演題詳細

シンポジウム / Symposium

シンポジウム2 (Symposium 2) : Progression of thrombosis and hemostasis

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日程
2013年10月11日(金)
時間
09:00 - 11:30
会場
第2会場 / Room No.2 (さっぽろ芸文館 3F 瑞雪)
座長・司会
松下 正 (Tadashi Matsushita):1、江藤 浩之 (Koji Eto):2
1:名古屋大学医学部付属病院 輸血部、2:京都大学iPS細胞研究所 臨床応用研究部門
 
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Induction of Megakaryocytes and Platelets from Fibroblasts by p45NF-E2/Maf

演題番号 : SY2-1

松原 由美子 (Yumiko Matsubara):1

1:慶應義塾大学医学部 発生・分化生物学

 

 The mechanism of megakaryocytes (MK) differentiation from stem cells and the subsequent release of platelets is only partially understood. Recently, we reported that a combination of transcription factors p45NF-E2, MafG, and MafK converts mouse fibroblast cell line 3T3 and adult human dermal fibroblasts (AHDFs) into MKs. The human p45NF-E2, MafG, and MafK-driven MKs have a pathway that is different from pluripotent cells. We first performed screening for MK-inducing factors using the preadipocyte cell line 3T3-L1 known to be able to differentiate into MKs in a previously defined MK lineage induction media (MKLI) containing thrombopoietin. Gene expression levels of candidate transcription factors were compared between 3T3 cells that do not differentiate into MKs and 3T3-L1 cells using quantitative real-time PCR. Among 3T3 cells transfected with candidate factors, cells transfected with p45NF-E2, Maf G, and Maf K had the highest frequency of CD41-positive cells. AHDFs transfected with these genes were cultured in MKLI medium. Cultured cells had megakaryocytic features, including surface markers, ploidy, and morphology. More than 99% of MK-sized cells expressed CD41, designated induced MK (iMK). Infusion of these iMK cells into immunodeficient mice led to a time-dependent appearance of human CD41-positive platelet-sized particles. Blood samples from iMK-infused into thrombocytopenic immunodeficient mice were perfused on a collagen-coated chip under flow condition (1,000seconds-1), and human CD41-positive platelets were incorporated into thrombi on the chip demonstrating their functionality. These findings demonstrate that a combination of p45NF-E2, Maf G, and Maf K is a key determinant of both megakaryopoiesis and thrombopoiesis.
 To elucidate a molecular mechanism of reprogramming of fibroblasts into iMK, we compared microRNA (miR) expression between AHDFs and AHDFs after 2 days of transfection with p45NF-E2, Maf G, and Maf K. Of 715 miRs analyzed using 3D-Gene chip (Toray), 4 were up-regulated with at least 4-fold change and 75 were down-regulated with at least 4-fold change. The main finding was down-regulation of hsa-miR-708 (8.9-fold change), hsa-miR-28 (5.3-fold change), and hsa-miR-151 (4.6-fold change) that target c-mpl, compatible with our observation that c-mpl was expressed on AHDFs transfected with p45NF-E2, Maf G, and Maf K, but not on AHDFs. We also observed marked down-regulation of has-miR-130a (4.7-fold change) that regulates CD41 expression. Regarding pluripotency of AHDFs transfected with p45NF-E2, Maf G, and Maf K, we observed little expression of hsa-miR-200c, hsa-miR-302a, hsa-miR-302b, hsa-miR-302c, hsa-miR-302d, hsa-miR-369-3p, and hsa-miR-369-5p that reprogram AHDFs to pluripotency. These observations suggest that AHDFs transfected with p45NF-E2, Maf G, and Maf K were converted into iMKs, along with gene expression related to megakaryopoiesis, without involving a pluripotent state.

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