Generation of Human Induced Pluripotent Stem Cells Using RNA-Based Sendai Virus System and Pluripotency Validation of the Resulting Cell Population

Methods Mol Biol. 2016;1353:285-307. doi: 10.1007/7651_2015_205.

Abstract

Human induced pluripotent stem cells (hiPSCs) provide a platform for studying human disease in vitro, increase our understanding of human embryonic development, and provide clinically relevant cell types for transplantation, drug testing, and toxicology studies. Since their discovery, numerous advances have been made in order to eliminate issues such as vector integration into the host genome, low reprogramming efficiency, incomplete reprogramming and acquisition of genomic instabilities. One of the ways to achieve integration-free reprogramming is by using RNA-based Sendai virus. Here we describe a method to generate hiPSCs with Sendai virus in both feeder-free and feeder-dependent culture systems. Additionally, we illustrate methods by which to validate pluripotency of the resulting stem cell population.

Keywords: Pluripotency; Sendai; hiPSC.

MeSH terms

  • Animals
  • Cell Culture Techniques / methods
  • Cell Differentiation
  • Cellular Reprogramming*
  • Feeder Cells / cytology
  • Feeder Cells / metabolism
  • Fibroblasts / cytology*
  • Fibroblasts / drug effects
  • Fibroblasts / metabolism
  • Gene Expression
  • Genetic Vectors / chemistry
  • Genetic Vectors / metabolism
  • Homeodomain Proteins / genetics
  • Homeodomain Proteins / metabolism
  • Humans
  • Induced Pluripotent Stem Cells / cytology*
  • Induced Pluripotent Stem Cells / drug effects
  • Induced Pluripotent Stem Cells / metabolism
  • Intercellular Signaling Peptides and Proteins / pharmacology
  • Kruppel-Like Transcription Factors / genetics
  • Kruppel-Like Transcription Factors / metabolism
  • Mice
  • Models, Biological
  • Nanog Homeobox Protein
  • Octamer Transcription Factor-3 / genetics
  • Octamer Transcription Factor-3 / metabolism
  • RNA / genetics*
  • RNA / metabolism
  • SOXB1 Transcription Factors / genetics
  • SOXB1 Transcription Factors / metabolism
  • Sendai virus / genetics*
  • Transgenes*

Substances

  • GKLF protein
  • Homeodomain Proteins
  • Intercellular Signaling Peptides and Proteins
  • Kruppel-Like Transcription Factors
  • NANOG protein, human
  • Nanog Homeobox Protein
  • Octamer Transcription Factor-3
  • POU5F1 protein, human
  • SOX2 protein, human
  • SOXB1 Transcription Factors
  • RNA