Feeder-free Derivation of human-induced pluripotent stem cells with synthetic messenger RNA

A method for dedifferentiating or reprogramming feeder-free, xeno-free and integration-free mammalian somatic cells to produce feeder-free, xeno-free and integration-free induced pluripotent stem cells comprising:

• a) seeding mammalian somatic cells in a volume of 0.5 ml to 1 ml of xeno-free culture medium on a feeder-free and xeno-free surface;
• b) plating the mammalian somatic cells from step a) at a cell density of 15,000 cells/ml to 500,000 cells/ml;
• c) transfecting the plated mammalian somatic cells from step b) with a composition comprising:
• 1) one ml or less of xeno-free culture medium:
• 2) an effective amount of a fusion product between a synthetic mRNA encoding OCT4 and an N-terminal-linked transactivation domain, and
• 3) synthetic mRNA reprogramming factors encoding SOX2 KLF4, NANOG, and optionally c-MYC or LIN28, wherein the synthetic mRNA reprogramming factors are provided at a concentration of up to about 800 ng/ml;
• d) culturing said somatic cells with the composition from step c) under feeder-free and xeno-free culture conditions until the media is changed once daily;
• and
• e) repeating step d) until the mammalian somatic cells are reprogrammed or de-differentiated to produce feeder-free, xeno-free and integration-free induced pluripotent stem cells.