Department of Pediatric Hematology and Oncology, Hannover Medical School, Hannover, Germany.
Ann Hematol. 2012 Nov;91(11):1673-84. doi: 10.1007/s00277-012-1517-z. Epub 2012 Jul 5.
The process of megakaryopoiesis culminates in the release of platelets, the pivotal cellular component for hemostasis and wound healing. The regulatory architecture including the modulatory role of microRNAs, which underlies megakaryocytic maturation and platelet formation, is incompletely understood, precluding the ex vivo generation of sufficient platelet numbers for transfusion medicine. We derived a highly efficient differentiation protocol to produce mature polyploid megakaryocytes and functional platelets from CD34⁺-hematopoietic stem and progenitor cells by comparing previously published approaches. Our megakaryocytic culture conditions using the cytokines SCF, TPO, IL-9, and IL-6 include nicotinamide and Rho-associated kinase (ROCK) inhibitor Y27632 as contextual additives. The potency of our novel megakaryocytic differentiation protocol was validated using cord blood and peripheral blood human hematopoietic stem and progenitor cells. Using this novel megakaryocytic differentiation protocol, we characterized the modulatory capacity of several miRNAs highly expressed in normal megakaryocytic cells or malignant blasts from patients with megakaryoblastic leukemia. Overexpression of candidate microRNAs was achieved by lentiviral transduction of CD34⁺-hematopoietic stem and progenitor cells prior to differentiation. We revealed miR-125b and miR-660 as enhancers of polyploidization, as well as platelet output of megakaryocytes. The oncogene miR-125b markedly expanded the number of megakaryocytes during in vitro culture. Conversely, the miR-23a/27a/24-2 cluster, which is highly expressed in normal megakaryocytes, blocked maturation and platelet formation. Our study on the utilization of microRNAs in conjunction with a highly efficient differentiation protocol constitutes another step towards ex vivo platelet manufacturing on a clinically relevant scale.
巨核细胞生成过程以血小板的释放为终点，血小板是止血和伤口愈合的关键细胞成分。调控巨核细胞成熟和血小板形成的调节结构，包括 microRNAs 的调节作用，尚未完全了解，这妨碍了用于输血医学的足够数量血小板的体外生成。我们通过比较以前发表的方法，从 CD34+造血干细胞和祖细胞中衍生出一种高效的分化方案，以产生成熟的多倍体巨核细胞和功能性血小板。我们的巨核细胞培养条件使用细胞因子 SCF、TPO、IL-9 和 IL-6，并包括烟酰胺和 Rho 相关激酶（ROCK）抑制剂 Y27632 作为背景添加剂。使用脐带血和外周血人类造血干细胞和祖细胞验证了我们新型巨核细胞分化方案的效力。使用这种新型巨核细胞分化方案，我们对几种在正常巨核细胞或恶性母细胞白血病患者的恶性母细胞中高度表达的 microRNAs 的调节能力进行了特征描述。候选 microRNAs 的过表达是通过在分化之前用慢病毒转导 CD34+造血干细胞和祖细胞来实现的。我们发现 miR-125b 和 miR-660 可增强巨核细胞的多倍体化和血小板输出。癌基因 miR-125b 在体外培养期间显著增加巨核细胞的数量。相反，在正常巨核细胞中高度表达的 miR-23a/27a/24-2 簇阻止了成熟和血小板形成。我们关于利用 microRNAs 与高效分化方案相结合的研究，是朝着在临床相关规模上进行体外血小板制造的又一步。
