OC 48.3 - Contractility of iPSC-derived megakaryocyte precursors at different stages of development

Background: We have recently shown that megakaryocytes (iMKs) derived from induced pluripotent stem cells (iPSCs) contract fibrin similar to platelets, suggesting the use of iMKs as a model system to study platelet contractility and pathophysiological and therapeutic implications of MK contractility. However, the contractile potential of megakaryocyte precursor cells has not been studied.

Aims: To compare the contractility of undifferentiated iPSCs, megakaryocyte precursor hematopoietic progenitor cells (HPCs), and iMKs as a quantitative measure of iMK maturation.

Methods: iPSCs, iPSC-derived HPCs at day 8 of differentiation, and iMKs differentiated from HPCs at day 5 were studied. Flow cytometry of untreated and thrombin-treated cells was used to measure the expression of total αIIbβ3 (CD41a) and its active form (by PAC1 binding). HPCs were detected by Glycophorin-A (CD235) surface marker. To assess contractility, the cells were suspended in human platelet-free citrated plasma followed by thrombin-induced cell activation and plasma clotting. The time-lapse cell-driven shrinkage of the clot was monitored optically.

Results: The average fraction of cells expressing total αIIbβ3 was zero for iPSCs, 30% for HPCs, and 78% for iMKs. The expression level of the active αIIbβ3 on HPCs was 2.4-fold lower than on iMKs (14% vs 34% of total cell number). iPSCs exhibited only a minimal contraction of fibrin (nonspecific background) with the final extent of clot contraction measuring less than 5%. Meanwhile, the average extent of clot contraction induced by HPCs was 2.3-fold lower than by iMKs (26% vs 60%).

Conclusion(s): Overall, the flow cytometry data combined with the clot contraction assay suggest that the increasing contractility of iPSCs, HPCs, and MKs is determined by the growing expression of αIIbβ3-mediated cell attachment to fibrin. Our experiments identify the HPCs as the first stage of cellular development at which the cellular lineage partially acquires the ability to interact with fibrin fibers and induce clot contraction.