(F1273) GENERATION OF HUMAN iPSC-DERIVED HEMATOPOIETIC PROGENITOR CELLS AND DIFFERENTIATION TO FUNCTIONAL iNK CELLS IN A FLEXIBLE AND FEEDER-FREE CULTURE SYSTEM
Staff Scientist Thermo Fisher Scientific Frederick, Maryland, United States
Abstract: Having a reliable source of high-quality hematopoietic stem and progenitor cells (HSPCs) is critical for developing various autologous and allogeneic cell and gene therapies. iPSC-derived HSPCs (iHSPCs) can be a consistent source for generating a variety of clinically relevant cell types, including Natural Killer (NK) and T cells. However, these differentiation processes typically take weeks or months, making labor- and material-intensive testing costly during development. Here we describe a fit-for-purpose feeder-free iHSPC differentiation system that uses all cell therapy grade and regulation-compliant reagents. This flexible protocol supports cost-effective, small-scale testing in multi-well plates and can be scaled up through shake flasks. By avoiding steps that can limit culture scale up, like spin embryoid body formation and complicated handling, this protocol facilitates the translation of process development learnings to large-scale manufacturing. Using CTS™ StemPro™-34 serum-free xeno-free medium, we generated iHSPCs that were >65% CD34+CD43+ and >90% viable. To demonstrate functionality, the iHSPCs were differentiated into iNK cells in 100 mL cultures in shake flasks, followed by two weeks of expansion resulting in >10e9 CD56+ CD3- iNK cells, with >90% purity and >80% viability. NK cells have great potential as an “off the shelf” allogeneic therapeutic product, as they can target cancer cells in an antigen- and HLA-independent manner. We therefore tested the functional ability of the expanded iNK cells to mediate anti-tumor activity against the K562 erythroleukemia cell line and cancer organoids (“tumoroids”) derived from human colorectal adenocarcinoma. The iNK cells displayed significant cytotoxic activity, killing >80% of target K562 cells and adenocarcinoma tumoroids in a dose-dependent manner, without requiring enrichment prior to use. In conclusion, we demonstrate the use of a versatile differentiation workflow to generate clinically relevant cell types, including functional iHSPCs and iNK cells. Utilizing a cost-effective differentiation system during process development can aid in effectively characterizing both the therapeutic cells and production processes, to facilitate a smooth transition to scale-up or scale-out for clinical applications.
