A Versatile In Vivo System to Study Myc in Cell Reprogramming

Methods Mol Biol. 2021:2318:267-279. doi: 10.1007/978-1-0716-1476-1_14.

Abstract

Cellular reprogramming is a process by which adult differentiated cells lose their identity and are converted into pluripotent stem cells, known as induced pluripotent stem (iPS) cells. This process can be achieved in vitro and in vivo and is relevant for many fields including regenerative medicine and cancer. Cellular reprogramming is commonly induced by the ectopic expression of a transcription factor cocktail composed by Oct4, Sox2, Klf4, and Myc (abbreviated as OSKM), and its efficiency and kinetics are strongly dependent on the presence of Myc. Here, we describe a versatile method to study reprogramming in vivo based on the use of adeno-associated viral (AAV) vectors, which allows the targeting of specific organs and cell types. This method can be used to test Myc mutations or genes that may replace Myc, or be combined with different Myc regulators. In vivo reprogramming can be scored by the presence of teratomas and the isolation of in vivo iPS, thereby providing a simple surrogate for the function of Myc in dedifferentiation and stemness. Our protocol can be divided into five steps: (1) intravenous inoculation of AAV vectors; (2) monitoring the animals until the appearance of teratomas; (3) analysis of teratomas; (4) histopathological analysis of mouse organs; and (5) isolation of in vivo-generated iPS cells from teratomas, blood, and bone marrow. The information obtained by this in vivo testing platform may provide relevant information on the role of Myc in tissue regeneration, stemness, and cancer.

Keywords: AAV vectors; Cellular reprogramming; Induced pluripotent stem cells; Klf4; Myc; Oct4; Sox2; Teratomas; iPS cells; in vivo.

MeSH terms

  • Animals
  • Cell Differentiation / physiology
  • Cell Line
  • Cells, Cultured
  • Cellular Reprogramming / genetics*
  • Cellular Reprogramming / physiology
  • Cellular Reprogramming Techniques / methods*
  • DNA / genetics
  • Dependovirus / genetics
  • Fibroblasts / cytology
  • Genes, myc / genetics
  • Genes, myc / physiology
  • Genetic Engineering / methods
  • Genetic Vectors / genetics
  • Humans
  • Induced Pluripotent Stem Cells / cytology
  • Kruppel-Like Factor 4
  • Kruppel-Like Transcription Factors / metabolism
  • Mice
  • Octamer Transcription Factor-3 / metabolism
  • Pluripotent Stem Cells / cytology
  • Proto-Oncogene Proteins c-myc / genetics
  • Proto-Oncogene Proteins c-myc / metabolism*
  • SOXB1 Transcription Factors / metabolism
  • Transcription Factors / metabolism
  • Transduction, Genetic

Substances

  • KLF4 protein, human
  • Klf4 protein, mouse
  • Kruppel-Like Factor 4
  • Kruppel-Like Transcription Factors
  • Octamer Transcription Factor-3
  • POU5F1 protein, human
  • Proto-Oncogene Proteins c-myc
  • SOX2 protein, human
  • SOXB1 Transcription Factors
  • Transcription Factors
  • DNA