title: Generation of Induced Pluripotent Stem Cells from Frozen Buffy Coats using Non-integrating Episomal Plasmids.
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.
authors.affiliation: Center for Biomedicine, European Academy Bozen/Bolzano (EURAC).
authors.forename: Viviana
authors.initials: V
authors.lastname: Meraviglia
authors.affiliation: Center for Biomedicine, European Academy Bozen/Bolzano (EURAC).
authors.forename: Alessandra
authors.initials: A
authors.lastname: Zanon
authors.affiliation: Center for Biomedicine, European Academy Bozen/Bolzano (EURAC).
authors.forename: Alexandros A
authors.initials: AA
authors.lastname: Lavdas
authors.affiliation: Center for Biomedicine, European Academy Bozen/Bolzano (EURAC).
authors.forename: Christine
authors.initials: C
authors.lastname: Schwienbacher
authors.affiliation: Laboratory of Medical Genetics, Fondazione IRCCS Ca´ Granda, Ospedale Maggiore Policlinico.
authors.forename: Rosamaria
authors.initials: R
authors.lastname: Silipigni
authors.affiliation: Laboratory of Medical Genetics, Fondazione IRCCS Ca´ Granda, Ospedale Maggiore Policlinico.
authors.forename: Marina
authors.initials: M
authors.lastname: Di Segni
authors.affiliation: Del E. Webb Center for Neuroscience, Aging & Stem Cell Research, Sanford-Burnham Medical Research Institute.
authors.forename: Huei-Sheng Vincent
authors.initials: HS
authors.lastname: Chen
authors.affiliation: Center for Biomedicine, European Academy Bozen/Bolzano (EURAC).
authors.forename: Peter P
authors.initials: PP
authors.lastname: Pramstaller
authors.affiliation: Center for Biomedicine, European Academy Bozen/Bolzano (EURAC).
authors.forename: Andrew A
authors.initials: AA
authors.lastname: Hicks
authors.affiliation: Center for Biomedicine, European Academy Bozen/Bolzano (EURAC); Alessandra.Rossini@eurac.edu.
authors.forename: Alessandra
authors.initials: A
authors.lastname: Rossini
mesh_terms: D065150:Cellular Reprogramming / Q000235:genetics; D002498:Centrifugation / Q000379:methods; D015925:Cryopreservation / Q000379:methods; D006801:Humans; D057026:Induced Pluripotent Stem Cells / Q000166:cytology / Q000502:physiology*; D007963:Leukocytes, Mononuclear / Q000166:cytology / Q000502:physiology; D010957:Plasmids / Q000235:genetics*