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Poster

Use of Non-modified RNAs for the Derivation of Clinically-relevant iPS Cell Lines from Human Blood, Urine and Skin Cells using GMP-compliant Reagents

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Poster

Use of Non-modified RNAs for the Derivation of Clinically-relevant iPS Cell Lines from Human Blood, Urine and Skin Cells using GMP-compliant Reagents

In 2015 we published the unique application of a non-modified RNA technology to the reprogramming of human adult fibroblasts and human blood-derived endothelial progenitor cells (EPCs). Human blood provides easy access to adult human cell types for reprogramming purposes. Notably, EPCs can be clonally isolated from fresh or frozen mononuclear cell (MNC) preparations from only 10 mL of either human peripheral or cord blood (Figure 1B). The adherent nature and high proliferative capacity of EPCs makes them highly desirable for repeated transfection with RNA when compared to commonly isolated hematopoietic suspension cell types. Furthermore, urine sampling provides perhaps the most non-invasive form of cell procurement. Urine-derived epithelial cells (UDCs) can be highly reproducibly isolated from only 30 mL of urine (Figure 1C). Here we present a flexible, yet powerful, RNA-based reprogramming method that combines a novel cocktail of synthetic, non-modified reprogramming [OCT4, SOX2, KLF4, cMYC, NANOG and LIN28 (OSKMNL)] and immune evasion mRNAs [E3, K3, B18] with reprogramming-enhancing mature, double-stranded microRNAs from the 302/367 cluster. Inclusion of the E3, K3, and B18 immune evasion mRNAs in the RNA transfection cocktail eliminates the need to supplement cell culture medium with recombinant B18 protein during the reprogramming process. This unique combination of different RNAs results in a highly efficient and robust reprogramming protocol using only GMP-compliant substrates (iMatrix-511 and vitronectin), media compositions (xeno-free medium or human serum supplementation) and RNA to produce clinically-relevant iPS cells (Figure 2A-C). Elevated Oct4 transcript levels in the RNA cocktail resulted in transfection protocols that efficiently generated TRA-1-60 positive iPS cell colonies from human adult and neonatal fibroblasts (up to 4%), blood-derived EPCs (up to 0.04%), HUVECs (up to 3%) UDCs (up to 0.5%) within 10 days (Figure 3 A-C and Table 1). Additionally, these different iPS cell lines demonstrate highly consistent cardiomyocyte, neural and early endoderm differentiation potential (Figure 4 A-C). The unique combined application of non-modified RNAs, using GMP-compliant reagents, for the cellular reprogramming of different human cell lines results in clinically-relevant iPS cells that are well suited for consistent application of in vitro differentiation protocols.
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