Abstract: Objective To optimize a four-stage induction system for the in vitro directed differentiation of CD34⁺ stem cells from umbilical cord blood into functional red blood cells (RBCs), and to systematically characterize cell proliferation, morphological changes, surface marker expression, and hemoglobin synthesis under this system, providing an experimental basis for improving in vitro RBC preparation technology. Methods Umbilical cord blood was collected from 6 healthy newborns. Mononuclear cells were isolated by density gradient centrifugation, and CD34⁺ stem cells were selected using immunomagnetic beads. A 28-day four-stage induction differentiation system was established, with precise regulation of the combination and concentration of specific inducing factors at each stage. Cell counts were performed at different time points, morphology was assessed by Wright-Giemsa staining, erythroid surface markers (CD45, CD71, CD235a) were analyzed via flow cytometry, color changes of cell pellets were observed, and hemoglobin content was measured using a hemoglobin test kit. The differentiation process was systematically analyzed. Results Cells continuously proliferated throughout the differentiation process, reaching a peak at day 14. Morphologically, cells gradually progressed from primitive progenitors to erythroid cells, with enucleated or nuclear-shifted cells of similar size to mature RBCs observed at day 28. Surface marker expression showed a typical trajectory of erythroid differentiation: CD45 progressively decreased (28-day positivity rate 24.93%), CD71 peaked at day 14 (84%) and then decreased, and CD235a steadily increased (28-day positivity 71.64%). Red pigmentation appeared in cell pellets at day 21, and deepened at day 28, with a hemoglobin content of 70.7 g/L. Conclusion The optimized induction system efficiently directs CD34⁺umbilical cord blood stem cells towards erythroid differentiation and functional hemoglobin production through precise stage-specific factor regulation. This study could provide specific schemes and experimental data to support the advancement of in vitro RBC generation systems.

Key words: Umbilical cord blood, CD34⁺ stem cells, Red blood cells, In vitro differentiation, Four-stage induction