells), ionized atelo As a result of using the collagen-containing hydrogel, it was confirmed that insulin producing cells with a more mature degree of differentiation compared to the two-dimensional culture were produced. By confirming the superiority, it is possible to provide a method for inducing differentiation of insulin-producing cells using an ionized atelocollagen-containing hydrogel, and an insulin-producing cell and artificial pancreas prepared therefor.
도 1은 본 발명에 따라 제조된 이온화 아텔로콜라겐 함유 수화젤의 점도를 분석한 결과이다.1 is a result of analyzing the viscosity of the hydrogel containing ionized atelocollagen prepared according to the present invention.
도 2는 본 발명에 따라 제조된 이온화 아텔로콜라겐 함유 수화젤의 점탄성을 분석한 결과이다.2 is a result of analyzing the viscoelasticity of the hydrogel containing ionized atelocollagen prepared according to the present invention.
도 3은 본 발명에 따라 제조된 이온화 아텔로콜라겐 함유 수화젤의 구조를 주사전자현미경으로 관찰한 결과이다.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.
도 4는 본 발명에 따라 제조된 이온화 아텔로콜라겐 함유 수화젤을 이용하여 인슐린 생성세포를 분화하는 방법을 나타내는 계략도이다.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.
도 5는 본 발명에 따라 제조된 이온화 아텔로콜라겐 함유 수화젤을 이용하여 인슐린 생성세포를 분화하는 단계에 따른 세포의 형태를 현미경으로 관찰한 결과이다.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.
도 6은 본 발명에 따라 제조된 이온화 아텔로콜라겐 함유 수화젤을 이용하여 인슐린 생성세포의 분화 정도를 평가하기 위해 췌장관련 유전자의 발현 수준을 확인한 결과이다.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.
도 7은 본 발명에 따라 제조된 이온화 아텔로콜라겐 함유 수화젤을 이용하여 인슐린 생성세포의 분화 정도를 평가하기 위해 인슐린 및 C-peptide의 분비 수준을 확인한 결과이다.7 is a result of confirming the secretion levels of insulin and C-peptide in order to evaluate the degree of differentiation of insulin-producing cells using the hydrogel containing ionized atelocollagen prepared according to the present invention.
도 8은 본 발명에 따라 제조된 이온화 아텔로콜라겐 함유 수화젤을 이용하여 인슐린 생성세포의 분화 정도를 평가하기 위해 글루코즈 농도에 따른 인슐린 분비 반응성 (glucose stimulated insulin secretion:GSIS)을 확인한 결과이다.8 is a result of confirming the insulin 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.
도 9는 본 발명에 따라 제조된 이온화 아텔로콜라겐 함유 수화젤을 이용하여 인슐린 생성세포의 분화 정도를 평가하기 위해 면역화학적 염색을 이용하여 인슐린 생성세포를 관찰한 결과이다.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.
도 10은 본 발명에 따라 제조된 이온화 아텔로콜라겐 함유 수화젤에 봉입된 인슐린 생성세포를 당뇨병 NSG 마우스(NOD scid gamma mouse)의 피하에 이식하고 3개월 후의 조직 사진이다. 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).
도 11은 BME(Basal Medium Eagle) 수화젤에 봉입된 인슐린 생성세포를 당뇨병 NSG 마우스(NOD scid gamma mouse)의 피하에 이식하고 3개월 후의 조직 사진이다.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).
본 명세서에서 사용되는 용어는 본 발명에서의 기능을 고려하면서 가능한 현재 널리 사용되는 일반적인 용어들을 선택하였으나, 이는 당 분야에 종사하는 기술자의 의도 또는 판례, 새로운 기술의 출현 등에 따라 달라질 수 있다. 또한, 특정한 경우는 출원인이 임의로 선정한 용어도 있으며, 이 경우 해당되는 발명의 설명 부분에서 상세히 그 의미를 기재할 것이다. 따라서 본 발명에서 사용되는 용어는 단순한 용어의 명칭이 아닌, 그 용어가 가지는 의미와 본 발명의 전반에 걸친 내용을 토대로 정의되어야 한다.The terms used in this specification have been selected as currently widely used general terms as possible while considering the functions in the present invention, which may vary depending on the intention or precedent of a person skilled in the art, the emergence of new technology, and the like. In addition, in a specific case, there is a term arbitrarily selected by the applicant, and in this case, the meaning will be described in detail in the description of the corresponding invention. Therefore, the term used in the present invention should be defined based on the meaning of the term and the overall content of the present invention, rather than the name of a simple term.
다르게 정의되지 않는 한, 기술적이거나 과학적인 용어를 포함해서 여기서 사용되는 모든 용어들은 본 발명이 속하는 기술 분야에서 통상의 지식을 가진 자에 의해 일반적으로 이해되는 것과 동일한 의미를 가지고 있다. 일반적으로 사용되는 사전에 정의되어 있는 것과 같은 용어들은 관련 기술의 문맥상 가지는 의미와 일치하는 의미를 가지는 것으로 해석되어야 하며, 본 출원에서 명백하게 정의하지 않는 한, 이상적이거나 과도하게 형식적인 의미로 해석되지 않는다.Unless defined otherwise, all terms used herein, including technical or scientific terms, have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Terms such as those defined in a commonly used dictionary should be interpreted as having a meaning consistent with the meaning in the context of the related art, and should not be interpreted in an ideal or excessively formal meaning unless explicitly defined in the present application. does not
수치 범위는 상기 범위에 정의된 수치를 포함한다. 본 명세서에 걸쳐 주어진 모든 최대의 수치 제한은 낮은 수치 제한이 명확히 쓰여져 있는 것처럼 모든 더 낮은 수치 제한을 포함한다. 본 명세서에 걸쳐 주어진 모든 최소의 수치 제한은 더 높은 수치 제한이 명확히 쓰여져 있는 것처럼 모든 더 높은 수치 제한을 포함한다. 본 명세서에 걸쳐 주어진 모든 수치 제한은 더 좁은 수치 제한이 명확히 쓰여져 있는 것처럼, 더 넓은 수치 범위 내의 더 좋은 모든 수치 범위를 포함할 것이다.Numerical ranges are inclusive of the values defined in that range. Every maximum numerical limitation given throughout this specification includes all lower numerical limitations as if the lower numerical limitation were expressly written. Every minimum numerical limitation given throughout this specification includes all higher numerical limitations as if the higher numerical limitation were expressly written. All numerical limitations given throughout this specification will include all numerical ranges that are better within the broader numerical limits, as if the narrower numerical limitations were expressly written.
이하, 본 발명을 보다 상세하게 설명한다.Hereinafter, the present invention will be described in more detail.
본 발명은 이온화 아텔로콜라겐 함유 수화젤을 이용하여 인슐린 생성세포의 분화 유도 방법으로서, (1) 인간 유도만능줄기세포(iPSC)를 액티빈 A(activin A), CHIR99021 및 Y-27632를 포함하는 배지에 배양하여 내배엽(definitive endoderm)으로 분화를 유도하는 단계; (2) 상기 (1) 단계에서 분화가 유도된 내배엽을 B27-insulin, 돌소몰핀(Dorsomorphin), 레티논산(Retinoic acid), SB431542, 및 SANT1을 포함하는 배지에 배양하여 췌장 전구세포(pancreatic progenitor cell)로 분화를 유도하는 단계; 및 (3) 상기 (2) 단계에서 분화가 유도된 췌장 전구세포를 B27-insulin, 덱사메타손(dexamethasone), 니코틴아미드(nicotinamide), 포스콜린(forskolin) 및 Exendin-4를 포함하는 이온화 아텔로콜라겐 함유 수화젤에 봉입하여 배양하여 췌장 내분비 세포(pancreatic endocrine cell)로 분화를 유도하는 단계;를 포함하는 인슐린 생성세포의 분화 유도 방법을 제공한다.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. 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.
상기 배지는 DMEM(Dulbecco's Modified Eagle's Medium), MEM(Minimal Essential Medium), 임프루브드 MEM(improved MEM), BME(Basal Medium Eagle), RPMI 1640(Roswell Park Memorial Institute medium 1640), 어드밴스드 RPMI 1640(Advanced RPMI1640), F-10, F-12, DMEM-F12, α -MEM(α -Minimal Essential Medium), G-MEM(Glasgow's Minimal Essential Medium) 및 IMDM(Iscove's Modified Dulbecco's Medium)으로 구성된 군으로부터 선택된 어느 하나이다.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, α-MEM (α-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 .
구체적으로 상기 (1) 단계는 인간 유도만능줄기세포(iPSC)를 10 ~ 400 ng/mL 액티빈 A(activin A), 1 ~ 5 μM CHIR99021, 및 5 ~ 50 μ M Y-27632이 포함된 배지에서 배양하여 내배엽으로 분화를 유도하고, 상기 (2) 단계는 내배엽을 1 ~ 5% B27 , 1 ~ 10 μM 돌소몰핀(Dorsomorphin), 1 ~ 5 μM 레티논산(Retinoic acid), 2 ~ 20 μM SB431542 , 및 0.1 ~ 1 μM SANT1을 포함하는 배지에 배양하여 췌장 전구세포로 분화를 유도하고, 상기 (3) 단계는 췌장 전구세포를 1 ~ 5% B27 , 0.01 ~ 2 μM 덱사메타손(dexamethasone), 2 ~ 10 mM 니코틴아미드(nicotinamide), 1 ~ 20 μ M 포스콜린(forskolin) 및 1 ~ 500 nM Exendin-4를 포함하는 이온화 아텔로콜라겐 함유 수화젤에 봉입하여 배양하여 췌장 내분비 세포로 분화를 유도한다.Specifically, in step (1), human induced pluripotent stem cells (iPSCs) were cultured in a medium containing 10 to 400 ng/mL activin A, 1 to 5 μM CHIR99021, and 5 to 50 μM Y-27632. Induction of differentiation into endoderm by culturing in (2), 1 ~ 5% B27, 1 ~ 10 μM dorsomorphin, 1 ~ 5 μM retinoic acid, 2 ~ 20 μM SB431542 , and culturing in a medium containing 0.1 to 1 μM SANT1 to induce differentiation into pancreatic progenitor cells, and in step (3), 1 to 5% B27, 0.01 to 2 μM dexamethasone, 2 to Induce differentiation into pancreatic endocrine cells by encapsulating in a hydrogel containing ionized atelocollagen containing 10 mM nicotinamide, 1 to 20 μM forskolin, and 1 to 500 nM Exendin-4.
상기 (3) 단계는, (3-1) 분화가 유도된 췌장 전구세포를 오목한 마이크로웰(concave micro-well)에서 B27-insulin, 덱사메타손(dexamethasone), 니코틴아미드(nicotinamide), 포스콜린(forskolin) 및 Exendin-4를 포함하는 배지에 배양하여 췌장 전구세포(pancreatic progenitor cell)의 세포 구상체(spheroid)를 제조하는 단계; 및 (3-2) 상기 (3-1) 단계에서 제조된 세포 구상체(spheroid)를 B27-insulin, 덱사메타손(dexamethasone), 니코틴아미드(nicotinamide), 및 Exendin-4를 포함하는 이온화 아텔로콜라겐 함유 수화젤에 봉입하여 배양하는 단계;를 추가로 포함한다.In the 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.
상기 이온화 아텔로콜라겐 함유 수화젤은 양이온화 아텔로콜라겐을 0.5~3% 중량%로 포함하며, 전단율(shear rate) 1s-1에서 1 Pa.s 내지 100 Pa.s의 범위의 점도를 갖고, 나노섬유 네트워크(nanofibrous network)를 형성한다.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.
또한, 본 발명은 상기 인슐린 생성세포의 분화 유도 방법에 따라 제조된 인슐린 생성세포를 제공한다.The present invention also provides insulin-producing cells prepared according to the method for inducing differentiation of insulin-producing cells.
또한, 본 발명은 상기 인슐린 생성세포의 분화 유도 방법에 따라 제조된 인슐린 생성세포를 포함하는 인공 췌장을 제공한다.In addition, the present invention provides an artificial pancreas comprising insulin-producing cells prepared according to the method for inducing differentiation of insulin-producing cells.
이하, 본 발명의 이해를 돕기 위하여 실시예를 들어 상세하게 설명하기로 한다. 다만 하기의 실시예는 본 발명의 내용을 예시하는 것일 뿐 본 발명의 범위가 하기 실시예에 한정되는 것은 아니다. 본 발명의 실시예는 당업계에서 평균적인 지식을 가진 자에게 본 발명을 보다 완전하게 설명하기 위해 제공되는 것이다.Hereinafter, to help the understanding of the present invention, examples will be described in detail. However, the following examples are merely illustrative of the content of the present invention, and the scope of the present invention is not limited to the following examples. The embodiments of the present invention are provided to more completely explain the present invention to those of ordinary skill in the art.
실시예 1. 유도만능줄기세포 (iPSCs)로부터 인슐린 생성세포 분화Example 1. Differentiation of insulin producing cells from induced pluripotent stem cells (iPSCs)
본 발명에서 사용한 인간 유도만능줄기세포(induced pluripotent stem cells: iPSC)는 아산 연구소의 줄기세포 연구센터에서 제공받았으며, iPSC는 필수 8TM 보충제(Gibco, 미국) 및 항생제(Gibco, 미국)가 포함된 8TM 기본 배지(Gibco, 미국)를 이용하여 비트로넥틴(vitronectin)으로 코팅된 배양 접시(culture dish)에서 배양하였다. 유도만능 줄기세포는 StemPro Accutase 배지(Gibco, 미국)를 이용하여 1 : 5 내지 1 : 6으로 계대배양 하였으며, 37℃ 5% CO2 조건에서 배양하였다.Human induced pluripotent stem cells (iPSCs) 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.
유도만능줄기세포(iPSC)를 인슐린 생성세포로 분화하기 위해, 유도만능줄기세포(iPSC)를 내배엽(definitive endoderm), 췌장 전구세포(pancreatic progenitor cell), 및 췌장 내분비 세포(pancreatic endocrine cell)로 순차적으로 분화를 유도하여 인슐린 생성세포를 제조하였다. 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.
구체적으로 비트로넥틴(vitronectin)으로 코팅된 배양 접시(culture dish)에서 2% FBS(Fetal Bovine Serum, Invitrogen, 미국)를 포함하는 RPMI(Roswell Park Memorial Institute medium) 배지를 이용하여 유도만능줄기세포(iPSC)를 배양하였으며, 1일 차에 100 ng/ml 액티빈 A(activin A, PeproTech, 미국), 3 μM CHIR99021(Sigma-Aldrich, 미국), 및 10 μM Y-27632(Sigma-Aldrich, 미국)를 처리하고, PBS로 세척한 다음 새로운 배지로 교체한 후, 100 ng/ml 액티빈 A(activin A, PeproTech, 미국), 및 3 μM CHIR99021(Sigma-Aldrich, 미국)를 2일 동안 처리하여 내배엽(definitive endoderm)으로 분화를 유도하였다.Specifically, using RPMI (Roswell Park Memorial Institute medium) medium containing 2% FBS (Fetal Bovine Serum, Invitrogen, USA) in a culture dish coated with vitronectin, induced pluripotent stem cells (iPSCs) ), and on day 1 100 ng/ml activin A (activin A, PeproTech, USA), 3 μM CHIR99021 (Sigma-Aldrich, USA), and 10 μM Y-27632 (Sigma-Aldrich, USA) were administered. After treatment, washing with PBS, and replacing with fresh medium, 100 ng/ml activin A (activin A, PeproTech, USA), and 3 μM CHIR99021 (Sigma-Aldrich, USA) were treated for 2 days for endoderm ( induced differentiation into definitive endoderm).
이 후, 비트로넥틴(vitronectin)으로 코팅된 배양 접시(culture dish)에서 1% B27(Invitrogen, 미국)를 포함하는 MEM 배지(Improved MEM Zinc Option culture medium, Invitrogen, 미국)를 이용하여 내배엽(definitive endoderm)으로 분화된 세포를 배양하였으며, 1 μM 돌소몰핀(Dorsomorphin; Sigma-Aldrich, 미국), 2 μM 레티논산(Retinoic acid, Tocris Bioscience, 영국), 10 μM SB431542(Tocris Bioscience, 영국), 및 0.25 μM SANT1(Sigma-Aldrich, 미국)을 7일 동안 처리하여 췌장 전구세포(pancreatic progenitor cell)로 분화를 유도하였다.Thereafter, using MEM medium (Improved MEM Zinc Option culture medium, Invitrogen, USA) containing 1% B27 (Invitrogen, USA) in a culture dish coated with vitronectin (vitronectin) endoderm (definitive endoderm) Cells differentiated with ) were cultured, 1 μM Dorsomorphin (Sigma-Aldrich, USA), 2 μM retinoic acid (Retinoic acid, Tocris Bioscience, UK), 10 μM SB431542 (Tocris Bioscience, UK), and 0.25 μM SANT1 (Sigma-Aldrich, USA) was treated for 7 days to induce differentiation into pancreatic progenitor cells.
이 후, 췌장 전구세포(pancreatic progenitor cell)로 분화된 세포를 오목한 마이크로웰(concave micro-well)에서 1% B27을 포함하는 MEM 배지를 이용하여 0.1 μM 덱사메타손(dexamethasone; SigmaAldrich, 미국), 10 mM 니코틴아미드(nicotinamide; Sigma-Aldrich, 미국), 10 μM 포스콜린(forskolin; Sigma-Aldrich, 미국), 및 10 nM Exendin-4(Sigma-Aldrich, 미국)를 처리하여 1000개의 세포를 포함하는 구상체 (spheroid) 형태로 제조하였다. 췌장 전구세포(pancreatic progenitor cell) 단일 세포 또는 상기 제조된 세포 구상체(spheroid)를 이온화 아텔로콜라겐 함유 수화젤(Hydrogel)로 옮겨서 상기와 동일한 배지 조건으로 장기간 배양하여 인슐린 생성세포로 분화를 유도하였다.Thereafter, the cells differentiated into pancreatic progenitor cells were in a concave micro-well using MEM medium containing 1% B27, 0.1 μM dexamethasone (SigmaAldrich, USA), 10 mM Spheroids containing 1000 cells treated with nicotinamide (Sigma-Aldrich, USA), 10 μM 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. .
상기 서술된 유도만능줄기세포(iPSC)를 인슐린 생성세포로 분화하는 방법은 하기 표 1 및 도 4와 같이 요약될 수 있으며, 인슐린 생성세포로 분화하는 과정에서 필요에 따라 KGF (Keratinocyte growth factor), LDN193189 (TGFß/Smad inhibitor), PDBu (Phorbol 12, 13-dibutyrate), T3 (3,5,3'-triiodothyronine), ALK5i (ALK5 inhibitor), Heparin Betacellulin, XXi (Y-secretase), Ascorbic acid 등을 추가적으로 사용하였다.The method for differentiating the above-described induced pluripotent stem cells (iPSCs) into insulin-producing cells can be summarized as shown in Table 1 and FIG. 4 below, and if necessary in the process of differentiation into insulin-producing cells, KGF (Keratinocyte growth factor), LDN193189 (TGFß/Smad inhibitor), PDBu (Phorbol 12, 13-dibutyrate), T3 (3,5,3'-triiodothyronine), ALK5i (ALK5 inhibitor), Heparin Betacellulin, XXi (Y-secretase), Ascorbic acid, etc. additionally used.
실시예 2. 이온화 아텔로콜라겐을 포함하는 수화젤 제조Example 2. Preparation of hydrogel containing ionized atelocollagen
이온화되지 않은 아텔로콜라겐은 당업계에 널리 알려진 동물조직의 전처리 과정, 텔로펩타이드 제거 및 아텔로콜라겐의 추출 과정을 통하여 제조하였으며, 이온화 아텔로콜라겐은 대한민국 공개특허공보 제10-2013-0101204호에 공지된 내용에 따라 제조하였다.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.
구체적으로, 이온화 아텔로콜라겐을 제조하기 위해, 상기의 아텔로콜라겐을 70~90% 에탄올(또는 메탄올)에 1~5 중량%로 넣고 0.5~1M 초산(acetic acid) 또는 0.1~0.5M HCl을 넣어 pH 2~4로 맞추고 4℃에서 4~10일 동안 교반 혼합하여 아텔로콜라겐 분산액을 제조하였다. 상기 아텔로콜라겐 분산액을 0.1~0.5M NaOH으로 pH 7.4로 맞추고, 원심분리하여 침전물을 수득하였다. 상기 수득된 침전물의 1g당 10~100mL 정도의 비율로 정제수에 희석한 후, 투석 멤브레인(dialysis membrane)에 넣어 정제수 (dialysis buffer) 내에서 투석(dialysis)하였다. 16~24시간 동안 교반한 후 정제수 (dialysis buffer)를 교체하고, 그 후에는 3~5시간 마다 정제수(dialysis buffer)를 3~12번 교체하였다. 상기의 과정으로 투석된 양이온화 아텔로콜라겐 침전물을 -70℃에서 30시간 이상 동결 건조하였다. 건조된 양이온화 아텔로콜라겐을 0.5~3%로 증류수에 녹이고, 세포 배양액을 추가하여 수화젤(Hydrogel)로 제조하였다.Specifically, 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 ℃ 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). After stirring for 16 to 24 hours, 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.
본 발명에 따라 제조된 이온화 아텔로콜라겐을 포함하는 수화젤의 물리적 특성을 평가하기 위해, 점도를 측정하고, 주사전자현미경으로 수화젤의 형태를 관찰하고, 단백질의 함량을 분석하였다. 또한, 세포 배양의 성능을 비교하기 위해 기존의 세포 3차원 배양에 많이 사용되고 있는 Cultrex Basement Membrane Extract, Type 2 (BME2)와 비교하였다.In order to evaluate the physical properties of the hydrogel containing ionized atelocollagen prepared according to the present invention, the viscosity was measured, the shape of the hydrogel was observed with a scanning electron microscope, and the protein content was analyzed. In addition, in order to compare the performance of cell culture, it was compared with Cultrex Basement Membrane Extract, Type 2 (BME2), which is widely used for conventional 3D cell culture.
점도는 Advanced Rheometric Expansion System (TA Instruments, USA)로 측정하였으며, 분석 결과, 도 1에 나타난 바와 같이, 이온화 아텔로콜라겐을 0.5%, 1%, 및 1.5% 로 포함하는 수화젤은 암세포 유래 수화젤인 BME(Matrigel)과 유사하게 전단율(shear rate) 1s-1에서 1 Pa.s 내지 100 Pa.s의 범위의 점도를 나타냈으며, 전단율(shear rate)이 증가할수록 점도가 감소하는 경향을 나타냈다. 상기 결과는 본 발명에 따라 제조된 이온화 아텔로콜라겐을 포함하는 수화젤은 습식 3D 세포 프린팅 방법에서 사용되는 세포와 혼합된 바이오 잉크와 유사한 점도를 갖으며, 다양한 세포를 배양하는 데에 사용될 수 있을 정도로 적합한 점도를 가질 뿐만 아니라, 주사기를 이용하여 원활하게 토출하는 것이 가능하기 때문에 3차원 프린팅 방법 등으로 다양한 형태의 3차원 구조체를 제조하여 사용할 수 있음을 입증한다.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. showed The above results show that 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.
또한, 이온화 아텔로콜라겐을 포함하는 수화젤의 점탄성을 측정한 결과, 도 2에 나타난 바와 같이 37℃에서 모듈러스(Modulus)가 급격하게 상전되어 증가하여 젤(gel)의 형태로 변하는 것을 확인하였으며, 상기의 결과는 본 발명에 따라 제조된 이온화 아텔로콜라겐을 포함하는 수화젤은 적합한 온도 감응성을 갖기 때문에 세포 배양 온도에서 일정한 온도로 유지하여 배양하는 것이 가능하다는 것을 입증한다.In addition, as a result of measuring the viscoelasticity of the hydrogel containing the ionized atelocollagen, as shown in FIG. 2, 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.
또한, 이온화 아텔로콜라겐을 포함하는 수화젤의 구조를 주사전자현미경으로 관찰한 결과, 도 3에 나타난 바와 같이 BME(Matrigel)와 유사한 나노섬유 네트워크(nanofibrous network)를 형성하여 수화젤 내외로 영양분 공급 및 노폐물 배출이 원활한 형태를 나타내며, 콜라겐의 단백질 성분으로 인하여 수화젤 구조체의 형태가 안정적으로 유지되는 것으로 관찰되었다.In addition, as a result of observing the structure of the hydrogel containing ionized atelocollagen with a scanning electron microscope, as shown in FIG. 3 , a nanofibrous network similar to BME (Matrigel) was formed to supply nutrients to and from the hydrogel. And it was observed that the shape of the hydrogel structure was stably maintained due to the protein component of collagen.
실시예 3. 분화된 인슐린 생성세포의 평가Example 3. Evaluation of differentiated insulin-producing cells
상기 실시예 1에 기술된 바와 같이, 유도만능줄기세포(iPSC)를 내배엽(definitive endoderm), 췌장 전구세포(pancreatic progenitor cell), 및 췌장 내분비 세포(pancreatic endocrine cell)로 순차적으로 분화를 유도하여 인슐린 생성세포를 제조하였다. 도 4에 나타난 바와 같이, 췌장의 주요 전사인자인 PDX1을 발현하는 췌장 내분비 세포(pancreatic endocrine cell)로 분화되는 단계에서 본 발명에 따라 제조된 이온화 아텔로콜라겐 함유 수화젤 또는 다른 수화젤에 봉입한 후, 장기간 배양하여 인슐린 생성세포로 분화를 유도하였다. 2차원 배양 접시에서 배양된 췌장 내분비 세포(pancreatic endocrine cell)는 Trypsin-EDTA, Accutase, TrypLE 등의 효소를 이용하여 단일 세포를 분리하였으며, 오목한 마이크로웰(concave micro-well)에 배양하여 1000개의 세포를 포함하는 구상체 (spheroid) 형태로 제조되었다. 췌장 내분비 세포(pancreatic endocrine cell) 단일 세포 또는 구상체 (spheroid)를 이온화 아텔로콜라겐을 포함하는 수화젤에 봉입하여 배양하였다. As described in Example 1 above, by sequentially inducing differentiation of induced pluripotent stem cells (iPSCs) into endoderm (definitive endoderm), pancreatic progenitor cells, and pancreatic endocrine cells (pancreatic endocrine cells), insulin Production cells were prepared. As shown in 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.
췌장 내분비 세포(pancreatic endocrine cell)에서 분화된 인슐린 생성세포 (Insulin producing cell: IPCs)를 2차원 배양 접시, 오목한 마이크로웰(concave micro-well), 구상체 (spheroid) 형태 확인 및 수화젤 봉입 단계로 배양하였고, 각 세포의 형태를 관찰하였다. 수화젤 봉입 단계에서 배양 형태를 비교하기 위해, 이온화 아텔로콜라겐 함유 수화젤 및 BME 수화젤에 단일 세포 또는 구상체 (spheroid) 형태를 각각 봉입하고, 배양 1일차 및 10일차에 배양 형태를 현미경으로 관찰하였다. 도 5에 나타난 바와 같이, 수화젤 봉입된 세포가 형태를 잘 유지하며 분화되는 것을 확인하였다.Insulin producing cells (IPCs) 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. In order to compare the culture morphology in the hydrogel encapsulation step, 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.
또한, 인슐린 생성세포 (Insulin producing cell: IPCs)의 분화 정도를 비교하기 위해, 췌장관련 유전자 발현, 인슐린 분비, C-peptide 분비, 글루코즈 농도에 따른 인슐린 분비 반응성 (glucose stimulated insulin secretion:GSIS)를 확인하였다. 실험군은 이온화 아텔로콜라겐 함유 수화젤에 단일세포로 봉입한 IPCs (Collagen) 및 구상체로 봉입한 IPC spheroid (Collagen)를 사용하였으며, 대조군으로 2차원 배양접시에서 분화한 IPCs (2D), BME에 단일세포로 봉입한 IPCs (BME), 및 BME에 구상체로 봉입한 IPC spheroid (BME)를 사용하였다. In addition, to compare the degree of differentiation of insulin producing cells (IPCs), pancreatic-related gene expression, insulin secretion, C-peptide secretion, and insulin secretion reactivity (glucose stimulated insulin secretion: GSIS) according to glucose concentration were confirmed. did. The experimental group used IPCs (Collagen) encapsulated as single cells and IPC spheroids (Collagen) encapsulated as spheroids in hydrogel containing ionized atelocollagen. Cell-encapsulated IPCs (BME) and IPC spheroids (BME) encapsulated in BME were used.
인슐린 유전자는 분화된 0일차, 5일차, 및 10일차 세포로부터 mRNA를 추출한 후, 췌장내분비세포 (Insulin, glucagon, somatostatin), 외분비세포 (amylase), 관세포 (CK19) 등의 췌장 구성세포관련 유전자, 췌장관련 전사인자 (PDX1, NGN3, NKX2.2, NKX6.1)등의 발현을 확인하였다. 총 RNA는 트리아졸(TRIzol reagent ; Invitrogen, 미국)을 이용하여 제품 매뉴얼에 따라 분리하였으며, 역전사중합효소 마스터 프리믹스(Reverse transcription master premix(Reverse transcription master premix; ELPiS, 한국)를 이용하여 cDNA를 합성하였다. 실시간 PCR은 사이버 그린 슈퍼믹스(SsoAdvancedTM Universal SYBR Green Supermix; BIO-RAD, 미국)를 사용하여 수행하였으며, 하기 표 2의 프라이머 세트를 이용하여 각각의 mRNA를 증폭하였다. PCR은 95℃에서 15초, 58℃에서 30초, 72℃에서 30초를 1 사이클로 하여 45사이클을 반복 수행하였다.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). Real-time PCR was performed using Cyber Green Supermix (SsoAdvanced™ 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.
| GeneGene | Sequence (5'→3')Sequence (5'→3') | Product size (bp)Product size (bp) | SEQ No.SEQ No. | ||
| AmylaseAmylase | ForwardForward | GGTTCAGGTCTCTCCACCAAGGTTCAGGTCTCTCCACCAA | 214214 | 1One | |
| ReverseReverse | TCCTGCACTCACAGCGTTACTCCTGCACTCACAGCGTTAC | 22 | |||
| CK19CK19 | ForwardForward | CAGACGCCGGAGAGAAATGAGCAGACGCCGGAGAGAAATGAG | 171171 | 33 | |
| ReverseReverse | CACTTCTTGAGGTTGACAGGTCCACTTCTTGAGGTTGACAGGTC | 44 | |||
| GAPDHGAPDH | ForwardForward | GAAGGTGAAGGTCGGAGTGAAGGTGAAGGTCGGAGT | 226226 | 55 | |
| ReverseReverse | GAAGATGGTGATGGGATTTCGAAGATGGTGATGGGATTTC | 66 | |||
| GlucagonGlucagon | ForwardForward | CCCAAGATTTTGTGCAGTGGTTCCCAAGATTTTGTGCAGTGGTT | 221221 | 77 | |
| ReverseReverse | GCGGCCAAGTTCTTCAACAATGCGGCCAAGTTCTTCAACAAT | 88 | |||
| InsulinInsulin | ForwardForward | GCAGCCTTTGTGAACCAACACGCAGCCTTTGTGAACCAACAC | 6767 | 99 | |
|
Reverse | CCCCGCACACTAGGTAGAGACCCCGCACACTAGGTAGAGA | 1010 | |||
| NKX2.2NKX2.2 | ForwardForward | CGGCGAGTGCTTTTCTCCAACGGCGAGTGCTTTTCTCCAA | 165165 | 1111 | |
| ReverseReverse | GCGCTTCATCTTGTAGCGGGCGCTTCATCTTGTAGCGG | 1212 | |||
| NGN3NGN3 | ForwardForward | GAAAGGACCTGTCTGTCGCTGAAAGGACCTGTCTGTCGCT | 124124 | 1313 | |
| ReverseReverse | AGGGAGAAGCAGAAGGAACAAGGGAGAAGCAGAAGGAACA | 1414 | |||
| NKX6.1NKX6.1 | ForwardForward | CACACGAGACCCACTTTTTCCACACGAGACCCACTTTTTC | 7676 | 1515 | |
| ReverseReverse | CCGCCAAGTATTTTGTTTCTCCGCCAAGTATTTTGTTTCT | 1616 | |||
| PDX1PDX1 | ForwardForward | GCATCCCAGGTCTGTCTTCTGCATCCCAGGTCTGTCTTCT | 140140 | 1717 | |
| ReverseReverse | CACTGCCAGAAAGGTTTGAACACTGCCAGAAAGGTTTGAA | 1818 | |||
| SomatostatinSomatostatin | ForwardForward | CTGTCTGAACCCAACCAGACCTGTCTGAACCCAACCAGAC | 9090 | 1919 | |
|
Reverse | CAGCTCAAGCCTCATTTCATCAGCTCAAGCCTCATTTCAT | 2020 |
도 6에 나타난 바와 같이, 2차원 배양에 비하여 수화젤에 봉입하였을 때 내분비세포 유전자 발현 및 췌장 전사인자의 발현이 증가하는 것으로 나타났다. 특히, 구상체 형태로 아텔로콜라겐 함유 수화젤에 배양된 경우, 분화 정도가 가장 성숙되는 것으로 나타났다.As shown in FIG. 6 , it was found that expression of endocrine cell gene expression and pancreatic transcription factor increased when encapsulated in hydrogel compared to two-dimensional culture. In particular, when cultured on atelocollagen-containing hydrogel in the form of spheroids, the degree of differentiation was found to be the most mature.
또한, 인슐린 및 C-peptide 분비는 세포 배양액을 모아서 각각의 ELISA (Enzyme-Linked Immunosorbent Assay)를 이용하여 분비 수준을 측정하였다. 도 7에 나타난 바와 같이, 인슐린 및 C-peptide의 분비 수준은 2차원 배양에 비하여 수화젤에 봉입하였을 때 증거하는 것으로 나타났으며, 특히, 구상체 형태로 아텔로콜라겐 함유 수화젤에 배양된 경우, 인슐린 및 C-peptide의 분비 수준이 가장 높게 나타났다.In addition, 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.
또한, 글루코즈 농도에 따른 인슐린 분비 반응성(GSIS)을 평가하였다. 글루코즈 농도에 따른 인슐린 분비 반응성(GSIS) 저 글루코즈(low glucose) 농도 (2mM) 또는 고 글루코즈(high glucose) 농도 (20mM)에서 한 시간 동안 분비되는 인슐린 양을 측정하였다. 인슐린 분비 수준은 인슐린 ELISA (Enzyme-Linked Immunosorbent Assay)로 측정하였으며, 반응성 (Glucose stimulation index)은 저 글루코즈(low glucose)에서 분비되는 인슐린 양 / 고 글루코즈(high glucose)에서 분비되는 인슐린 양으로 계산하였다. 도 8에 나타난 바와 같이, 2차원 배양에 비하여 글루코즈 농도에 따른 인슐린 분비 반응성이 증가하는 것으로 나타났으며, 특히, 단일 세포 및 구상체 형태로 아텔로콜라겐 함유 수화젤에 배양된 경우, BME 배양에 비하여 인슐린 분비 반응성이 우수한 것으로 나타났다.In addition, 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. . As shown in Figure 8, it was found that 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.
또한, 면역화학적 염색을 통하여 세포내 분화된 인슐린 생성세포를 확인하였다. 수화젤 내에 세포를 확인하기 위하여 세포를 포르말린으로 고정하였다. 이후 수화젤을 파라핀블럭을 만들어서 4um두께로 잘라서 조직 슬라이드를 제작하였다. 슬라이드를 deparaffin, dehydration 후 antigen retrieval 하였다. 이후 blocking을 1시간 동안 하고 Guinea pig anti-insulin (1:200; Abcam) and mouse anti-glucagon (1:1000; Abcam, MA, USA) rabbit anti-PDX1 (1:200; Abcam, MA, USA)에서 4℃ overnight 으로 반응하였다. 세척 후 secondary antibody로 anti-guinea pig IgG Alexa Fluor 647 (1:200; Abcam, MA, USA), anti-rabbit IgG Alexa Fluor 488 (1:200; Thermo Fisher Scientific, MA, USA) and anti-mouse IgG Alexa Fluor 488 (1:200; Thermo Fisher Scientific, MA, USA)로 상온에서 1시간 반응후 세척, DAPI 로 핵을 염새하였다. Prolong gold antifade mountant (Life technologies, Maryland, USA)로 마운팅 후 확인하였다. 도 9에 나타난 바와 같이, 아텔로콜라겐 함유 수화젤에 배양된 경우, 인슐린 생성세포가 cluster를 이루고 다량 존재하고 있음을 확인하였다.In addition, intracellularly differentiated insulin-producing cells were identified through immunochemical staining. In order to identify the cells in the hydrogel, the cells were fixed with formalin. After that, the hydrogel was cut to a thickness of 4 μm 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. After washing, anti-guinea pig IgG Alexa Fluor 647 (1:200; Abcam, MA, USA), anti-rabbit IgG Alexa Fluor 488 (1:200; Thermo Fisher Scientific, MA, USA) and anti-mouse IgG with secondary antibodies After reaction for 1 hour at room temperature with Alexa Fluor 488 (1:200; Thermo Fisher Scientific, MA, USA), the cells were washed, and the nuclei were stained with DAPI. It was confirmed after mounting with Prolong gold antifade mountant (Life technologies, Maryland, USA). As shown in FIG. 9 , when cultured on atelocollagen-containing hydrogel, it was confirmed that insulin-producing cells formed clusters and were present in large amounts.
실시예 4. 이식 조직학적 평가Example 4. Histological evaluation of transplantation
본 발명에 따라 제조된 이온화 아텔로콜라겐 함유 수화젤이 인슐린 생성세포의 분화에 미치는 영향을 in vivo에서 평가하였다. 췌장 내분비 세포(pancreatic endocrine cell) 구상체 (spheroid)를 이온화 이온화 아텔로콜라겐 함유 수화젤에 도입하고, 생성세포를 당뇨병 NSG 마우스(NOD scid gamma mouse)의 피하에 이식하였다. 대조군으로 암세포유래 세포외기질인 BME 수화젤에 췌장 내분비 세포(pancreatic endocrine cell) 구상체 (spheroid)를 도입하고 이식하였다. 마우스 모델은 면역반응이 조절된 NSG이고, 이식은 피하에 수화젤 형태로 이식하였다. 이식 후 3개월 뒤에 동물 희생 후 피부 조직을 얻어서 포르말린에 고정 후 파라핀 블록을 만들어서 조직슬라이드를 제작하였다. 이식 부위를 Hematoxylin & Eosin (H&E)과 PDX1, Insulin의 면역 염색을 통하여 이식된 세포 및 이식 주변부위를 확인하였다.The effect of the hydrogel containing ionized atelocollagen prepared according to the present invention on the differentiation of insulin-producing cells was evaluated in vivo. 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). As a control, 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. Three months after transplantation, after animal sacrifice, skin tissue was obtained, fixed in formalin, and paraffin blocks were made to prepare tissue slides. Immunostaining of the transplant site with Hematoxylin & Eosin (H&E), PDX1, and Insulin was used to confirm the transplanted cells and the surrounding area.
도 10 및 11에 나타난 바와 같이, 이온화 아텔로콜라겐 함유 수화젤에 봉입되어 이식된 경우, 이식세포가 체내에서 잘 성숙화 되어 인슐린을 발현하며, 주변에 혈관 조직이 많이 형성되는 것을 확인하였다. 하지만 BME 수화젤을 사용한 경우 소량 남아 있던 미분화 세포의 teratoma 형성을 유도하여 teratoma가 형성되어 있으며, 혈관이 유도되지 않았다.As shown in FIGS. 10 and 11 , when encapsulated in hydrogel containing ionized atelocollagen and transplanted, the transplanted cells matured well in the body to express insulin, and it was confirmed that a lot of vascular tissue was formed around. However, when BME hydrogel was used, teratoma formation was induced by the remaining undifferentiated cells in a small amount, and blood vessels were not induced.
이상으로 본 발명 내용의 특정한 부분을 상세히 기술하였는 바, 당업계의 통상의 지식을 가진 자에게 있어서, 이러한 구체적 기술은 단지 바람직한 실시양태일 뿐이며, 이에 의해 본 발명의 범위가 제한되는 것이 아닌 점은 명백하다. 즉, 본 발명의 실질적인 범위는 첨부된 청구항들과 그것들의 등가물에 의하여 정의된다.As described above in detail a specific part of the content of the present invention, for those of ordinary skill in the art, it is clear that this specific description is only a preferred embodiment, and the scope of the present invention is not limited thereby. do. That is, the substantial scope of the present invention is defined by the appended claims and their equivalents.
Claims (9)
- 이온화 아텔로콜라겐 함유 수화젤을 이용하여 인슐린 생성세포의 분화 유도 방법으로서,A method for inducing differentiation of insulin-producing cells using an ionized atelocollagen-containing hydrogel, comprising:(1) 인간 유도만능줄기세포(iPSC)를 액티빈 A(activin A), CHIR99021 및 Y-27632를 포함하는 배지에 배양하여 내배엽(definitive endoderm)으로 분화를 유도하는 단계;(1) inducing differentiation into endoderm (definitive endoderm) by culturing human induced pluripotent stem cells (iPSCs) in a medium containing activin A, CHIR99021 and Y-27632;(2) 상기 (1) 단계에서 분화가 유도된 내배엽을 B27-insulin, 돌소몰핀(Dorsomorphin), 레티논산(Retinoic acid), SB431542, 및 SANT1을 포함하는 배지에 배양하여 췌장 전구세포(pancreatic progenitor cell)로 분화를 유도하는 단계; 및(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) 상기 (2) 단계에서 분화가 유도된 췌장 전구세포를 B27-insulin, 덱사메타손(dexamethasone), 니코틴아미드(nicotinamide), 포스콜린(forskolin) 및 Exendin-4를 포함하는 이온화 아텔로콜라겐 함유 수화젤에 봉입하여 배양하여 췌장 내분비 세포(pancreatic endocrine cell)로 분화를 유도하는 단계;를 포함하는 인슐린 생성세포의 분화 유도 방법.(3) Hydration containing 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 them in a gel and culturing them;
- 제1항에 있어서, 상기 배지는 DMEM(Dulbecco's Modified Eagle's Medium), MEM(Minimal Essential Medium), 임프루브드 MEM(improved MEM), BME(Basal Medium Eagle), RPMI 1640(Roswell Park Memorial Institute medium 1640), 어드밴스드 RPMI 1640(Advanced RPMI1640), F-10, F-12, DMEM-F12, α -MEM(α -Minimal Essential Medium), G-MEM(Glasgow's Minimal Essential Medium) 및 IMDM(Iscove's Modified Dulbecco's Medium)으로 구성된 군으로부터 선택된 어느 하나인 것을 특징으로 하는 인슐린 생성세포의 분화 유도 방법.According to claim 1, wherein 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) , with Advanced RPMI 1640, F-10, F-12, DMEM-F12, α -MEM (α -Minimal Essential Medium), Glasgow's Minimal Essential Medium (G-MEM) and Iscove's Modified Dulbecco's Medium (IMDM) A method for inducing differentiation of insulin-producing cells, characterized in that any one selected from the group consisting of.
- 제1항에 있어서, 상기 (1) 단계는 인간 유도만능줄기세포(iPSC)를 10 ~ 400 ng/mL 액티빈 A(activin A), 1 ~ 5 μM CHIR99021, 및 5 ~ 50 μM Y-27632이 포함된 배지에서 배양하여 내배엽으로 분화를 유도하고,The method of claim 1, wherein in step (1), 10 ~ 400 ng/mL activin A, 1 ~ 5 μM CHIR99021, and 5 ~ 50 μM Y-27632 of human induced pluripotent stem cells (iPSCs) Inducing differentiation into endoderm by culturing in the containing medium,상기 (2) 단계는 내배엽을 1 ~ 5% B27, 1 ~ 10 μM 돌소몰핀(Dorsomorphin), 1 ~ 5 μM 레티논산(Retinoic acid), 2 ~ 20 μM SB431542, 및 0.1 ~ 1 μM SANT1을 포함하는 배지에 배양하여 췌장 전구세포로 분화를 유도하고,In step (2), endoderm containing 1 to 5% B27, 1 to 10 μM Dorsomorphin, 1 to 5 μM Retinoic acid, 2 to 20 μM SB431542, and 0.1 to 1 μM SANT1 Inducing differentiation into pancreatic progenitor cells by culturing in a medium,상기 (3) 단계는 췌장 전구세포를 1 ~ 5% B27, 0.01 ~ 2 μM 덱사메타손(dexamethasone), 2 ~ 10 mM 니코틴아미드(nicotinamide), 1 ~ 20 μM 포스콜린(forskolin) 및 1 ~ 500 nM Exendin-4를 포함하는 이온화 아텔로콜라겐 함유 수화젤에 봉입하여 배양하여 췌장 내분비 세포로 분화를 유도하는 것을 특징으로 하는 인슐린 생성세포의 분화 유도 방법.In step (3), 1 ~ 5% B27, 0.01 ~ 2 μM dexamethasone, 2 ~ 10 mM nicotinamide, 1 ~ 20 μM forskolin and 1 ~ 500 nM Exendin of pancreatic progenitor cells A method for inducing differentiation of insulin-producing cells, characterized in that the cells are encapsulated in a hydrogel containing ionized atelocollagen containing -4 and cultured to induce differentiation into pancreatic endocrine cells.
- 제1항에 있어서, 상기 (3) 단계는, According to claim 1, wherein the step (3),(3-1) 분화가 유도된 췌장 전구세포를 오목한 마이크로웰(concave micro-well)에서 B27-insulin, 덱사메타손(dexamethasone), 니코틴아미드(nicotinamide), 포스콜린(forskolin) 및 Exendin-4를 포함하는 배지에 배양하여 췌장 전구세포(pancreatic progenitor cell)의 세포 구상체(spheroid)를 제조하는 단계; 및 (3-1) containing B27-insulin, dexamethasone, nicotinamide, forskolin and Exendin-4 in a concave micro-well of differentiation-induced pancreatic progenitor cells preparing cell spheroids of pancreatic progenitor cells by culturing in a medium; and(3-2) 상기 (3-1) 단계에서 제조된 세포 구상체(spheroid)를 B27-insulin, 덱사메타손(dexamethasone), 니코틴아미드(nicotinamide), 및 Exendin-4를 포함하는 이온화 아텔로콜라겐 함유 수화젤에 봉입하여 배양하는 단계;를 추가로 포함하는 것을 특징으로 하는 인슐린 생성세포의 분화 유도 방법.(3-2) Hydrating the cell spheroid prepared in step (3-1) with ionized atelocollagen containing B27-insulin, dexamethasone, nicotinamide, and Exendin-4 The method of inducing differentiation of insulin-producing cells, characterized in that it further comprises; encapsulated in a gel and culturing.
- 제1항에 있어서, 상기 이온화 아텔로콜라겐 함유 수화젤은 양이온화 아텔로콜라겐을 0.5~3% 중량%로 포함하는 것을 특징으로 하는 인슐린 생성세포의 분화 유도 방법.The method of claim 1, wherein the hydrogel containing ionized atelocollagen contains 0.5 to 3% by weight of cationized atelocollagen by weight.
- 제1항에 있어서, 상기 이온화 아텔로콜라겐 함유 수화젤은 전단율(shear rate) 1s-1에서 1 Pa.s 내지 100 Pa.s의 범위의 점도를 갖는 것을 특징으로 하는 인슐린 생성세포의 분화 유도 방법.The induction of differentiation of insulin-producing cells according to claim 1, wherein the ionized atelocollagen-containing hydrogel has a viscosity in the range of 1 Pa.s to 100 Pa.s at a shear rate of 1s -1 Way.
- 제1항에 있어서, 상기 이온화 아텔로콜라겐 함유 수화젤은 나노섬유 네트워크(nanofibrous network)를 형성하는 것을 특징으로 하는 인슐린 생성세포의 분화 유도 방법.The method of claim 1, wherein the ionized atelocollagen-containing hydrogel forms a nanofibrous network.
- 제1항 내지 제7항 중 어느 한 항의 인슐린 생성세포의 분화 유도 방법에 따라 제조된 인슐린 생성세포.An insulin-producing cell prepared according to the method for inducing differentiation of an insulin-producing cell according to any one of claims 1 to 7.
- 제1항 내지 제7항 중 어느 한 항의 인슐린 생성세포의 분화 유도 방법에 따라 제조된 인슐린 생성세포를 포함하는 인공 췌장.An artificial pancreas comprising insulin-producing cells prepared according to the method for inducing differentiation of insulin-producing cells according to any one of claims 1 to 7.
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| KR20200049669A (en) * | 2018-10-30 | 2020-05-08 | 재단법인 아산사회복지재단 | Composition for inducing dedifferentiation for insulin producing cells from human induced pluripotent stem cells and method for inducing insulin producing cells using the same |
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| KR20200049669A (en) * | 2018-10-30 | 2020-05-08 | 재단법인 아산사회복지재단 | Composition for inducing dedifferentiation for insulin producing cells from human induced pluripotent stem cells and method for inducing insulin producing cells using the same |
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| S. KUMAR, ABDULLAH ALARFAJ, MURUGAN MUNUSAMY, A. SINGH, I-CHIA PENG, SIVAN PRIYA, RUKMAN HAMAT, AKON HIGUCHI: "Recent Developments in β-Cell Differentiation of Pluripotent Stem Cells Induced by Small and Large Molecules", INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES, vol. 15, no. 12, 1 January 2014 (2014-01-01) - 17 December 2014 (2014-12-17), pages 23418 - 23447, XP055167269, DOI: 10.3390/ijms151223418 * |
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