Red blood cells differentiated in vitro using sequential liquid and semi-solid culture as a pre-clinical model

The in vitro erythrocyte differentiation model remains a strong, clinically relevant tool to model erythroid development in normal and disease related hematopoiesis. This model also has application to developing therapeutics for diseases related to red blood cells such as sickle cell anemia where targeting increased expression of fetal hemoglobin has been a major emphasis. Since the original protocol's publication in 2002, many groups have published modified methodologies to address issues in efficiency of maturation and terminal enucleation, as well as in scalability. While all reports have merit and show efficient enucleation, the methodologies used vary widely in technique and cytokine content. Additionally, despite the strengths in these methods, reproducibility of efficient differentiation to the point of differentiation is difficult. To address these limitations, we developed a streamlined process where total PBMCs are first primed using the original liquid culture expansion phase (published in 2002) before being differentiated with minimal input via standardized, commercially purchased semi-solid medium culture pre-supplemented with erythropoietin. Our data show this methodology to produce similar levels of CD235/CD71 positivity as previous methods but with enhanced CD235 solo positivity and evidence of enucleated cells in comparison with other widely used methods. Given the difficulty and wide variation in in vitro differentiation techniques, we present this methodology as a streamlined methodology for production of mature erythroid cells with minimal input using easily purchased reagents.