Stem cell-derived erythroid cells hold great potential for the treatment of blood-loss anemia and for erythropoiesis research; however, cultures using conventional flat plates or bioreactors have failed to show promising results. By mimicking the in vivo bone marrow (BM) environment in which most erythroid cells are physically aggregated, we show that a three-dimensional (3D) aggregate culture system facilitates erythroid cell maturation and red blood cell (RBC) production more effectively than two-dimensional high-density cell cultivation. Late erythroblasts (polychromatic or orthochromatic erythroblasts) were differentiated from cord blood CD34 + cells over 15 days and then allowed to form tight aggregates at a minimum density of 1 · 10 7 cells/mL for 2-3 days. To scale up the cell culture and to make the media supply efficient throughout the cell aggregates, several macroporous microcarriers and porous scaffolds were applied to the 3D culture system. In comparison to control culture conditions, erythroid cells in 3D aggregates were significantly more differentiated toward RBCs with significantly reduced nuclear dysplasia. When 3D culture was performed inside macroporous microcarriers, the cell culture scale was increased and cells exhibited enhanced differentiation and enucleation. Microcarriers with a pore diameter of approximately 400 mm produced more mature cells than those with a smaller pore diameter. In addition, this aggregate culture method minimized the culture space and media volume required. In conclusion, a 3D aggregate culture system can be used to generate transfusable human erythrocytes at the terminal maturation stage, mimicking the in vivo BM microenvironment. Porous structures can efficiently maximize the culture scale, enabling large-scale production of RBCs. These results enhance our understanding of the importance of physical contact among late erythroblasts for their final maturation into RBCs.

Gelsolin is an actin binding protein present in blood plasma and in cytoplasm of cells including macrophages. Gelsolin has important functions in cell cycle regulation, apoptotic regulation, and morphogenesis. Even though bone marrow macrophages and serum factors are critical for regulating erythropoiesis, the role of gelsolin on human erythroblasts has not been studied. Here, we investigated the effects of human recombinant plasma gelsolin (pGSN) on human immature erythroblasts. CD34+ cells isolated from cord blood were differentiated into erythroid cells in serum-free medium. When pGSN was applied to the culture medium, it accelerated basophilic and polychromatic erythroblast maturation and increased the enucleation rate with highly expressed erythropoiesis-related mRNAs. Also, pGSN was effective in reducing dysplastic changes caused by vincristine, suggesting its role in cell cycle progression at G2/M checkpoints. Also, pGSN activated caspase-3 during maturation stages in which caspase-3 functions as a non-apoptotic maturational signal or a pro-apoptotic signal depending on maturation stages. Our results suggest that pGSN has a pivotal role in maturation of erythroblasts and this factor might be one of the way how bone marrow macrophages and previously unknown serum factors work to control erythropoiesis. pGSN might be used as additive for in vitro production of erythrocytes.

The aim of this study was to develop a robust, quality controlled, and reproducible erythroid culture system to obtain high numbers of mature erythroblasts and red blood cells (RBCs). This was achieved using a fully controlled stirred‐tank bioreactor by the design of experiments (DOE) methods in the serum‐free medium by defining the appropriate culture parameters. Human cord blood CD34+ cells were first cultured in static flasks and then inoculated to stirred‐tank bioreactors. Cell diameter was gradually decreased and final RBC yields were significantly higher when cells were inoculated at sizes smaller than 12 μm. The larger immature cells in the basophilic stage did not survive, while smaller mature erythroid cells were successfully expanded at high agitation speeds, demonstrating that appropriate seeding timing is critical. A high inoculation cell density of 5 × 106 cells/ml was achieved reaching 1.5 × 107 cells/ml. By using DOE analysis fitted to precise stages of erythropoiesis, we were able to acquire the optimal culture parameters for pH (7.5), temperature (37°C), dissolved oxygen, agitation speed (500 rpm), inoculation timing (cell diameter 12–13 μm), media feeding regimen, and cell seeding density (5 × 106 cells/ml). The final pure RBCs showed appropriate functions compared with fresh donor RBCs, confirming that manufacturing mature RBCs with reproducibility is possible.

No abstract

Gelsolin, an actin-remodeling protein, is involved in cell motility, cytoskeletal remodeling, and cytokinesis and is abnormally expressed in many cancers. Recently, human recombinant plasma gelsolin protein (pGSN) was reported to have important roles in cell cycle and maturation of primary erythroblasts. However, the role of human plasma gelsolin in late stage erythroblasts prior to enucleation and putative clinical relevance in patients with myelodysplastic syndrome (MDS) and hemato-oncologic diseases have not been reported. Polychromatic and orthochromatic erythroblasts differentiated from human cord blood CD34+ cells, and human bone marrow (BM) cells derived from patients with MDS, were cultured in serum-free medium containing pGSN. Effects of pGSN on mitochondria, erythroid dysplasia, and enucleation were assessed in cellular and transcriptional levels. With pGSN treatment, terminal maturation at the stage of poly- and ortho-chromatic erythroblasts was enhanced, with higher numbers of orthochromatic erythroblasts and enucleated red blood cells (RBCs). pGSN also significantly decreased dysplastic features of cell morphology. Moreover, we found that patients with MDS with multi-lineage dysplasia or with excess blasts-1 showed significantly decreased expression of gelsolin mRNA (GSN) in their peripheral blood. When BM erythroblasts of MDS patients were cultured with pGSN, levels of mRNA transcripts related to terminal erythropoiesis and enucleation were markedly increased, with significantly decreased erythroid dysplasia. Moreover, pGSN treatment enhanced mitochondrial transmembrane potential that is unregulated in MDS and cultured cells. Our findings demonstrate a key role for plasma gelsolin in erythropoiesis and in gelsolin-depleted MDS patients, and raises the possibility that pGSN administration may promote erythropoiesis in erythroid dysplasia.