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Integration-free human induced pluripotent stem cells from blood

a technology of human induced pluripotent stem cells and blood, which is applied in the direction of genetically modified cells, instruments, viruses/bacteriophages, etc., can solve the problems of slow expansion of fibroblasts from skin biopsy prior to cellular reprogramming, interfere with the use of hips cell lines in several applications, and inefficient and inconvenient use of hips cell lines

Inactive Publication Date: 2011-11-17
TRUE NORTH THERAPEUTICS
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Problems solved by technology

The resulting hiPS line contains one or more proviral integrations, which may interfere with the use of the hiPS cell lines in several applications including, for example, regenerative medicine applications.
Further, use of skin fibroblasts for generating hiPS cells presents a number of disadvantages: isolation of a skin biopsy is somewhat invasive for the patient; expansion of fibroblasts from a skin biopsy prior to cellular reprogramming is relatively slow and inefficient; and skin is often directly exposed to environmental mutagens (e.g., UV irradiation) that may compromise the genomic integrity of the fibroblasts and therefore that of fibroblast-derived hiPS cell lines.

Method used

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  • Integration-free human induced pluripotent stem cells from blood
  • Integration-free human induced pluripotent stem cells from blood
  • Integration-free human induced pluripotent stem cells from blood

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example 1

Generation of Integration-Free hiPS Cells by Transient Transfection of Nucleated Blood Cells with Reprogramming Factors Oct4, Sox2, K14, and c-Myc

[0072]The original objective of this work was to evaluate the ability of the piggyBac plasmid / transposon reprogramming system (as described in Woltjen et al (2009), Nature, 458(7239) 766-770) to reprogram PBMCs. This system, as reported in Woltejen et al (2009), utilizes piggyBac transposase to initially drive genomic integration of a transposon expression vector encoding four reprogramming factors (Klf4, c-Myc, Oct4, and Sox2) and a separate transposon vector encoding the reverse-tetracycline transactivator (rtTA) to drive expression in trans of the four reprogramming factors to induce reprogramming of somatic cells into iPS cells. Subsequently, the same transposase is transiently expressed to remove the integrated transposon(s) from the genome to obtain integration-free hiPS cells, although to date this system has only been demonstrated ...

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Abstract

Provided herein are methods for generating human induced pluripotent stem cells free from genomic integration of exogenous transgenes by transfecting into nucleated blood cells one or more DNA expression vectors (e.g., plasmid vectors) that do not contain a mammalian origin of replication, and encode and permit expression of one or more reprogramming factors (e.g., Oct4, Sox2, Klf4, and c-Myc). Also provided herein are the integration-free human induced pluripotent stem cells obtained by the methods described herein.

Description

BACKGROUND OF THE INVENTION[0001]The advent of cellular reprogramming technology to generate human induced pluripotent stem (hiPS) cells combined with directed differentiation in vitro has opened the door to vast opportunities for more effective drug discovery and regenerative medicine. Typically, an hiPS cell line is generated by transducing fibroblasts expanded from a small skin biopsy with one or more integrating retroviruses encoding reprogramming factors. The resulting hiPS line contains one or more proviral integrations, which may interfere with the use of the hiPS cell lines in several applications including, for example, regenerative medicine applications. Further, use of skin fibroblasts for generating hiPS cells presents a number of disadvantages: isolation of a skin biopsy is somewhat invasive for the patient; expansion of fibroblasts from a skin biopsy prior to cellular reprogramming is relatively slow and inefficient; and skin is often directly exposed to environmental ...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): G01N33/567C12N5/071
CPCC12N15/63C12N15/85C12N2800/24C12N5/0696C12N2501/125C12N2501/22C12N2510/00C12N2501/602C12N2501/603C12N2501/604C12N2501/606C12N2506/11C12N2501/2304
Inventor IRION, STEFAN
Owner TRUE NORTH THERAPEUTICS
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