iPSC-Derived KitD816V/Jak2V817F Double-Mutant Kitjak Cell Lines Allow Inexpensive Large-Scale Production of Enucleated Red Blood Cells in the Absence of SCF and Erythropoietin

Cultured Red Blood Cells (cRBCs) have the potential to supplement transfusion needs and to be used as drug carriers or immune tolerization agents. Several methods have been devised to amplify primary Hematopoietic Progenitor cells (HPCs) into cRBCs. Although these protocols have the potential to relieve local blood shortages, they are not ideal for large scale cRBC production because the primary HPCs must constantly be replenished.

Immortalized human erythroid cell lines have been developed by overexpressing the E6/E7 proteins of human papilloma virus 16, c-Myc, Bcl-XL, Spi1 or Bmi-1. Many of these lines can be cultured for months and in some cases indefinitely, and some retain their ability to differentiate into cRBCs with rates of enucleation varying between 0.1-30%. These lines are useful as research tools and are a potential source of cells for some translational applications. Nevertheless, karyotypic instability, the cost of the media and cytokines, and cumbersome culture procedure to maintain some of these lines remain major obstacles for their large-scale use.

We previously developed the Pluripotent Stem Cell Robust Erythroid Differentiation (PSC-RED) protocol to differentiate induced-pluripotent stem cells (iPSCs) into cRBCs. The PSC-RED protocol yields a large number of enucleated RBCs, does not require any albumin, and allows transferrin recycling. Despite these advantages, the PSC-RED protocol is not well-adapted for large-scale cRBC production because differentiation of an iPSC into a RBC takes up to 45 days and because of cytokine cost.

To eliminate the requirement for Stem Cell Factor (SCF) and Erythropoietin (Epo) which accounts for more than 95% of total cytokine cost in the PSC-RED protocol, we have used CRSPR/cas9 technology to introduce in iPSCs the kitD816V mutation that allows the SCF receptor (the kit gene) to constitutive signal in the absence of SCF, and the Jak2V617F mutation that allows constitutive signaling through the JAK/STAT pathway in the absence of Epo.

We report here that kitD816V, Jak2V617F double mutants iPSCs readily differentiate into self-renewing erythroid progenitors cells that can grow in the absence of any cytokines for >60 population doubling ( >10¹⁹ fold expansion for >5 months) in an inexpensive culture medium containing little more than two small molecules and low amounts of recombinant transferrin. Importantly, KitJak cell lines can be reproducibly generated from iPSCs by a two-week differentiation procedure without any time lag, suggesting that no other mutations are necessary to generate these self-renewing erythroid progenitors. Indeed, we have shown using a next-generation sequencing approach that KitJak cells appear to be remarkably karyotypically stable since multiple experiments fail to detect any chromosomal aneuploidy after long-term culture.

KitJak cells have an immuno-phenotype of late CFU-E/pro-Erythroblast, can be cultured at high density and can be induced to differentiate into cRBCs using a 10- to 12-day differentiation protocol that leads to a 20 to 40-fold cell amplification. The rate of enucleation is 30-40%, providing an overall yield of 7-14 RBCs per self-renewing KitJak cell. The cRBCs produced express mostly fetal hemoglobin, are slightly larger that adult cells, and have normal hemoglobin content.

The development of these KitJak cell lines greatly facilitates the production of cRBCs and will pave the way to large-scale erythroid cell production for transfusion and other translational applications. As a first application, we show that the RBCs produced are fully compatible with the Immucor red blood cell typing system and can be used as reagent red blood cells.