EHA Library - The official digital education library of European Hematology Association (EHA)

Donor-derived red blood cells (RBC) are the most common form of cellular therapy. However the source of cells is dependent on donor availability with a potential risk of allo-immunization and blood borne diseases.

The in vitro production of RBC has proven to be successful, however there are barriers to overcome prior to clinical application. e.g: xeno-free culturing methods, scale up cultures to obtain transfusion units (1-2*10¹² erythrocytes), and for iPSC we need virus- and transgene-free reprogramming protocols.

To solve the above mentioned issues a customized humanized GMP-grade medium (Cellquin) was generated in order to control erythroid culture parameters and to reduce culture costs. This medium allowed 1*10⁸ times erythroid expansion from PBMCs to pure adult EBL cultures within 25 days, comparable to non-GMP commercial media. To generate iPSC, a non-integrative polycistronic episomal vector containing (OCT4-SOX2-KLF4-cMYC-LIN28) was used to reprogram PBMC-expanded EBLs to iPSC, displaying pluripotency potential and normal karyotype. iPSCs were adapted to single cell passage allowing directed colony differentiation using a feeder-free monolayer approach. From day 6 of differentiation Cellquin was applied with lineage-specific growth factors, resulted iPSC differentiation to EBLs which was initiated by the appearance of hemogenic endothelium following hematopoietic specification. Our differentiation method resulted in ~150*10⁶ CD41- CD34- CD71+ CD235+ CD36+ expanded EBLs from 1200 iPSCs within 21 days (12 days of iPSC diff. + 9 days of expansion). Further maturation of iPSC-EBLs yielded CD71+ CD235+ CD36- pure orthochromatic normoblasts expressing mainly gamma globin chains (fetal) and small amount of beta globins (adult) in agreement with literature. Currently we are testing enucleation potential of matured iPSC-EBLs.

Here we showed that our monolayer approach is simple, highly controlled and compatible with upscaling. Avoiding virus-, integrative reprogramming, feeders and with our GMP-grade media we maintained a cost effective system moving toward clinical application.