title: Rapid and Efficient Generation of Transgene-Free iPSC from a Small Volume of Cryopreserved Blood.
abstract: Human peripheral blood and umbilical cord blood represent attractive sources of cells for reprogramming to induced pluripotent stem cells (iPSCs). However, to date, most of the blood-derived iPSCs were generated using either integrating methods or starting from T-lymphocytes that have genomic rearrangements thus bearing uncertain consequences when using iPSC-derived lineages for disease modeling and cell therapies. Recently, both peripheral blood and cord blood cells have been reprogrammed into transgene-free iPSC using the Sendai viral vector. Here we demonstrate that peripheral blood can be utilized for medium-throughput iPSC production without the need to maintain cell culture prior to reprogramming induction. Cell reprogramming can also be accomplished with as little as 3000 previously cryopreserved cord blood cells under feeder-free and chemically defined Xeno-free conditions that are compliant with standard Good Manufacturing Practice (GMP) regulations. The first iPSC colonies appear 2-3 weeks faster in comparison to previous reports. Notably, these peripheral blood- and cord blood-derived iPSCs are free of detectable immunoglobulin heavy chain (IGH) and T cell receptor (TCR) gene rearrangements, suggesting they did not originate from B- or T- lymphoid cells. The iPSCs are pluripotent as evaluated by the scorecard assay and in vitro multi lineage functional cell differentiation. Our data show that small volumes of cryopreserved peripheral blood or cord blood cells can be reprogrammed efficiently at a convenient, cost effective and scalable way. In summary, our method expands the reprogramming potential of limited or archived samples either stored at blood banks or obtained from pediatric populations that cannot easily provide large quantities of peripheral blood or a skin biopsy.
authors.affiliation: The New York Stem Cell Foundation Research Institute, New York, NY, 10032, USA, mzhou@nyscf.org.
authors.forename: Hongyan
authors.initials: H
authors.lastname: Zhou
authors.forename: Hector
authors.initials: H
authors.lastname: Martinez
authors.forename: Bruce
authors.initials: B
authors.lastname: Sun
authors.forename: Aiqun
authors.initials: A
authors.lastname: Li
authors.forename: Matthew
authors.initials: M
authors.lastname: Zimmer
authors.forename: Nicholas
authors.initials: N
authors.lastname: Katsanis
authors.forename: Erica E
authors.initials: EE
authors.lastname: Davis
authors.forename: Joanne
authors.initials: J
authors.lastname: Kurtzberg
authors.forename: Scott
authors.initials: S
authors.lastname: Lipnick
authors.forename: Scott
authors.initials: S
authors.lastname: Noggle
authors.forename: Mahendra
authors.initials: M
authors.lastname: Rao
authors.forename: Stephen
authors.initials: S
authors.lastname: Chang
mesh_terms: D015703:Antigens, CD / Q000378:metabolism; D015415:Biomarkers / Q000378:metabolism; D001782:Blood Donors; D018826:CD13 Antigens / Q000378:metabolism; D018929:Cell Culture Techniques / Q000379:methods; D002454:Cell Differentiation; D019070:Cell Lineage; D065150:Cellular Reprogramming; D015925:Cryopreservation; D005312:Fetal Blood / Q000166:cytology*; D015870:Gene Expression; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D007150:Immunohistochemistry; D057026:Induced Pluripotent Stem Cells / Q000166:cytology* / Q000378:metabolism; D000090062:Kruppel-Like Factor 4; D051741:Kruppel-Like Transcription Factors / Q000235:genetics / Q000378:metabolism; D007963:Leukocytes, Mononuclear / Q000166:cytology*; D050814:Octamer Transcription Factor-3 / Q000235:genetics / Q000378:metabolism; D039904:Pluripotent Stem Cells / Q000166:cytology / Q000378:metabolism; D016271:Proto-Oncogene Proteins c-myc / Q000235:genetics / Q000378:metabolism; D011990:Receptors, Transferrin / Q000378:metabolism; D055748:SOXB1 Transcription Factors / Q000235:genetics / Q000378:metabolism; D029082:Sendai virus / Q000235:genetics; D019076:Transgenes / Q000235:genetics
chemical_list: D015703:Antigens, CD; D015415:Biomarkers; C068126:CD71 antigen; D000090062:Kruppel-Like Factor 4; D051741:Kruppel-Like Transcription Factors; C489427:MYC protein, human; D050814:Octamer Transcription Factor-3; C494173:POU5F1 protein, human; D016271:Proto-Oncogene Proteins c-myc; D011990:Receptors, Transferrin; C527782:SOX2 protein, human; D055748:SOXB1 Transcription Factors; D018826:CD13 Antigens