Large-scalein-vitroproduction of red blood cells from human peripheral blood mononuclear cells

Abstract

Transfusion of donor-derived red blood cells is the most common form of cellular therapy. Donor availability and the potential risk of alloimmunization and other transfusion-related complications may, however, limit the availability of transfusion units especially for chronically transfused patients.In-vitrocultured, customizable red blood cells would negate these concerns and introduce precision medicine. Large-scale, cost effective production depends on optimization of culture conditions. We developed a defined medium and adapted our protocols to GMP culture requirements, which reproducibly provided pure erythroid cultures from peripheral blood mononuclear cells without prior CD34+isolation, and a 3×107-fold increase in erythroblasts in 25 days. Expanded erythroblast cultures could be differentiated to CD71dimCD235a+CD44+CD117−DRAQ5−red blood cells in 12 days. More than 90% of the cells enucleated and expressed adult hemoglobin as well as the correct blood group antigens. Deformability and oxygen binding capacity of cultured red blood cells was comparable toin-vivoreticulocytes. Daily RNA sampling during differentiation followed by RNA-seq provided a high-resolution map/resource of changes occurring during terminal erythropoiesis. The culture process was compatible with upscaling using a G-Rex bioreactor with a capacity of 1L per reactor, allowing transition towards clinical studies and small-scale applications.

Circular RNAs exhibit limited evidence for translation, or translation regulation of the mRNA counterpart in terminal hematopoiesis

“…This in vitro differentiation model resulted in overall gene expression patterns that closely resembled that of ex vivo isolated cells ( Supplemental Fig. S1A ; Supplemental Table S1 ; Heshusius et al 2019 ). Also, physical properties such as hemoglobin content, oxygen association, and dissociation capacity and deformability matched those of ex vivo red blood cells ( Heshusius et al 2019 ).…”

“…S1A ; Supplemental Table S1 ; Heshusius et al 2019 ). Also, physical properties such as hemoglobin content, oxygen association, and dissociation capacity and deformability matched those of ex vivo red blood cells ( Heshusius et al 2019 ).…”

“… CircRNAs are abundant during in vitro erythroid differentiation. ( A ) Erythroblast progenitors were cultured for 12 d under differentiation conditions, as in Heshusius et al (2019) . Samples were taken at indicated days of differentiation, and RNA-seq was performed ( n = 4 donors).…”

“…We first investigated if and how circRNA expression alters during terminal erythropoiesis. To this end, we examined paired-end RNA sequencing data of erythroblasts isolated from 4 donors that were cultured for 12 days under differentiating conditions, and that were harvested on each day between day 0 to 7, in addition to day 9 and 12 (Figure 1A; (Heshusius et al 2019)). This in vitro differentiation model resulted in overall gene expression patterns that closely resembled that of ex vivo isolated cells ((Figure S1A; Table S1; (Heshusius et al 2019)).…”

“…To this end, we examined paired-end RNA sequencing data of erythroblasts isolated from 4 donors that were cultured for 12 days under differentiating conditions, and that were harvested on each day between day 0 to 7, in addition to day 9 and 12 (Figure 1A; (Heshusius et al 2019)). This in vitro differentiation model resulted in overall gene expression patterns that closely resembled that of ex vivo isolated cells ((Figure S1A; Table S1; (Heshusius et al 2019)). Also physical properties such as hemoglobin content, oxygen association and dissociation capacity and deformability matched those of ex vivo red blood cells (Heshusius et al 2019).…”