title: Transgene-free iPSCs generated from small volume peripheral blood nonmobilized CD34+ cells.
abstract: A variety of somatic cells can be reprogrammed to induced pluripotent stem cells (iPSCs), but CD34(+) hematopoietic stem cells (HSCs) present in nonmobilized peripheral blood (PB) would be a convenient target. We report a method for deriving iPSC from PB HSCs using immunobead purification and 2- to 4-day culture to enrich CD34(+) HSCs to 80% ± 9%, followed by reprogramming with loxP-flanked polycistronic (human Oct4, Klf4, Sox2, and c-Myc) STEMCCA-loxP lentivector, or with Sendai vectors. Colonies arising with STEMCCA-loxP were invariably TRA-1-60(+), yielding 5.3 ± 2.8 iPSC colonies per 20 mL PB (n = 17), where most colonies had single-copy STEMCCA-loxP easily excised by transient Cre expression. Colonies arising with Sendai were variably reprogrammed (10%-80% TRA-1-60(+)), with variable yield (6 to >500 TRA-1-60(+) iPSC colonies per 10 mL blood; n = 6). Resultant iPSC clones expressed pluripotent cell markers and generated teratomas. Genomic methylation patterns of STEMCCA-loxP-reprogrammed clones closely matched embryonic stem cells. Furthermore, we showed that iPSCs are derived from the nonmobilized CD34(+) HSCs enriched from PB rather than from any lymphocyte or monocyte contaminants because they lack somatic rearrangements typical of T or B lymphocytes and because purified CD14(+) monocytes do not yield iPSC colonies under these reprogramming conditions.
authors: Laboratory of Host Defenses, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892-1456, USA.
authors: Randall K
authors: RK
authors: Merling
authors: Colin L
authors: CL
authors: Sweeney
authors: Uimook
authors: U
authors: Choi
authors: Suk See
authors: SS
authors: De Ravin
authors: Timothy G
authors: TG
authors: Myers
authors: Francisco
authors: F
authors: Otaizo-Carrasquero
authors: Jason
authors: J
authors: Pan
authors: Gilda
authors: G
authors: Linton
authors: Lifeng
authors: L
authors: Chen
authors: Sherry
authors: S
authors: Koontz
authors: Narda L
authors: NL
authors: Theobald
authors: Harry L
authors: HL
authors: Malech
mesh_terms: D018952:Antigens, CD34 / Q000378:metabolism; D001483:Base Sequence; D018929:Cell Culture Techniques / Q000379:methods; D002460:Cell Line; D019070:Cell Lineage / Q000235:genetics*; D002469:Cell Separation / Q000379:methods; D065150:Cellular Reprogramming / Q000235:genetics*; D016172:DNA Fingerprinting; D053595:Embryonic Stem Cells / Q000166:cytology / Q000502:physiology; D005347:Fibroblasts / Q000166:cytology / Q000502:physiology; D015322:Gene Rearrangement, B-Lymphocyte / Q000235:genetics; D015329:Gene Rearrangement, T-Lymphocyte / Q000235:genetics; D055106:Genome-Wide Association Study; D006801:Humans; D007153:Immunologic Deficiency Syndromes / Q000473:pathology; D057026:Induced Pluripotent Stem Cells / Q000166:cytology* / Q000502:physiology*; D019426:Integrases / Q000235:genetics; D000090062:Kruppel-Like Factor 4; D016086:Lentivirus / Q000235:genetics; D008214:Lymphocytes / Q000166:cytology / Q000502:physiology; D008969:Molecular Sequence Data; D009000:Monocytes / Q000166:cytology / Q000502:physiology; D029082:Sendai virus / Q000235:genetics; D013724:Teratoma / Q000473:pathology; D014161:Transduction, Genetic / Q000379:methods; D019076:Transgenes / Q000235:genetics*
chemical_list: D018952:Antigens, CD34; C000715467:KLF4 protein, human; D000090062:Kruppel-Like Factor 4; C045073:Cre recombinase; D019426:Integrases
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