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临床输血与检验

临床输血与检验 ›› 2026, Vol. 28 ›› Issue (3): 339-344.DOI: 10.3969/j.issn.1671-2587.2026.03.007

• 基础研究 • 上一篇    下一篇

脐带血CD34+干细胞四阶段诱导分化为功能性红细胞的体系优化与特征分析*

凌亚亭1,2, 张春2, 智渊1,2, 张钰1,2, 杜海林1,2, 傅强2, 何成涛1,2   

  1. 1南京红十字血液中心研究室,江苏南京 210003;
    2南京红十字血液中心输血医学重点实验室,江苏南京 210003
  • 收稿日期:2025-12-01 出版日期:2026-06-20 发布日期:2026-07-07
  • 通讯作者: 何成涛,主要从事免疫血清学检测方面研究,（E-mail）hechengtao413@126.com。共同通信作者：傅强,主要从事献血服务,管理和血液安全工作,（E-mail）fuqiangnj@hotmail.com。
  • 作者简介:凌亚亭,主要从事免疫血清学检测方面研究,（E-mail）17719450825@163.com。并列第一作者：张春,主要从事输血安全管理工作,（E-mail）18951670988@189.cn。
  • 基金资助:
    *本项目受南京红十字血液中心输血医学重点实验室开放课题（No.2024009）资助

Experimental Optimization and Characterization of A Four-stage Induction System for Differentiating Umbilical Cord Blood CD34+ Stem Cells into Functional Red Blood Cells

LING Yating1,2, ZHANG Chun2, ZHI Yuan1,2, ZHANG Yu1,2, DU Hailin1,2, FU Qiang2, HE Chengtao1,2   

  1. 1Laboratory, Nanjing Red Cross Blood Center, Nanjing 210003;
    2Key Laboratory of Transfusion Medicine, Nanjing Red Cross Blood Center, Nanjing 210003
  • Received:2025-12-01 Online:2026-06-20 Published:2026-07-07

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摘要： 目的 优化脐带血来源CD34+干细胞体外定向分化为功能性红细胞的四阶段诱导体系,明确该体系下细胞增殖、形态演变、表面标志物表达及血红蛋白合成的特征,为体外红细胞制备技术提供实验依据。方法 收集6名健康新生儿脐带血,通过密度梯度离心法从脐带血中分离单个核细胞,免疫磁珠法分选出CD34+干细胞。建立四阶段诱导分化体系（28天）,按阶段精准调控特异性诱导因子组合及浓度。在不同时间点进行细胞计数,瑞氏-吉姆萨染色观察细胞形态、流式细胞术检测细胞表面标志物（CD45、CD71、CD235a）表达、肉眼观察细胞沉淀颜色、血红蛋白检测试剂盒检测血红蛋白含量,系统分析分化过程特征。结果 分化全程细胞持续增殖,14天达增殖高峰;形态上从原始细胞逐步向红系细胞过渡,28天可见与成熟红细胞体积相似的脱核或核偏位细胞;表面标志物呈现红系分化典型轨迹：CD45持续降低（28天阳性率24.93%）,CD71 14天达峰值（84%）后下降,CD235a持续升高（28天达71.64%）;21天细胞沉淀出现红色,28天红色加深,血红蛋白含量达70.7 g/L。结论 本研究优化的四阶段诱导体系通过精准的阶段化因子调控,可高效驱动脐带血CD34+干细胞向红系定向分化并合成功能性血红蛋白,为体外红细胞制备的体系优化提供具体方案和实验数据支撑。

关键词: 脐带血, CD34+干细胞, 红细胞, 体外分化, 四阶段诱导

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

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