Integration-free human induced pluripotent stem cells from blood
Provided herein are methods for generating human induced pluripotent stem cells free from genomic integration of exogenous transgenes by transfecting into nucleated blood cells one or more DNA expression vectors (e.g., plasmid vectors) that do not contain a mammalian origin of replication, and encode and permit expression of one or more reprogramming factors (e.g., Oct4, Sox2, Klf4, and c-Myc). Also provided herein are the integration-free human induced pluripotent stem cells obtained by the methods described herein.
1. A method for generating integration-free human induced pluripotent stem cells, comprising transfecting human nucleated blood cells with one or more plasmid DNA expression vectors encoding reprogramming factors (a) Oct4, Sox2, Klf4, and c-Myc; (b) Oct4, Sox2, and Klf4; (c) Oct4, Sox2, Klf4, c-Myc, and Nanog; or (d) Oct 4, Sox2, Lin-28, and Nanog, expressing the encoded reprogramming factors in the transfected nucleated blood cells, and culturing the transfected nucleated blood cells under conditions adapted to growth of human induced pluripotent stem cells, identifying human induced pluripotent stem cell colonies, identifying integration-free human induced pluripotent stem cells that do not comprise exogenous DNA from the one or more plasmid DNA expression vectors, wherein:
(i) at least two of the encoded reprogramming factors are linked by an intervening self-cleaving peptide sequence;
(ii) the transfected human nucleated blood cells do not express an exogenous trans-acting factor that binds to a replication origin of an extra-chromosomal template or the one or more plasmid DNA expression vectors do not comprise a mammalian origin of replication sequence; and
(iii) the transfection is performed only one time, or is performed only within a single 24 hour period.
2. The method of claim 1 , wherein the one or more plasmid DNA expression vectors encode the reprogramming factors Oct4, Sox2, Klf4, and c-Myc.
3. The method of claim 1 , wherein the one or more plasmid DNA expression vectors encode the reprogramming factors Oct4, Sox2, Klf4, c-Myc, and Nanog.
4. The method of claim 1 , wherein the transfection comprises transfecting the nucleated blood cells with only one transfection method.
5. The method of claim 1 , wherein the one or more plasmid DNA expression vectors further encode a reporter protein that is expressed in the transfected nucleated blood cells.