title: Efficient iPS cell generation from blood using episomes and HDAC inhibitors.
abstract: This manuscript illustrates a protocol for efficiently creating integration-free human induced pluripotent stem cells (iPSCs) from peripheral blood using episomal plasmids and histone deacetylase (HDAC) inhibitors. The advantages of this approach include: (1) the use of a minimal amount of peripheral blood as a source material; (2) nonintegrating reprogramming vectors; (3) a cost effective method for generating vector free iPSCs; (4) a single transfection; and (5) the use of small molecules to facilitate epigenetic reprogramming. Briefly, peripheral blood mononuclear cells (PBMCs) are isolated from routine phlebotomy samples and then cultured in defined growth factors to yield a highly proliferative erythrocyte progenitor cell population that is remarkably amenable to reprogramming. Nonintegrating, nontransmissible episomal plasmids expressing OCT4, SOX2, KLF4, MYCL, LIN28A, and a p53 short hairpin (sh)RNA are introduced into the derived erythroblasts via a single nucleofection. Cotransfection of an episome that expresses enhanced green fluorescent protein (eGFP) allows for easy identification of transfected cells. A separate replication-deficient plasmid expressing Epstein-Barr nuclear antigen 1 (EBNA1) is also added to the reaction mixture for increased expression of episomal proteins. Transfected cells are then plated onto a layer of irradiated mouse embryonic fibroblasts (iMEFs) for continued reprogramming. As soon as iPSC-like colonies appear at about twelve days after nucleofection, HDAC inhibitors are added to the medium to facilitate epigenetic remodeling. We have found that the inclusion of HDAC inhibitors routinely increases the generation of fully reprogrammed iPSC colonies by 2 fold. Once iPSC colonies exhibit typical human embryonic stem cell (hESC) morphology, they are gently transferred to individual iMEF-coated tissue culture plates for continued growth and expansion.
authors.affiliation: Clinical Research Division, Fred Hutchinson Cancer Research Center.
authors.forename: Jesse J
authors.initials: JJ
authors.lastname: Hubbard
authors.affiliation: Division of Hematology, The Children's Hospital of Philadelphia.
authors.forename: Spencer K
authors.initials: SK
authors.lastname: Sullivan
authors.affiliation: Department of Pathology, The Children's Hospital of Philadelphia.
authors.forename: Jason A
authors.initials: JA
authors.lastname: Mills
authors.affiliation: Clinical Research Division, Fred Hutchinson Cancer Research Center.
authors.forename: Brian J
authors.initials: BJ
authors.lastname: Hayes
authors.affiliation: Clinical Research Division, Fred Hutchinson Cancer Research Center.
authors.forename: Beverly J
authors.initials: BJ
authors.lastname: Torok-Storb
authors.affiliation: Clinical Research Division, Fred Hutchinson Cancer Research Center; aramakri@fhcrc.org.
authors.forename: Aravind
authors.initials: A
authors.lastname: Ramakrishnan
mesh_terms: D000369:Aged, 80 and over; D000818:Animals; D001773:Blood Cells / Q000166:cytology* / Q000187:drug effects*; D004900:Erythroblasts / Q000166:cytology / Q000187:drug effects; D056572:Histone Deacetylase Inhibitors / Q000494:pharmacology*; D006801:Humans; D057026:Induced Pluripotent Stem Cells / Q000166:cytology*; D000090062:Kruppel-Like Factor 4; D051379:Mice
chemical_list: D056572:Histone Deacetylase Inhibitors; C000715467:KLF4 protein, human; C000715468:Klf4 protein, mouse; D000090062:Kruppel-Like Factor 4