Generation of Enucleated Erythrocytes From Lin^-CD45^-CD133⁺ Cells Isolated From Human Umbilical Cord Blood In Vitro

At present, healthcare facilities often face blood shortages because of the low supply of donated blood relative to the high demand. Therefore, efforts to develop red blood cell (RBC) production methods have gained traction. In this work, Lin^-CD45^-CD133⁺ cells were isolated from human umbilical cord blood (UCB) and subsequently differentiated into erythrocytes in vitro in serum-free culture medium.

Lin^-CD45^-CD133⁺ cells were prepared from mononuclear cells (MNCs) using magnetic-activated cell sorting (MACS). The characteristics of Lin^-CD45^-CD133⁺ cells were confirmed using flow cytometry analysis, colony-forming unit (CFU) assays, morphological analysis, immunocytochemistry (ICC) analysis, and real-time fluorescent quantitative polymerase chain reaction (RT-PCR). Erythrocytes were differentiated in serum-free medium supplemented with stem cell factor (SCF), interleukin-3 (IL-3), erythropoietin (EPO), and FK506 for 13 days, after which autoplasma derived from UCB was added at a concentration of 5% beginning on day 14. Erythroid differentiation and maturation were examined using electron microscopy and flow cytometric analysis.

Lin^-CD45^-CD133⁺ cells were successfully obtained from UCB. These cells were slightly smaller than normal RBCs and had a high nucleus-to-cytoplasm ratio. Oct-4 and Nanog were expressed at both the mRNA and protein levels in Lin^-CD45^-CD133⁺ cells. Most of the colonies were burst-forming unit-erythroid (BFU-E). After 7 days of in vitro culture, the Lin^-CD45^-CD133⁺ cells were negative for CD133 expression and positive for CD45 expression. The percentage of CD71⁺ cells gradually increased, peaked on day 10, and then started decreasing on day 13. The percentage of CD235a⁺ cells increased gradually after day 7 and peaked on day 13. CD240 expression was detected on day 18, with the highest level detected on day 20. The number of erythroid cells increased persistently during differentiation, and their morphology was consistent with that of normal erythrocytes.

An ex vivo culture system was developed that can generate human erythrocytes from Lin^-CD45^-CD133⁺ cells isolated from human UCB.