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23386128
Transgene-free iPSCs generated from small volume peripheral blood nonmobilized CD34+ cells.

Blood, 2013/4/04;121(14):e98-107.

Merling RK[1], Sweeney CL, Choi U, De Ravin SS, Myers TG, Otaizo-Carrasquero F, Pan J, Linton G, Chen L, Koontz S, Theobald NL, Malech HL

Affiliations

PMID: 23386128DOI: 10.1182/blood-2012-03-420273

Impact factor: 25.476

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.
MeSH terms
Antigens, CD34; Base Sequence; Cell Culture Techniques; Cell Line; Cell Lineage; Cell Separation; Cellular Reprogramming; DNA Fingerprinting; Embryonic Stem Cells; Fibroblasts; Gene Rearrangement, B-Lymphocyte; Gene Rearrangement, T-Lymphocyte; Genome-Wide Association Study; Humans; Immunologic Deficiency Syndromes; Induced Pluripotent Stem Cells; Integrases; Kruppel-Like Factor 4; Lentivirus; Lymphocytes; Molecular Sequence Data; Monocytes; Sendai virus; Teratoma; Transduction, Genetic; Transgenes
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