Transgene-Free iPSCs Generated from Small Volume Peripheral Blood Non-mobilized CD34+ Cells

A variety of somatic cells can be reprogrammed to induced pluripotent stem cells (iPSCs), but the small number of CD34+ hematopoietic stem cells (HSCs) present in non-mobilized peripheral blood (PB) would be a convenient and desirable starting target. We report here a simple method for targeting der...

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Bibliographic Details
Published in:Molecular therapy 2012-09, Vol.20 (9), p.50-50
Main Authors: Merling, Randall K, Sweeney, Colin L, Choi, Uimook, DeRavin, Suk See, Myers, Timothy G, Otaizo-Carrasquero, Francisco, Pan, Jason, Linton, Gilda, Chen, Lifeng, Koontz, Sherry, Theobald, Narda L, Malech, Harry L
Format: Article
Language:English
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Summary:A variety of somatic cells can be reprogrammed to induced pluripotent stem cells (iPSCs), but the small number of CD34+ hematopoietic stem cells (HSCs) present in non-mobilized peripheral blood (PB) would be a convenient and desirable starting target. We report here a simple method for targeting derivation of iPSC from non-mobilized PB CD34+ HSCs using immunobead purification and 2-4 day culture to achieve enrichment of CD34+ HSCs to 80+ or -9%, followed by reprogramming transduction with loxP-flanked polycistronic (Oct4, Klf4, Sox2, and c-Myc) STEMCCA-loxP lentivector at an MOI of 2. Our yield was 4.7+ or -2.2 iPSC colonies (n=12) per 20 mL non-mobilized peripheral blood, where most colonies had single copy STEMCCA-loxP that was easily excised by transient transfection expression of Cre. Resultant iPSC clones expressed pluripotent cell markers, had genomic methylation pattern closely matching embryonic stem cells, and generated teratomas containing tissues of all three germ lineages i n immunodeficient mice. Furthermore, we conclude that these iPSC are derived from the non-mobilized CD34+ HSCs enriched from PB rather than from any lymphocyte or monocyte contaminants because they lacked somatic rear-rangements typical of T or B lymphocytes, and because we demonstrated that purified CD14+ monocytes do not yield iPSC colonies under these reprogramming conditions.
ISSN:1525-0016
1525-0024