Scientist STEMCELL Technologies Inc. Vancouver, British Columbia, Canada
Abstract: Allogeneic cell-based immunotherapy (e.g. stem cell transplantation) has become increasingly relevant in the treatment of hematologic malignancies. Human pluripotent stem cell (hPSC)-derived allogeneic therapy allows for genetic manipulation and unlimited expansion of the starting material. However, the development of stem cell-based immunotherapy is limited by high costs of optimization and manufacturing challenges, and requires robust scalable protocols to produce high-quality immune cells in large quantities up to 6 x 10^9 per dose. We developed a robust serum- and feeder-free culture system for large-scale generation of multipotent hematopoietic stem and/or progenitor cells (HSPCs) from hPSCs. hPSCs from five cell lines maintained in 2D or 3D systems were aggregate passaged into a suspension culture system, such as the 6-well plate orbital shaker cultures (2 mL) or the PBS-MINI MagDrive bioreactor (e.g. 100 mL), where embryoid bodies (EBs) were spontaneously formed and cultured in STEMdiff™ Hematopoietic - EB Media. After 12 days of culture, CD34+ HSPCs suspended in culture were isolated from EBs using a cell strainer, yielding 1.3 x 10^6 ± 1.6 x 10^5 CD34+ HSPCs per 1 mL culture vessel volume, 7.0 ± 0.6 CD34+ HSPCs per input hPSC, with a frequency of 73.1 ± 6.9% CD34+ cells (mean ± SEM; n = 36). Bioreactor cultures of 100 mL performed similarly to small-scale 3D cultures, and produced 1.6 x 10^7 - 1.1 x 10^8 live CD34+ cells (n = 4), using three different PSC lines. Multipotency of the resulting HSPCs was demonstrated by successful 2D differentiation to natural killer (NK), T, B, microglia, erythroid, and megakaryocyte lineages. For immune applications, CD56+ NK cells were produced using STEMdiff™ NK reagents under 2D scalable conditions with a frequency of 76 ± 5% and a yield of 1.0 x 10^3 ± 3.0 x 10^2 NK cells per input hPSC (n = 8). Additionally, differentiation parameters can be optimized in a small 2 mL culture before scaling up to ≥ 100 mL bioreactor systems, significantly reducing costs and barriers associated with large-scale generation of multipotent CD34+ hPSC-derived HSPCs for allogeneic immunotherapies and other applications.
