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

Merling RK1, Sweeney CL, Choi U, De Ravin SS, Myers TG, Otaizo-Carrasquero F, Pan J, Linton G, Chen L, Koontz S, Theobald NL, Malech HL.
Source: Blood
Publication Date: (2013)
Issue: 121(14): e98-107
Research Area:
Immunotherapy / Hematology
Stem Cells
Basic Research
Cells used in publication:
CD34+ cell, human
Species: human
Tissue Origin: blood
Culture Media:

Nonmobilized CD34+ cells were expanded for 2 to 4 days to obtain approximately 50 000 cells in X-VIVO 10 media  with 1% human serum albumin; 50 ng/mL each of human stem cell factor, Flt3-ligand, and thrombopoietin; and 5 or 50 ng/mL of human interleukin-3.


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.