Generation of induced pluripotent stem cells from frozen buffy coats using non-integrating episomal plasmids

Viviana Meraviglia, Alessandra Zanon, Alexandros A. Lavdas, Christine Schwienbacher, Rosamaria Silipigni, Marina Di Segni, Huei Sheng Vincent Chen, Peter P. Pramstaller, Andrew A. Hicks, Alessandra Rossini

Research output: Contribution to journalArticle

Abstract

Somatic cells can be reprogrammed into induced pluripotent stem cells (iPSCs) by forcing the expression of four transcription factors (Oct-4, Sox-2, Klf-4, and c-Myc), typically expressed by human embryonic stem cells (hESCs). Due to their similarity with hESCs, iPSCs have become an important tool for potential patient-specific regenerative medicine, avoiding ethical issues associated with hESCs. In order to obtain cells suitable for clinical application, transgene-free iPSCs need to be generated to avoid transgene reactivation, altered gene expression and misguided differentiation. Moreover, a highly efficient and inexpensive reprogramming method is necessary to derive sufficient iPSCs for therapeutic purposes. Given this need, an efficient non-integrating episomal plasmid approach is the preferable choice for iPSC derivation. Currently the most common cell type used for reprogramming purposes are fibroblasts, the isolation of which requires tissue biopsy, an invasive surgical procedure for the patient. Therefore, human peripheral blood represents the most accessible and least invasive tissue for iPSC generation. In this study, a cost-effective and viral-free protocol using non-integrating episomal plasmids is reported for the generation of iPSCs from human peripheral blood mononuclear cells (PBMNCs) obtained from frozen buffy coats after whole blood centrifugation and without density gradient separation.

Original languageEnglish
Article numbere52885
JournalJournal of visualized experiments : JoVE
Volume2015
Issue number100
DOIs
Publication statusPublished - Jun 5 2015

    Fingerprint

Keywords

  • Cellular biology
  • Developmental biology
  • Episomal plasmids
  • Induced pluripotent stem cells
  • Issue 100
  • Molecular biology
  • Peripheral blood mononuclear cells
  • Reprogramming
  • Stem cell biology

ASJC Scopus subject areas

  • Biochemistry, Genetics and Molecular Biology(all)
  • Chemical Engineering(all)
  • Immunology and Microbiology(all)
  • Neuroscience(all)
  • Medicine(all)

Cite this

Meraviglia, V., Zanon, A., Lavdas, A. A., Schwienbacher, C., Silipigni, R., Di Segni, M., Chen, H. S. V., Pramstaller, P. P., Hicks, A. A., & Rossini, A. (2015). Generation of induced pluripotent stem cells from frozen buffy coats using non-integrating episomal plasmids. Journal of visualized experiments : JoVE, 2015(100), [e52885]. https://doi.org/10.3791/52885