Lipid-stripped albumin enables high-efficiency mRNA reprogramming of adult human fibroblasts

Jasmine K. Harris1, Mitchell Kopacz1, Franklin Kostas1, Mackenzie Parmenter2, Christopher B. Rohde1, Matthew Angel1

1Factor Bioscience Inc., Cambridge, MA, 2Novellus, Inc., Cambridge, MA

Mol Ther, Vol 29, No 4S1, 2021

Induced pluripotent stem cells (iPSCs) have emerged as an exciting platform for developing personalized cell therapies. However, clinical applications are limited by the low efficiency of viral and episomal reprogramming methods and safety concerns related to vector integration and genomic scarring. Using messenger RNA (mRNA) to reprogram cells avoids these issues, however previously reported mRNA-based reprogramming methods required lengthy transfection schedules and suffer from the need to use proprietary media formulations. Here we report a seven-day, high-efficiency, immunosuppressant-free mRNA reprogramming protocol for dedifferentiating adult human dermal fibroblasts into iPSCs. To achieve this, we designed a chemically defined, animal component-free and xeno-free reprogramming medium that contains specially treated albumin, which we show enhances transfection efficiency and supports efficient reprogramming. We measured reprogramming efficiency by counting the number of SSEA4 positive colonies generated from a starting culture of 1×103 adult human dermal fibroblasts. Using this approach, we measured a 6% reprogramming efficiency, which is a 60-fold improvement to the <0.1% efficiency typically obtained with viral and episomal reprogramming methods. We analyzed iPSCs for expression of the pluripotency markers OCT4 (POU5F1), SSEA4, and NANOG, as well as by differentiating the cells in vitro into mesenchymal stem cells, hematopoietic stem cells, osteoblasts, adipocytes, chondrocytes, T cells, natural killer (NK) cells, cardiomyocytes, and neurons. We also explored the role of other media components, including TGF-β, in mRNA reprogramming and evaluated protein expression, immunogenicity, and reprogramming efficiency of various novel mRNA chemistries and molecular designs.

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