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

Abstract

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.

Immunoregulatory properties of erythroid nucleated cells induced from CD34+ progenitors from bone marrow

“…Our findings are relevant and important, since CD71+ erythroid cells can have both a positive regulatory effect (in inflammatory and autoimmune diseases, pregnancy, and the neonatal period) and a negative regulatory effect (in cancer, infectious diseases, and anemia) on the mechanisms of the immune system [15]. Differentiation of erythrocytes in vitro is an important clinically significant process for modeling the study of the development of erythrocytes in normal and pathological hematopoiesis, for the development of therapeutic protocols aimed at the treatment of diseases associated with erythrocytes, such as sickle cell anemia, thalassemia, various anemias [1,50,51]. In addition to immunoregulatory properties, in vitro erythropoiesis is a good model for studying translational control in cell differentiation and physiology [52], antibody screening tools, disease models or developmental research tools [53].…”

Current status of red blood cell manufacturing in 3D culture and bioreactors

“…Additional additives, such as thrombopoietin, IL-6, feline McDonough sarcoma-like tyrosine kinase 3, heparin, glucocorticoids, insulin-like growth factor-1, transforming growth factor β agonists, and peroxisome proliferator-activated receptor α agonists, can be added and further modified; with or without serum or feeder layers [ 8 - 10 ].…”