Enhanced Ex Vivo Generation of Erythroid Cells from Human Induced Pluripotent Stem Cells in a Simplified Cell Culture System with Low Cytokine Support
Enhanced Ex Vivo Generation of Erythroid Cells from Human Induced Pluripotent Stem Cells in a Simplified Cell Culture System with Low Cytokine Support
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DOI：
10.1089/scd.2019.0132
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发表时间：
2019-11-19
影响因子：
4
通讯作者：
Dorn, Isabel
中科院分区：
文献类型：
--
作者：
Bernecker, Claudia;Ackermann, Mania;Dorn, Isabel
人类诱导多能干细胞 (hiPSC) 的红细胞 (RBC) 分化为发育研究和创新疗法提供了巨大潜力。然而，hiPSC 的离体红细胞生成目前受到常见培养系统的低效率和非生理条件的限制。特别是，生理生态位的缺乏可能会损害细胞生长和谱系特异性分化。我们在这里描述了一种简化的、无异源和无饲养层的培养系统，用于延长红细胞生成，该系统使用少量的支持细胞因子[干细胞因子 (SCF)、促红细胞生成素 (EPO) 和白细胞介素 3 (IL-3)]，并且基于“造血细胞形成复合物 (HCFC)”的中间开发。从该 HCFC 中，CD43(+) 造血细胞（纯度 >95%）持续释放到上清液中，并且可以在 6 周的时间内重复收集以进行进一步的红系分化。释放的细胞主要是具有高红系集落形成潜力的CD34(+)/CD45(+)祖细胞和CD36(+)红系前体细胞。从一个六孔板的上清液中总共可以收获 1.5 x 10(7) 个细胞，在随后同质分化为 GPA(+) 红系细胞的过程中显示出 100 至 1000 倍的扩增。接近 40%（高达 60%）的平均去核率进一步证实了该系统的效力。这些益处可以通过在 HCFC 内产生一个生态位来解释，该生态位模仿发生红细胞生成的生理微环境的时空信号。与其他方案相比，该方法的复杂性更低，细胞因子和培养基消耗更少，细胞输出更高，去核效果更好。此外，细胞因子添加的轻微修改使系统转向连续生成粒细胞和巨噬细胞。
Red blood cell (RBC) differentiation from human induced pluripotent stem cells (hiPSCs) offers great potential for developmental studies and innovative therapies. However, ex vivo erythropoiesis from hiPSCs is currently limited by low efficiency and unphysiological conditions of common culture systems. Especially, the absence of a physiological niche may impair cell growth and lineage-specific differentiation. We here describe a simplified, xeno- and feeder-free culture system for prolonged RBC generation that uses low numbers of supporting cytokines [stem cell factor (SCF), erythropoietin (EPO), and interleukin 3 (IL-3)] and is based on the intermediate development of a "hematopoietic cell forming complex (HCFC)." From this HCFC, CD43(+) hematopoietic cells (purity >95%) were continuously released into the supernatant and could be collected repeatedly over a period of 6 weeks for further erythroid differentiation. The released cells were mainly CD34(+)/CD45(+) progenitors with high erythroid colony-forming potential and CD36(+) erythroid precursors. A total of 1.5 x 10(7) cells could be harvested from the supernatant of one six-well plate, showing 100- to 1000-fold amplification during subsequent homogeneous differentiation into GPA(+) erythroid cells. Mean enucleation rates near 40% (up to 60%) further confirmed the potency of the system. These benefits may be explained by the generation of a niche within the HCFC that mimics the spatiotemporal signaling of the physiological microenvironment in which erythropoiesis occurs. Compared to other protocols, this method provides lower complexity, less cytokine and medium consumption, higher cellular output, and better enucleation. In addition, slight modifications in cytokine addition shift the system toward continuous generation of granulocytes and macrophages.