Integration-free human induced pluripotent stem cells from blood

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 ...

AI Technical Summary

Benefits of technology

This patent describes a method for generating human induced pluripotent stem cells (hiPS cells) that are free from genomic integration of exogenous nucleic acid sequences. The method involves transfecting nucleated blood cells with one or more DNA expression vectors containing reprogramming factors, such as Oct4, Sox2, Klf4, and c-Myc, and culturing the cells under specific conditions to allow for the growth of hiPS cells. The resulting hiPS cells lack exogenous DNA sequences and are useful for various applications, such as regenerative medicine and drug development. The method can be performed using electroporation, nucleofection, or lipofection, and can involve the use of a single DNA expression vector or multiple vectors. The nucleated blood cells used for transfection can be from adult peripheral blood or fetal cells.

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.

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