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Induced Hepatic Stem Cell And Process For Production Thereof, And Applications Of The Cell

A technology for hepatic stem cells and hepatocytes is applied in the fields of induced hepatic stem cells and their manufacture, as well as the uses of the cells, and can solve the problems of inability to manufacture cells and the like

Inactive Publication Date: 2013-01-02
NAT CANCER CENT
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0018] That is, it is generally considered that the factors important for maintaining undifferentiated properties, NANOG gene, POU5F1 gene, SOX2 gene, ZFP42 gene, SALL4 gene, LIN28 gene, and TERT gene, and various properties of differentiated cells (multiple differentiation genes expression) cells expressing together cannot make

Method used

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  • Induced Hepatic Stem Cell And Process For Production Thereof, And Applications Of The Cell
  • Induced Hepatic Stem Cell And Process For Production Thereof, And Applications Of The Cell
  • Induced Hepatic Stem Cell And Process For Production Thereof, And Applications Of The Cell

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Experimental program
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Embodiment 1

[0135] 1. Preparation of pantropic retroviral vector

[0136] Using Fugene HD (manufactured by Roche; Cat no.4709691), the retroviral vector plasmids of the three genes POU5F1-pMXs, KLF4-pMXs, and SOX2-pMXs were introduced into packaging cells for the preparation of pantropic retroviral vectors Plat-GP cells to prepare retroviral vector solution. The genes POU5F1-pMXs, KLF4-pMXs, SOX2-pMXs were used in a ratio of 4:2:1 in sequence. The ratio of 4:2:1 can be used to introduce genes into packaging cells. In addition, it can also be used to prepare retroviral vector solutions of the three genes POU5F1-pMXs, KLF4-pMXs, and SOX2-pMXs separately and use 4: Mix at a ratio of 2:1. details as follows.

[0137]

[0138] The addgene vector was used for the genes POU5F1-pMXs, KLF4-pMXs, and SOX2-pMXs (Table 5 below).

[0139] The amount of each vector was set to POU5F1-pMXs (manufactured by addgene) 2μg, KLF4-pMXs (manufactured by addgene) 1μg, SOX2-pMXs (manufactured by addgene) 0.5μg, Ve...

Embodiment 2

[0157] 2. Produce human-induced liver stem cells from cells derived from neonatal skin tissue

[0158] Human-induced hepatic stem cells are produced from cells derived from human newborn skin tissues (trade name: neonatal normal human skin fibroblasts; primary culture; Lot no. 7F 3956) belonging to tissues just after birth.

[0159] One freeze-preserved cell derived from human newborn skin tissue (primary culture; manufactured by Lonza; CC-2511; Lot no.7F3956) was thawed in a constant temperature bath at 37°C and suspended in a thermostat containing 1X antibiotic-antifungal (Invitrogen; Cat no. 15240-062) and 10% FBS D-MEM (high glucose) (Invitrogen; Cat no. 11965-092) medium to obtain 10 ml of cell suspension.

[0160] Next, the obtained cell suspension was centrifuged at 1000 rpm and 4°C for 5 minutes. After removing the supernatant, it was suspended in Fibroblast Medium Kit-2 (2% FBS) (hereinafter also referred to as registered trademark FGM) -2BulletKit) (manufactured by Lonza...

Embodiment 3

[0192] 3. Produce human-induced liver stem cells from cells derived from cancer tissues of patients with gastric cancer

[0193] Cells are separated from cancerous tissues of human gastric cancer (advanced cancer) patients. Three genes (the ratio of POU5F1, KLF4, and SOX2 are 4:2:1) were added to the obtained cells to carry out gene transfer to produce human induced hepatic stem cells. details as follows.

[0194] Use Hank's balanced salt solution (without phenol red) (manufactured by Invitrogen; Cat no.14175-095) to wash a part of the fresh tissue of gastric cancer (advanced cancer: male, 67 years old, Japanese) obtained during surgery, thin with scissors Cut into about 1mm 2 . Furthermore, wash with Hank's balanced salt solution (without phenol red) until the supernatant becomes transparent, remove the supernatant, and add 5 ml of 0.1% collagenase (manufactured by Wako Pure Chemical Industries, Ltd.; Cat no.034-10533) / 1X antibiotic to the tissue pellet. An antifungal agent (...

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Abstract

Disclosed are: an induced hepatic stem cell useful for safety tests, toxicity tests, metabolism tests, drug interaction tests, anti-viral activity tests, screening tests for medicinal agents such as hyperlipemia therapeutic agents, hypertension therapeutic agents, pharmaceutical low-molecular-weight compound agents and pharmaceutical antibody agents, the screening for potential drug targets, the production of animal models, the production of hepatocyte-produced proteins, and regenerative medicine; a process for producing the cell; and applications of the cell. The induced hepatic stem cell is characterized by fulfilling at least the following requirements (1) to (3): (1) at least 15 genes selected from specific genes that are marker genes for embryonic stem cells are expressed in the cell; (2) the cell has properties of hepatocytes; and (3) the cell can be proliferatively cultured or sub-cultured for at least three days.

Description

Technical field [0001] The invention relates to the screening test of drugs such as safety test, toxicity test, metabolism test, drug interaction test, antiviral activity test, hyperlipidemia drugs, hypertension treatment drugs, low molecular compound drugs, antibody drugs, etc., and new drug discovery Target screening, animal model production, production of hepatocyte-produced proteins, and induced hepatic stem cells useful in regenerative medicine, especially related to the characteristics of hepatocytes and the expression of embryonic stem cells in the same amount as embryonic stem cells Induced hepatic stem cells that are genetically grouped and capable of proliferating and subculturing for a long period of time, their production methods, and uses of the cells. Background technique [0002] The research and development of new drugs in pharmaceutical companies have problems of long research and development cycles and high research and development costs. Moreover, the current ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/10A01K67/027A61K35/12C12N5/071C12P21/02C12Q1/02C12N15/12A61K35/407
CPCC12N2501/603C12N5/067C12N5/0696C12N2506/45C12N2501/604C12N2510/00A61K35/407C12N2501/60C12N2501/602A61K35/12A61P1/16C12N5/16
Inventor 石川哲也萩原启太郎落谷孝广
Owner NAT CANCER CENT
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