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. The present findings provide the first evidence for the differentiation of OP9 cells into MK lineages. Regarding the efficiency of the MK and platelet production from OP9 cells, approximately 4×104
MKs and 1×105
platelets were generated from 1×106
OP9 cells. On the other hand, 1×106
human bone marrow mononuclear cells produced approximately 6×103
MKs and 3×103
platelets in a similar culture scale using MKLI medium 
. Although it is difficult to compare precisely the efficiency of the MK and platelet production among various stem cell sources, our observations suggested that OP9 cells possess high capacity of the differentiation into MK lineages in vitro
OP9 cells cultured in maintenance medium express specific surface marker for MSC and were reported to be pre-adipocytes 
. These cell lineage fate of mesenchymal cells is distinct from that of HSCs, and OP9 cells are widely used as feeder cells for differentiation of iPS cells and ES cells into hematopoietic cells and MK lineages 
. However, the present study shows that OP9 cells themselves are the source of MKs and platelets. OP9-derived MKs and platelets were characterized by specific surface markers, DNA polyploidy, morphology using electron microscopy, and immunohistochemistry. These analyses have been performed on in vitro
-generated MKs and platelets beginning with cell sources including HSCs, ES cells, and iPS cells. When MKs and platelets derived from iPS cells or ES cells are harvested in the differentiation study using OP9 co-culture system, there is a possibility that some of MK lineage cells are derived from OP9 cells.
The gene expression analyses indicated that differentiation of OP9 cells into MK lineages did not share the common mechanism with pluripotent cells. OP9 cells possess the important factors related to megakaryopoiesis and thrombopoiesis, and these observations are compatible with our previous findings that 3T3-L1 pre-adipocytes possess GATA2, RUNX1, Fli1, FOG1, and p45NF-E2. The expression of GATA1 was not detected in OP9 cells and 3T3-L1 cells 
. Furthermore, we did not observe the GATA1 expression during differentiation of OP9 cells into MKs. Although GATA1 was reported to be a critical factor for the erythroid and MK development, previous studies demonstrated that GATA2 coordinates MK differentiation in GATA1 deficient and mutant cells 
. Also, the present study revealed that the differentiation of OP9 cells into MK lineages involves in a p45NF-E2-mediated mechanism. The NF-E2 transcriptional factor is a basic-leucine zipper hetero-dimer complex consisting of p45 subunit, known as tissue-restricted subunit, and the small Maf proteins, Maf K and Maf G, known as widely expressed in many cells 
. Observations in p45NF-E2 deficient MKs suggested that p45NF-E2 is important in the MK terminal differentiation and platelet release 
. On the other hand, the in vitro
and in vivo
study using p45NF-E2-overexpressing bone marrow cells showed additional roles of p45NF-E2 in early megakaryopoiesis 
. We previously reported that fibroblasts transfected with p45NF-E2, Maf G and Maf K differentiated into MKs and platelets, whereas fibroblast did not differentiate into MK lineage cells. These observations support p45NE-E2, Maf G, and Maf K as critical factors for megakaryopoiesis and thrombopoiesis. In the present study, OP9 cells have Maf G and Maf K, and thus cells were transfected with P45NF-E2. The present findings provide additional information for the importance of p45NF-E2 in megakaryopoiesis and thrombopoiesis. Further studies are definitely needed to elucidate the detailed pathways that cause OP9 cells to differentiate into the MK lineage ultimately leading to platelet production.
In summary, OP9 cells differentiated into MKs and platelets, although OP9 cells have been wildly used as feeder cells in differentiation of ES cells and iPS cells into MKs and platelets. OP9 cells possess critical factors related to megakaryopoiesis and thrombopoiesis. The generation of MKs and platelets from OP9 cells could have important implications for study on the underlying mechanisms of megakaryopoiesis and thrombopoiesis.