Generation of Red Blood Cells from Human Induced Pluripotent Stem Cells
Generation of Red Blood Cells from Human Induced Pluripotent Stem Cells
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DOI：
10.1089/scd.2011.0078
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发表时间：
2011-09-01
影响因子：
4
通讯作者：
Slukvin, Igor I.
中科院分区：
文献类型：
--
作者：
Dias, Jessica;Gumenyuk, Marina;Slukvin, Igor I.
人诱导多能干细胞（hiPSC）和胚胎干细胞（hESC）分化成红系细胞谱系为研究红系发育、珠蛋白转换调节、药物测试和体外红细胞（RBC）疾病建模提供了新的机会。在这里，我们描述了一种从hiPSC/hESC有效生成RBC的方法，其使用0 P9共培养系统诱导造血分化，然后在具有红细胞生成支持细胞因子的无血清培养基中选择性扩增红系细胞。我们表明，使用基于慢病毒的载体产生的成纤维细胞衍生的转基因hiPSC和使用附加型载体产生的无转基因hiPSC可以分化成RBC，其效率与H1 hESC相似。用这种方法建立的红系细胞培养物由基本上纯的CD 235 a（+）CD 45（-）无白细胞RBC群体组成，具有强大的扩增潜力和长寿命（长达90天）。与hESC类似，hiPSC衍生的RBC主要表达胎儿γ球蛋白和胚胎e球蛋白，表明在成纤维细胞转变为多能状态后β-球蛋白基因座的完全重编程。虽然在hiPSC/hESC衍生的红系细胞中检测到β-珠蛋白表达，但其表达显著低于胚胎和胎儿珠蛋白。总体而言，这些结果证明了从成纤维细胞衍生的hiPSC大规模生产红系细胞的可行性，如已经针对hESC所描述的。由于由无转基因hiPSC产生的RBC缺乏基因组整合和重编程基因的背景表达，因此它们将是用于疾病建模和基因功能研究的优选细胞来源。
Differentiation of human induced pluripotent stem cells (hiPSCs) and embryonic stem cells (hESCs) into the erythroid lineage of cells offers a novel opportunity to study erythroid development, regulation of globin switching, drug testing, and modeling of red blood cell (RBC) diseases in vitro. Here we describe an approach for the efficient generation of RBCs from hiPSC/hESCs using an OP9 coculture system to induce hematopoietic differentiation followed by selective expansion of erythroid cells in serum-free media with erythropoiesis-supporting cytokines. We showed that fibroblast-derived transgenic hiPSCs generated using lentivirus-based vectors and transgene-free hiPSCs generated using episomal vectors can be differentiated into RBCs with an efficiency similar to that of H1 hESCs. Erythroid cultures established with this approach consisted of an essentially pure population of CD235a(+)CD45(-) leukocyte-free RBCs with robust expansion potential and long life span (up to 90 days). Similar to hESCs, hiPSC-derived RBCs expressed predominately fetal gamma and embryonic e globins, indicating complete reprogramming of beta-globin locus following transition of fibroblasts to the pluripotent state. Although beta-globin expression was detected in hiPSC/hESC-derived erythroid cells, its expression was substantially lower than the embryonic and fetal globins. Overall, these results demonstrate the feasibility of large-scale production of erythroid cells from fibroblast-derived hiPSCs, as has been described for hESCs. Since RBCs generated from transgene-free hiPSCs lack genomic integration and background expression of reprogramming genes, they would be a preferable cell source for modeling of diseases and for gene function studies.