Forming megakaryocytes from murine-induced pluripotent stem cells by the inducible overexpression of supporting factors.
Forming megakaryocytes from murine-induced pluripotent stem cells by the inducible overexpression of supporting factors.
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DOI：
10.1002/rth2.12453
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发表时间：
2021-01
影响因子：
4.6
通讯作者：
Modlich U
中科院分区：
文献类型：
--
作者：
Cullmann K;Jahn M;Spindler M;Schenk F;Manukjan G;Mucci A;Steinemann D;Boller K;Schulze H;Bender M;Moritz T;Modlich U
血小板是小的无核细胞，在静止状态下在血液中循环，但可以被外部信号激活。如有需要，可输注献血者的血小板。作为一种替代来源，血小板可以从诱导多能干细胞（iPSC）中产生;然而，回收的数量很低。为了优化小鼠iPSC的巨核细胞（MK）和血小板输出，我们研究了转录因子加塔结合因子1（GATA 1）、核因子红细胞2和前B细胞白血病转录因子1（Pbx 1）以及小鸟苷三磷酸酶RhoA（RhoAhc）的过度活性变体的过表达。为了避免脱靶效应，我们产生了在Rosa 26基因座中携带反向四环素应答反式激活因子M2（rtTA‐M2）的iPSC，并表达了来自泰特诱导型γ逆转录病毒载体的因子。iPSC的分化由胚状体（EB）形成启动。EB解离后，早期造血祖细胞富集并与血小板生成素和干细胞因子共培养在OP 9饲养细胞上以诱导巨核细胞（MK）分化。GATA 1和Pbx 1的过表达使MK输出增加2至2.5倍，并允许延长MK的收集时间。细胞学和超微结构分析确定了典型的MK与扩大的细胞，多小叶核，颗粒结构，和内部膜系统。然而，GATA 1和Pbx 1的表达并没有改善MK成熟或血小板释放，尽管体外生成的血小板在纤维蛋白原或胶原相关肽上的扩散中起作用。我们证明了使用用泰特诱导型逆转录病毒载体转导的rtTA-M2转基因iPSC允许在分化期间的稍后时间点进行基因表达。通过这种策略，我们可以确定增加体外MK生产的因素。
Platelets are small anucleate cells that circulate in the blood in a resting state but can be activated by external cues. In case of need, platelets from blood donors can be transfused. As an alternative source, platelets can be produced from induced pluripotent stem cells (iPSCs); however, recovered numbers are low. To optimize megakaryocyte (MK) and platelet output from murine iPSCs, we investigated overexpression of the transcription factors GATA‐binding factor 1 (GATA1); nuclear factor, erythroid 2; and pre–B‐cell leukemia transcription factor 1 (Pbx1) and a hyperactive variant of the small guanosine triphosphatase RhoA (RhoAhc). To avoid off‐target effects, we generated iPSCs carrying the reverse tetracycline‐responsive transactivator M2 (rtTA‐M2) in the Rosa26 locus and expressed the factors from Tet‐inducible gammaretroviral vectors. Differentiation of iPSCs was initiated by embryoid body (EB) formation. After EB dissociation, early hematopoietic progenitors were enriched and cocultivated on OP9 feeder cells with thrombopoietin and stem cell factor to induce megakaryocyte (MK) differentiation. Overexpression of GATA1 and Pbx1 increased MK output 2‐ to 2.5‐fold and allowed prolonged collection of MK. Cytologic and ultrastructural analyses identified typical MK with enlarged cells, multilobulated nuclei, granule structures, and an internal membrane system. However, GATA1 and Pbx1 expression did not improve MK maturation or platelet release, although in vitro–generated platelets were functional in spreading on fibrinogen or collagen‐related peptide. We demonstrate that the use of rtTA‐M2 transgenic iPSCs transduced with Tet‐inducible retroviral vectors allowed for gene expression at later time points during differentiation. With this strategy we could identify factors that increased in vitro MK production.
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影响因子：
16.6
作者：
Duetting, Sebastian;Gaits-Iacovoni, Frederique;Nieswandt, Bernhard
通讯作者：
Nieswandt, Bernhard
影响因子：
20.3
作者：
Lecine, P;Villeval, JL;Shivdasani, RA
通讯作者：
Shivdasani, RA
影响因子：
20.3
作者：
Mikkola, HKA;Fujiwara, Y;Orkin, SH
通讯作者：
Orkin, SH
影响因子：
5.3
作者：
KELLER, G;KENNEDY, M;WILES, MV
通讯作者：
WILES, MV
影响因子：
3.3
作者：
Mookerjee, S.;Foster, H. R.;Ghevaert, C. J.
通讯作者：
Ghevaert, C. J.