Cullmann Katharina, Jahn Magdalena, Spindler Markus, Schenk Franziska, Manukjan Georgi, Mucci Adele, Steinemann Doris, Boller Klaus, Schulze Harald, Bender Markus, Moritz Thomas, Modlich Ute
RG Gene Modification in Stem Cells, Division of Veterinary Medicine Paul-Ehrlich-Institut Langen Germany.
Institute of Experimental Biomedicine I University Hospital and Rudolf Virchow Center University of Würzburg Würzburg Germany.
Res Pract Thromb Haemost. 2020 Dec 3;5(1):111-124. doi: 10.1002/rth2.12453. eCollection 2021 Jan.
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.
血小板是循环于血液中的无核小细胞，处于静息状态，但可被外部信号激活。必要时，可输注献血者的血小板。作为替代来源，血小板可由诱导多能干细胞（iPSC）产生；然而，获得的数量较低。
为了优化小鼠iPSC来源的巨核细胞（MK）和血小板产量，我们研究了转录因子GATA结合因子1（GATA1）、核因子红细胞2和前B细胞白血病转录因子1（Pbx1）以及小GTP酶RhoA的高活性变体（RhoAhc）的过表达情况。
为避免脱靶效应，我们在Rosa26位点生成了携带反向四环素应答反式激活因子M2（rtTA-M2）的iPSC，并从四环素诱导的γ逆转录病毒载体表达这些因子。通过形成胚状体（EB）启动iPSC的分化。EB解离后，富集早期造血祖细胞，并与血小板生成素和干细胞因子一起在OP9饲养细胞上共培养，以诱导巨核细胞（MK）分化。
GATA1和Pbx1的过表达使MK产量增加了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，其细胞增大、核分叶、有颗粒结构和内膜系统。然而，GATA1和Pbx1的表达并未改善MK的成熟或血小板释放，尽管体外生成的血小板在纤维蛋白原或胶原相关肽上的铺展功能正常。
我们证明，使用经四环素诱导逆转录病毒载体转导的rtTA-M2转基因iPSC可在分化后期实现基因表达。通过这种策略，我们可以鉴定出增加体外MK产量的因子。
