Generation of Adult Human Induced Pluripotent Stem Cells Using Nonviral Minicircle DNA Vectors

Nat Protoc. 2011 Jan;6(1):78-88. doi: 10.1038/nprot.2010.173. Epub 2010 Dec 23.

Abstract

Human induced pluripotent stem cells (hiPSCs) derived from patient samples have tremendous potential for innovative approaches to disease pathology investigation and regenerative medicine therapies. However, most hiPSC derivation techniques use integrating viruses, which may leave residual transgene sequences as part of the host genome, thereby unpredictably altering cell phenotype in downstream applications. In this study, we describe a protocol for hiPSC derivation by transfection of a simple, nonviral minicircle DNA construct into human adipose stromal cells (hASCs). Minicircle DNA vectors are free of bacterial DNA and thus capable of high expression in mammalian cells. Their repeated transfection into hASCs, abundant somatic cell sources that are amenable to efficient reprogramming, results in transgene-free hiPSCs. This protocol requires only readily available molecular biology reagents and expertise, and produces hiPSC colonies from an adipose tissue sample in ∼4 weeks.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adipocytes / cytology
  • Cell Culture Techniques*
  • Cell Dedifferentiation*
  • DNA / chemistry*
  • Flow Cytometry
  • Genetic Vectors*
  • Humans
  • Induced Pluripotent Stem Cells / cytology*
  • Stromal Cells / cytology
  • Transfection / methods
  • Transgenes

Substances

  • DNA