Efficient Generation of Megakaryocytes From Human Induced Pluripotent Stem Cells Using Food and Drug Administration-Approved Pharmacological Reagents
Efficient Generation of Megakaryocytes From Human Induced Pluripotent Stem Cells Using Food and Drug Administration-Approved Pharmacological Reagents
复制标题
DOI：
10.5966/sctm.2014-0183
复制
发表时间：
2015-04-01
影响因子：
6
通讯作者：
Wang, Zack Z.
中科院分区：
文献类型：
--
作者：
Liu, Yanfeng;Wang, Ying;Wang, Zack Z.
巨核细胞（MK）是成人骨髓中罕见的造血细胞，并产生对血管止血和伤口愈合至关重要的血小板。从人诱导多能干细胞（hiPSC）离体产生MK提供了用于治疗血小板减少症患者的血小板的可再生细胞来源，并允许更好地理解MK/血小板生物学。这种方法的关键要求包括开发一种稳健和一致的方法来生产功能性子代细胞，如来自hiPSC的MK，并最大限度地减少细胞培养物中动物源性产品的风险和变异。在这项研究中，我们开发了一种有效的系统，在无饲养层和无异种条件下从hiPSC产生MK，其中所有动物源性产物都被消除。对几种关键试剂进行了评估，并替换为食品和药物管理局批准的药理学试剂，包括romiplostim（Nplate，血小板生成素类似物），oprelvekin（重组白细胞介素-11）和Plasbumin（人白蛋白）。我们使用该方法以两个步骤从来自23个个体的hiPSC诱导MK生成：生成CD 34（+）CD 45（+）造血祖细胞（HPC）14天;以及从HPC生成和扩增CD 41（+）CD 42 a（+）MK另外5天。19天后，我们观察到大量的CD 41（+）CD 42 a（+）MK，它们也表达MK标记物CD 42 b和CD 61，并显示出多倍性（>= 16%的衍生细胞的DNA含量> 4 N）。转录组RNA测序分析显示，巨核细胞相关基因的高表达。对hiPSC衍生的MK的额外成熟和研究应提供对MK生物学的深入了解，并导致离体产生大量血小板。
Megakaryocytes (MKs) are rare hematopoietic cells in the adult bone marrow and produce platelets that are critical to vascular hemostasis and wound healing. Ex vivo generation of MKs from human induced pluripotent stem cells (hiPSCs) provides a renewable cell source of platelets for treating thrombocytopenic patients and allows a better understanding of MK/platelet biology. The key requirements in this approach include developing a robust and consistent method to produce functional progeny cells, such as MKs from hiPSCs, and minimizing the risk and variation from the animal-derived products in cell cultures. In this study, we developed an efficient system to generate MKs from hiPSCs under a feeder-free and xeno-free condition, in which all animal-derived products were eliminated. Several crucial reagents were evaluated and replaced with Food and Drug Administration-approved pharmacological reagents, including romiplostim (Nplate, a thrombopoietin analog), oprelvekin (recombinant interleukin-11), and Plasbumin (human albumin). We used this method to induce MK generation from hiPSCs derived from 23 individuals in two steps: generation of CD34(+)CD45(+) hematopoietic progenitor cells (HPCs) for 14 days; and generation and expansion of CD41(+)CD42a(+) MKs from HPCs for an additional 5 days. After 19 days, we observed abundant CD41(+)CD42a(+) MKs that also expressed the MK markers CD42b and CD61 and displayed polyploidy (>= 16% of derived cells with DNA contents >4N). Transcriptome analysis by RNA sequencing revealed that megakaryocytic-related genes were highly expressed. Additional maturation and investigation of hiPSC-derived MKs should provide insights into MK biology and lead to the generation of large numbers of platelets ex vivo.