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| Status |
Public on Apr 26, 2013 |
| Title |
Transgene-free iPSCs generated from small volume peripheral blood nonmobilized CD34+ cells |
| Organism |
Homo sapiens |
| Experiment type |
Methylation profiling by genome tiling array
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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±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±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 in 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 rearrangements typical of T or B lymphocytes, and because we demonstrated that purified CD14+ monocytes do not yield iPSC colonies under these reprogramming conditions.
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| Overall design |
Comparison of pluripipotent, induced pluripotent, and differentiated cell types
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| Contributor(s) |
Merling RK, Sweeney CL, Choi U, DeRavin SS, Myers TG, Otaizo-Carrasquero F, Pan J, Linton G, Chen L, Koontz S, Theobald NL, Malech HL |
| Citation(s) |
23386128 |
| Submission date |
Sep 11, 2012 |
| Last update date |
Mar 22, 2019 |
| Contact name |
Timothy G Myers |
| E-mail(s) |
tgm@nih.gov
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| Organization name |
National Institute of Allergy and Infectious Diseases
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| Department |
Research Technologies Branch
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| Lab |
Genomic Technologies Section
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| Street address |
50 South Drive, Room 5509
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| City |
Bethesda |
| State/province |
MD |
| ZIP/Postal code |
20892-8005 |
| Country |
USA |
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| Platforms (1) |
| GPL13534 |
Illumina HumanMethylation450 BeadChip (HumanMethylation450_15017482) |
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| Samples (21)
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| Relations |
| BioProject |
PRJNA174964 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSE40790_RAW.tar |
183.1 Mb |
(http)(custom) |
TAR |
| GSE40790_unmethylated_methylated_intensities.txt.gz |
50.5 Mb |
(ftp)(http) |
TXT |
| Processed data included within Sample table |
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