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Method for induction of differentiation of stem cells into hepatocytes

A technology for induction of differentiation and stem cells, applied in biochemical equipment and methods, embryonic cells, animal cells, etc., can solve problems such as the method of induction of stem cell differentiation that has not been disclosed

Inactive Publication Date: 2012-09-12
HEALTH SCI TECH TRANSFER CENT JAPAN HEALTH SCI FOUND
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, these non-patent documents do not disclose the method of inducing differentiation from stem cells to hepatocytes

Method used

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  • Method for induction of differentiation of stem cells into hepatocytes
  • Method for induction of differentiation of stem cells into hepatocytes
  • Method for induction of differentiation of stem cells into hepatocytes

Examples

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Embodiment 1

[0105] (Example 1) Method for inducing differentiation from human iPS cells into hepatocytes

[0106] In this example, the case where the HEX gene was introduced into human iPS cells using an Ad vector and induced to differentiate into hepatocytes from the human iPS cells into which the gene was introduced will be described. The experimental route of this embodiment is shown in figure 1 .

[0107] 1) Construction of Ad vector for HEX gene introduction

[0108] An Ad vector was prepared in which an introduced gene was carried in the E1-deficient site of the E1-deficient type 5 Ad genome (pAdHM41-K7) and an EF-1α promoter was carried upstream thereof. The introduced gene is a HEX gene composed of the sequence shown in GenBank Accession No. BC014336.

[0109] 2) Culture of human iPS cells

[0110] In this example, human iPS cells (Tic (JCRB1331), Dotcom (JCRB1327), Squeaky (JCRB1329)) were used. Using mouse embryonic fibroblasts (MEF) as feeder cells, culture medium 1 shown ...

experiment example 1-1

[0116] (Experimental example 1-1) Results of antibody immunostaining method

[0117] On the 12th day of culture, immunological antibody staining was performed using an anti-alpha-fetoprotein (AFP) antibody (manufactured by Dako) labeled with Alexa (trademark) 594 and an anti-CK7 antibody (manufactured by Invitrogen) labeled with Alexa (trademark) 488 The expression of each gene was confirmed by the method. AFP is a marker of liver stem cells, and CK7 is a marker of bile duct epithelial cells. Such as figure 2 As shown, in the system into which the HEX gene was introduced using the Ad vector, stronger reactions of various markers were observed compared to the system without the gene. From this, it was confirmed that induction of differentiation from human iPS cells into hepatocytes was promoted by introducing the HEX gene using the Ad vector.

experiment example 1-2

[0118] (Experimental example 1-2) the result of real-time PCR method

[0119] For cells cultured on day 0 (undifferentiated cells), day 5 (endoderm cells) and day 12, the TaqMan (trademark) gene expression analysis kit (Applied Biosystems, catalog number: Hs01040607_m1 for AFP) was used. For albumin (Hs00910225_m), the expression levels of AFP and albumin, which can be markers of liver stem cells, were examined by real-time PCR. The expression level was calculated based on the expression level of each gene in human fetal liver total RNA (Clontech Company, catalog number 636540) as a benchmark (100).

[0120] As a result, both AFP and albumin were significantly increased in the HEX gene-introduced system, and it was found that the HEX gene-introduced system showed a tendency to induce differentiation ( image 3 , 4 ).

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Abstract

Disclosed are: a gene transduction method for use in the induction of the differentiation of stem cells such as ES cells or iPS cells into hepatocytes effectively; stem cells into each of which a gene useful for the induction of the differentiation into hepatocytes is introduced; and hepatocytes produced from stem cells each having the gene introduced therein. A specific gene can be introduced into stem cells such as ES cells or iPS cells using an adenovirus vector. The effective induction of the differentiation into hepatocytes can be achieved by introducing the gene. Specifically, the effective induction of the differentiation of stem cells such as ES cells or iPS cells into hepatocytes can be achieved by introducing at least one gene selected from HEX gene, HNF4A gene and SOX17 gene into the stem cells.

Description

technical field [0001] The present invention relates to a method for inducing differentiation of pluripotent stem cells such as embryonic stem cells (hereinafter also referred to as "ES cells") and induced pluripotent stem cells (hereinafter also referred to as "iPS cells") into hepatocytes. In addition, the present invention relates to stem cells into which genes useful for inducing differentiation into hepatocytes have been introduced. [0002] This application claims the priority of Japanese Patent Application No. 2009-247342, Japanese Patent Application No. 2010-121282, and Japanese Patent Application No. 2010-154225, which are incorporated herein by reference. Background technique [0003] Pluripotent stem cells are undifferentiated cells with multidirectional differentiation potential and self-renewal ability, which have been revealed to have tissue repair ability after tissue injury. Therefore, pluripotent stem cells are useful in the screening of therapeutic substan...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/09C12N5/10
CPCC12N5/067C12N2501/60C12N2506/02C12N2506/45C12N2510/00C12N2500/25C12N2500/36C12N2500/44C12N2500/46C12N2501/115C12N2501/119C12N2501/155C12N2501/16C12N2501/91
Inventor 水口裕之川端健二稻村充古江美保
Owner HEALTH SCI TECH TRANSFER CENT JAPAN HEALTH SCI FOUND
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