Miltenyi Biotec B.V. & Co. KG Bergisch Gladbach, Nordrhein-Westfalen, Germany
Abstract: iPSCs with their potential to differentiate into numerous different cell types hold great potential for cellular therapy. Innovative therapeutic approaches often entail the application of clearly defined cells types or subsets that need to be isolated and enriched from heterogenous cell populations. Conventional flow sorters were predominantly designed for research applications. Open systems and harsh sorting conditions therefore often prevent the translation of protocols into the clinic. We hereby show how a gentle microchip-based sorting technology can enable enrichment of PSCs and their derivatives in the safety of a sterile cartridge.
Human fibroblasts were reprogrammed via repeated mRNA transfections and resulted in a mixture of successfully generated iPSCs and non- or partly reprogrammed undefined cells. This heterogenous cell culture was stained with Tra-1-60-PE and sorted immediately after reprogramming. The iPSCs could be successfully enriched from purities of 4-41% to 85-99 %. Furthermore transgenic iPSC constitutively expressing GFP to a differing degree could be effectively enriched from 13-51% to a population of 72-98%. To obtain monoclonal transgenic iPSCs the sorted fraction was applied to a single-cell dispenser and seeded in 96-well plates. The cells showed high plating efficiency after the combination of sorting and subsequent single-cell dispension while maintaining typical iPSC morphology. Next, various iPSC-derived cell types were enriched after differentiation. Retinal pigmented epithelial cells could be enriched from a purity of 51-69% to 93-98% and showed a strongly pigmented phenotype after sorting. In other approaches cardiomyocytes were enriched from 56% to 96% and CD144-positive endothelial cells could be enriched from 48-49% to 90-96%. In all applications a high viability of over 85% was maintained after sorting.
In summary, this gentle, sterile sorting technology allows for efficient, reliable enrichment of PSCs and their derivatives and therefore facilitates working with clearly defined cell populations in a wide variety of PSC applications.
