Diamond-Blackfan anemia (DBA) is associated with developmental defects and profound anemia. Mutations in genes encoding a ribosomal protein of the small (eg, RPS19) or large (eg, RPL11) ribosomal subunit are found in more than half of these patients. The mutations cause ribosomal haploinsufficiency, which reduces overall translation efficiency of cellular mRNAs. We reduced the expression of Rps19 or Rpl11 in mouse erythroblasts and investigated mRNA polyribosome association, which revealed deregulated translation initiation of specific transcripts. Among these were Bag1, encoding a Hsp70 cochaperone, and Csde1, encoding an RNA-binding protein, and both were expressed at increased levels in erythroblasts. Their translation initiation is cap independent and starts from an internal ribosomal entry site, which appeared sensitive to knockdown of Rps19 or Rpl11. Mouse embryos lacking Bag1 die at embryonic day 13.5, with reduced erythroid colony forming cells in the fetal liver, and low Bag1 expression impairs erythroid differentiation in vitro. Reduced expression of Csde1 impairs the proliferation and differentiation of erythroid blasts. Protein but not mRNA expression of BAG1 and CSDE1 was reduced in erythroblasts cultured from DBA patients. Our data suggest that impaired internal ribosomal entry site–mediated translation of mRNAs expressed at increased levels in erythroblasts contributes to the erythroid phenotype of DBA.

Hematopoietic stem cells differentiate into a broad range of specialized blood cells. This process is tightly regulated and depends on transcription factors, micro-RNAs, and long non-coding RNAs. Recently, also circular RNA (circRNA) were found to regulate cellular processes. Their expression pattern and their identity is however less well defined. Here, we provide the first comprehensive analysis of circRNA expression in human hematopoietic progenitors, and in differentiated lymphoid and myeloid cells. We here show that the expression of circRNA is cell-type specific, and increases upon maturation. CircRNA splicing variants can also be cell-type specific. Furthermore, nucleated hematopoietic cells contain circRNA that have higher expression levels than the corresponding linear RNA. Enucleated blood cells, i.e. platelets and erythrocytes, were suggested to use RNA to maintain their function, respond to environmental factors or to transmit signals to other cells via microvesicles. Here we show that platelets and erythrocytes contain the highest number of circRNA of all hematopoietic cells, and that the type and numbers of circRNA changes during maturation. This cell-type specific expression pattern of circRNA in hematopoietic cells suggests a hithero unappreciated role in differentiation and cellular function.

Transfusion of donor-derived red blood cells (RBC) 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 vitro cultured, customizable RBC would negate these concerns and further increase precision medicine. Large-scale, cost-effective production depends on optimization of culture conditions. We developed a defined medium and adapted our protocols to good manufacturing practice (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 (or from 100 million peripheral blood mononuclear cells, 2 to 4 mL packed red cells can be produced). Expanded erythroblast cultures could be differentiated to CD71^dimCD235a⁺CD44⁺CD117⁻DRAQ5⁻ RBC 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 RBC was comparable to in vivo reticulocytes. Daily RNA sampling during differentiation followed by RNA-sequencing 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 1 L per reactor, allowing transition toward clinical studies and small-scale applications.

Effective T cell responses against invading pathogens require the concerted production of three key cytokines: TNF-α, IFN-γ, and IL-2. The cytokines functionally synergize, but their production kinetics widely differ. How the differential timing of expression is regulated remains, however, poorly understood. We compared the relative contribution of transcription, mRNA stability, and translation efficiency on cytokine production in murine effector and memory CD8⁺ T cells. We show that the immediate and ample production of TNF-α is primarily mediated by translation of preformed mRNA through protein kinase C (PKC)-induced recruitment of mRNA to polyribosomes. Also, the initial production of IFN-γ uses translation of preformed mRNA. However, the magnitude and subsequent expression of IFN-γ, and of IL-2, depends on calcium-induced de novo transcription and PKC-dependent mRNA stabilization. In conclusion, PKC signaling modulates translation efficiency and mRNA stability in a transcript-specific manner. These cytokine-specific regulatory mechanisms guarantee that T cells produce ample amounts of cytokines shortly upon activation and for a limited time.