Method of producing pancreatic hormone-producing cells

一种细胞、胰腺的技术，应用在制备胰腺激素产生细胞领域，能够解决胰岛素产生效率低等问题

Inactive Publication Date: 2013-06-12
TAKEDA PHARMA CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0010] However, since the insulin-producing cells obtained by the above-mentioned methods have considerably lower insulin-producing efficiency compared with normal pancreatic β cells, there is still a need to develop a method for efficiently obtaining insulin-producing cells applicable to cell therapy applications.

Method used

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  • Method of producing pancreatic hormone-producing cells

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preparation example Construction

[0080] 1. Preparation method of pancreatic hormone-producing cells

[0081] The preparation method of the present invention is a method for preparing pancreatic hormone-producing cells from stem cells. The preparation method of the present invention is also a method of inducing differentiation of cells with a lower differentiation state (stem cells) into cells with a higher differentiation state (pancreatic hormone-producing cells).

[0082] The preparation method of the present invention includes the following steps (1) to (6).

[0083] (1) Step of culturing stem cells in a medium containing a Rho kinase inhibitor

[0084] (2) A step of culturing the cells obtained in the above step (1) in a medium containing a GSK3 inhibitor

[0085] (3) A step of culturing the cells obtained in the above step (2) in a medium containing a GSK3 inhibitor and an activator of activin receptor-like kinase-4, 7

[0086] (4) After the cells obtained in (3) above are formed into a cell mass, t...

Embodiment approach 1）

[0253] (a) Pancreatic hormone-producing cells were cultured in the presence of the test compound and (b) pancreatic hormone-producing cells were cultured in the absence of the test compound, and the amount of pancreatic hormone expression inside the cell or the pancreas outside the cell was measured and compared, respectively. Hormone secretion.

[0254] The expression level of pancreatic hormone may, for example, be the expression level of pancreatic hormone protein, the expression level of a polynucleotide (for example, mRNA, etc.) encoding pancreatic hormone protein, or the like. The expression and secretion of pancreatic hormones can be measured by known methods, for example, by using antibodies that recognize pancreatic hormones, by Western blot analysis, ELISA method, etc. or methods similar thereto, measuring in cell extracts, in culture medium, etc. The presence of pancreatic hormones described above is carried out by the method.

[0255] The amount of mRNA can be mea...

Embodiment 1（1）

[0277] Induce human iPS cells to differentiate into endoderm cells (steps (1)-(3))

[0278] Induction of differentiation of human iPS cells into endoderm cells was carried out by the following method.

[0279] First, human iPS cells (iPS cells obtained by introducing Oct3 / 4 gene, Klf4 gene, and Sox2 gene: see Nat Biotechnol2008; 26: 101-106) maintained by culturing the state of cell clusters together with feeder cells were used for Ling Cell dissociation solution (ReproCELL Inc.) for long-like ES cells was dissociated in the state of cell clumps, and these cells were transferred to a 15ml centrifuge tube, left to stand for 5 minutes, and the feeder cells were removed by removing the supernatant.

[0280] To the human iPS cells pelleted in the centrifuge tube, add 0.25% trypsin-1 mM EDTA solution (GIBCO) until dissociated to form single cells. Then, press each 100mm Petri dish 15×10 4 The density of cells, the human iPS cells dispersed in the culture medium were plated on t...

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Abstract

The purpose of the present invention is to provide a method of producing pancreatic hormone-producing cells that have a form more similar to that of normally developed pancreatic cells from stem cells. The method of producing pancreatic hormone-producing cells is characterized by subjecting stem cells to the following steps (1)-(6): (1) a step for culturing stem cells in a culture medium that includes a Rho kinase inhibitor; (2) a step for culturing the cells obtained in step (1) in a culture medium including a GSK3 inhibitor; (3) a step for culturing the cells obtained in step (2) in a culture medium including a GSK3 inhibitor and an agent that activates activin receptor-like kinases 4 and 7; (4) a step for forming a cell aggregate of the cells obtained in step (3) and culturing said cell aggregate in a floating state within a culture medium; (5) a step for culturing the cells obtained in step (4) in a culture medium including a retinoic acid receptor agonist, an AMP-activated protein kinase and / or an activin receptor-like kinase 2, 3 and 6 inhibitor, an activin receptor-like kinase 4, 5 and 7 inhibitor and a cell growth factor; and (6) a step for culturing the cells obtained in step (5) in a culture medium.

Description

technical field [0001] The present invention relates to a method for producing pancreatic hormone-producing cells, and a medicament comprising the pancreatic hormone-producing cells obtained by the production method. Background of the invention [0002] The pancreas has endocrine glands (endocrine cells) and exocrine glands (exocrine cells), and is an organ that plays an important role through two types of cells. Exocrine cells mainly function to secrete digestive enzymes such as pancreatic lipase, trypsin, elastase, and pancreatic amylase, for example. [0003] Endocrine cells play a role in secreting pancreatic hormones. It is known that pancreatic α cells secrete glucagon, pancreatic β cells secrete insulin, pancreatic δ cells secrete somatostatin, and PP cells secrete pancreatic polypeptide (sometimes in this specification abbreviated as PP). Furthermore, in recent years, it has been reported that ghrelin, which is a hormone secreted by the stomach, is also secreted by...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0735A61K35/12A61P3/10C12N5/10C12Q1/02A61K35/39
CPCA61K35/39A61P3/10C12N5/0676C12N2501/115C12N2501/16C12N2501/385C12N2501/727C12N2506/02C12N2506/45C12N2513/00G01N33/507A61K35/00A61K35/12C12N5/00C12N5/066C12N5/069
Inventor 细谷昌树庄司昌伸
Owner TAKEDA PHARMA CO LTD
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