人多能干细胞向红细胞的诱导分化

Differentiation of human pluripotent stem cells into red blood cells

目前,体外生成人红细胞的实验技术较为复杂,为优化诱导步骤,采用两步法将人多能干细胞诱导分化为红细胞。首先,利用人多能干细胞(包括Rh阴性A型的脐带间充质干细胞(hUCMSC^(Rh-A))和人iPS(hiPS)细胞)在BVF培养液中进行诱导分化得到CD31~^+和CD34^+的阳性细胞群。经PCR和流式细胞仪检测CD31和CD34的表达发现,hUCMSC^(Rh-A)细胞诱导得到的CD31和CD34阳性细胞率分别是5.3%和22.7%;hiPS细胞诱导得到的CD31和CD34阳性细胞率分别是31.2%和8.2%。第二步,将获得的CD31^+和CD34^+的阳性细胞群在多种生长因子的作用下经过36 d诱导,分化为成熟红细胞。经吉姆萨染色检测得到的红细胞在形态和大小上与正常人红细胞相近,且存在血细胞去核的现象。荧光定量RT-PCR检测到了globin的表达,其中β-globin的表达量占20%以上。将得到的红细胞收集到离心管中,自然沉降后可见红色的红细胞沉淀。上述研究为大量制备人红细胞提供了新的有效的技术方法。

At present, the experimental technique to produce human red blood cells in vitro is complicated, and in order to optimize the induction steps, human pluripotent stem cells were differentiated into red blood cells through two induction steps. First, human pluripotent stem cells（including Rh negative A type umbilical cord mesenchymal stem cells（hUCMSC^（Rh-A）） and human i PS cells（hi PS）） were differentiated into CD31~＋ and CD34~＋ cells in BVF medium. PCR and flow cytometry were used to exam the expression of CD31 and CD34. We found that hUCMSC^（Rh-A） derived CD31~＋ and CD34~＋ cells were 5.3% and 22.7%, respectively; hi PS derived CD31~＋ and CD34~＋ cells were 31.2% and 8.2%, respectively. For the second induction step, the obtained CD31~＋ and CD34~＋ cells were differentiated into mature erythrocytes for 36 days under the addition of various growth factors. Through Giemsa staining, we found that the obtained mature erythrocytes were similar in morphology and size to normal human erythrocytes, and some obtained erythrocytes were enucleated. Globin expression was detected by real time RT-PCR, and the expression of β-globin was more than 20%. The obtained erythrocytes are collected into the centrifuge tube, and then erythrocytes were naturally settled and showed the red color. Our findings provide a novel and effective method for the quantity generation of human red blood cells in vitro.