Abstract

Platelets are essential for hemostatic plug formation and thrombosis. The mechanisms of megakaryocyte (MK) differentiation and subsequent platelet production from stem cells remain only partially understood. The manufacture of megakaryocytes (MKs) and platelets from cell sources including hematopoietic stem cells and pluripotent stem cells have been highlighted for studying the platelet production mechanisms as well as for the development of new strategies for platelet transfusion. The mouse bone marrow stroma cell line OP9 has been widely used as feeder cells for the differentiation of stem cells into MK lineages. OP9 cells are reported to be pre-adipocytes. We previously reported that 3T3-L1 pre-adipocytes differentiated into MKs and platelets. In the present study, we examined whether OP9 cells differentiate into MKs and platelets using MK lineage induction (MKLI) medium previously established to generate MKs and platelets from hematopoietic stem cells, embryonic stem cells, and pre-adipocytes. OP9 cells cultured in MKLI medium had megakaryocytic features, i.e., positivity for surface markers CD41 and CD42b, polyploidy, and distinct morphology. The OP9-derived platelets had functional characteristics, providing the first evidence for the differentiation of OP9 cells into MKs and platelets. We then analyzed gene expressions of critical factors that regulate megakaryopoiesis and thrombopoiesis. The gene expressions of p45NF-E2, FOG, Fli1, GATA2, RUNX1, thrombopoietin, and c-mpl were observed during the MK differentiation. Among the observed transcription factors of MK lineages, p45NF-E2 expression was increased during differentiation. We further studied MK and platelet generation using p45NF-E2-overexpressing OP9 cells. OP9 cells transfected with p45NF-E2 had enhanced production of MKs and platelets. Our findings revealed that OP9 cells differentiated into MKs and platelets in vitro. OP9 cells have critical factors for megakaryopoiesis and thrombopoiesis, which might be involved in a mechanism of this differentiation. p45NF-E2 might also play important roles in the differentiation of OP9 cells into MK lineages cells.

Highlights

  • Platelets play critical roles in hemostatic plug formation and thrombosis [1,2,3]

  • Differentiation of OP9 Cells into MKs and Platelets We examined whether OP9 cells differentiate into MKs and platelets by using MK lineage induction (MKLI) medium previously established to generate MKs and platelets from hematopoietic stem cells (HSCs), embryonic stem (ES) cells, and preadipocytes

  • This study demonstrated that OP9 cells differentiated into MKs and platelets in vitro using MKLI medium previously established to differentiate HSC, ES cells, pre-adipocytes into MK lineages

Read more

Summary

Introduction

Platelets play critical roles in hemostatic plug formation and thrombosis [1,2,3]. Platelets are released from terminally differentiated megakaryocytes (MKs). Current advances in a culture system to generate MKs and platelets in vitro help us to clarify the mechanism underlying MK differentiation and platelet production [11]. We reported the generation of MKs and functional platelets from both normal human subcutaneous adipose tissues and mouse pre-adipocyte cell line 3T3-L1 [20,21,22]. Differentiation of pre-adipocytes into MKs and platelets has been observed when the culture medium is switched from maintenance medium to MK lineage induction (MKLI) medium previously established to generate platelets from HSCs and ES cells [21,22,23,24]

Methods
Results
Conclusion
Full Text
Published version (Free)

Talk to us

Join us for a 30 min session where you can share your feedback and ask us any queries you have

Schedule a call