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OP9 bone marrow stroma cells differentiate into megakaryocytes and platelets.

Matsubara Y, Ono Y, Suzuki H, Arai F, Suda T, Murata M, Ikeda Y - PLoS ONE (2013)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Laboratory Medicine, Keio University School of Medicine, Tokyo, Japan. yumikoma@a7.keio.jp

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.

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Analyses of gene expression of OP9 cells during megakaryocyte differentiation.Gene expression assessed by reverse transcription-PCR.
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pone-0058123-g005: Analyses of gene expression of OP9 cells during megakaryocyte differentiation.Gene expression assessed by reverse transcription-PCR.

Mentions: We then analyzed gene expression of candidate factors to elucidate mechanisms for the differentiation of OP9 cells into MKs and platelets. To examine whether this conversion goes through the status of cell pluripotency, the expressions of OCT3/4, specifically expressed in pluripotent cells, and SOX2, a key factor for the maintenance of cell pluripotency [34], [35], were analyzed. Expression of both OCT3/4 and SOX2 was not detected in OP9 cells (day 0) and OP9-derived cells by RT-PCR (Figure 5). We also analyzed the gene expressions for transcription factors, p45NF-E2, FOG, Fli1, GATA1, GATA2, and RUNX1, that regulate megakaryopoiesis and thrombopoiesis [5]–[11]. These transcription factors, except for GATA1, were clearly detected in OP9 (day 0) and OP9-derived cells (days 4 and 8) (Figure 5). Because major cell population of BMMNCs on day 4 morphologically resembled that of OP9-derived cells on day 8, we used BMMNCs on days 0 and 4 as a control. OP9 cells (day 0) also showed gene expression of TPO and c-mpl, receptor for TPO (Figure 5). Regarding the key transcription factors for other hematopoietic cell lineages, PU.1 for leukocytes [36] and KLF1 for erythrocytes [37], were not detected in OP9 cells (day 0) and OP9-derived cells (days 4 and 8) (Figure 5). Among the observed transcription factors, p45NF-E2 expression was increased during the differentiation of OP9 cells into MKs, and expression levels of p45NF-E2 in OP9 cells (day 0) and OP9-derived cells (day 4) were measured by quantitative real-time PCR analysis. It was found that expression levels of OP9-derived cells (day 4) had 3.91±0.08-fold higher than that of OP9 cells (day 0). Other transcription factors were also measured. It was for 1.54±0.02-fold higher for FOG, 0.03±0.00 (0.0007)-fold higher for Fli1, 0.20±0.03-fold higher for GATA2, and 1.00±0.16-fold higher for RUNX1, as compared with those of OP9 cells (day 0). TPO expression levels of OP9-derived cells (day 4) had 3.59±0.70-fold higher than that of OP9 cells (day 0). c-mpl expression levels of OP9-derived cells (day 4) had 0.182±0.11-fold higher than that of OP9 cells (day 0). These results indicate that OP9 cells possess critical factors for MK differentiation and platelet production. Among them, the p45NF-E2 expression was increased during the differentiation of OP9 cells into MK lineages.


OP9 bone marrow stroma cells differentiate into megakaryocytes and platelets.

Matsubara Y, Ono Y, Suzuki H, Arai F, Suda T, Murata M, Ikeda Y - PLoS ONE (2013)

Analyses of gene expression of OP9 cells during megakaryocyte differentiation.Gene expression assessed by reverse transcription-PCR.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3585802&req=5

pone-0058123-g005: Analyses of gene expression of OP9 cells during megakaryocyte differentiation.Gene expression assessed by reverse transcription-PCR.
Mentions: We then analyzed gene expression of candidate factors to elucidate mechanisms for the differentiation of OP9 cells into MKs and platelets. To examine whether this conversion goes through the status of cell pluripotency, the expressions of OCT3/4, specifically expressed in pluripotent cells, and SOX2, a key factor for the maintenance of cell pluripotency [34], [35], were analyzed. Expression of both OCT3/4 and SOX2 was not detected in OP9 cells (day 0) and OP9-derived cells by RT-PCR (Figure 5). We also analyzed the gene expressions for transcription factors, p45NF-E2, FOG, Fli1, GATA1, GATA2, and RUNX1, that regulate megakaryopoiesis and thrombopoiesis [5]–[11]. These transcription factors, except for GATA1, were clearly detected in OP9 (day 0) and OP9-derived cells (days 4 and 8) (Figure 5). Because major cell population of BMMNCs on day 4 morphologically resembled that of OP9-derived cells on day 8, we used BMMNCs on days 0 and 4 as a control. OP9 cells (day 0) also showed gene expression of TPO and c-mpl, receptor for TPO (Figure 5). Regarding the key transcription factors for other hematopoietic cell lineages, PU.1 for leukocytes [36] and KLF1 for erythrocytes [37], were not detected in OP9 cells (day 0) and OP9-derived cells (days 4 and 8) (Figure 5). Among the observed transcription factors, p45NF-E2 expression was increased during the differentiation of OP9 cells into MKs, and expression levels of p45NF-E2 in OP9 cells (day 0) and OP9-derived cells (day 4) were measured by quantitative real-time PCR analysis. It was found that expression levels of OP9-derived cells (day 4) had 3.91±0.08-fold higher than that of OP9 cells (day 0). Other transcription factors were also measured. It was for 1.54±0.02-fold higher for FOG, 0.03±0.00 (0.0007)-fold higher for Fli1, 0.20±0.03-fold higher for GATA2, and 1.00±0.16-fold higher for RUNX1, as compared with those of OP9 cells (day 0). TPO expression levels of OP9-derived cells (day 4) had 3.59±0.70-fold higher than that of OP9 cells (day 0). c-mpl expression levels of OP9-derived cells (day 4) had 0.182±0.11-fold higher than that of OP9 cells (day 0). These results indicate that OP9 cells possess critical factors for MK differentiation and platelet production. Among them, the p45NF-E2 expression was increased during the differentiation of OP9 cells into MK lineages.

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Laboratory Medicine, Keio University School of Medicine, Tokyo, Japan. yumikoma@a7.keio.jp

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.

Show MeSH
Related in: MedlinePlus