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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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Flow cytometric analyses for megakaryocytes and platelets differentiated from OP9 cells. A, Representative flowcytometry histogram of CD41 and CD42b expression on megakaryocyte-sized cells. Dot-line shows data using isotype control. B, Representative flowcytometry histogram of CD41 and CD42b expression on platelet-sized cells. Dot-line shows data using isotype control. C, DNA ploidy analysis in OP9-derived CD41+ cells on day 8.
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pone-0058123-g001: Flow cytometric analyses for megakaryocytes and platelets differentiated from OP9 cells. A, Representative flowcytometry histogram of CD41 and CD42b expression on megakaryocyte-sized cells. Dot-line shows data using isotype control. B, Representative flowcytometry histogram of CD41 and CD42b expression on platelet-sized cells. Dot-line shows data using isotype control. C, DNA ploidy analysis in OP9-derived CD41+ cells on day 8.

Mentions: By flow cytometric analysis with MK lineage specific markers, approximately 95% and 60% of these OP9-derived MK-sized cells on day 8 expressed CD41, a surface marker throughout MK differentiation, and CD42b, a surface marker for the late stage of MK differentiation, respectively (Figure 1A). Approximately 70% and 60% of OP9-derived platelet-sized cells on day 12 expressed CD41 and CD42b, respectively (Figure 1B). Regarding the number of the OP9-derived MKs and platelets, approximately 4×104 MKs and 1×105 platelets were generated from 1×106 OP9 cells before the MK induction. OP9 cells before the MK induction (day 0) did not express CD41 and CD42b (Figure 1A, 1B). DNA ploidy of OP9-derived CD41+ cells ranged from 2N to 32N (Figure 1C). Immunohistochemical analyses showed that VWF and P-selectin, cytoplasmic proteins of MK lineage cells, were positive in OP9-derived CD41+ cells (Figure 2A, 2B). Under electron microscopic observation, OP9-derived MK-sized CD41+ cells had typical organelles for MKs, such as granules, demarcation membrane system, and lobulated nuclei, and OP9-derived platelet-sized CD41+ cells showed typical features for platelets, such as granules, mitochondria, and open canalicular system (Figure 2C, 2D). The present observations were similar to what described in MKs derived from mBMMNCs (Figure S3). Also, the present observations were similar to what described in MKs and platelets derived from mouse ES cells, mouse pre-adipocyte cell line 3T3-L1, human bone marrow CD34-positive cells, and human adipose tissues [19], [20]. OP9-derived MK-sized CD41+ cells were examined for proplatelet formation under scanning electron microscopic observation. We observed proplatelets forming OP9-derived MK-sized CD41+ cells (Figure 3A). Moreover, proplatelet-forming OP9-derived MK showing CD41 and alpha-tubulin was observed (Figure 3B). To examine whether OP9-derived platelets are functional, fibrinogen-binding assay was performed on OP9-derived platelet-sized cells on day 12. Binding of Alexa Fluor 488-labeled fibrinogen to OP9-derived platelet-sized cells was increased upon stimulation when assessed by mean fluorescence (mean±S.D.): 9.7±0.9 (no stimulation), 25.7±0.3 (10 µM ADP), p<0.0001 (vs no stimulation), 28.6±0.4 (1.5 mM PAR4-activating peptide), p<0.0001 (vs no stimulation), and 29.2±1.6 (0.5 U/ml thrombin), p<0.0001 (vs no stimulation) (Figure 4A). The representative data of dot-plots in this assay are shown in Figure S4. Moreover, P-selectin surface exposure, a marker for platelet activation, on OP9-derived platelet-sized cells on day 12 was examined in the presence or absence of stimulation, and mean fluorescence (mean±S.D.) was 7.8±1.1 (no stimulation), 17.9±1.5 (10 µM ADP), p = 0.0159 (vs no stimulation), 19.1±1.8 (1.5 mM PAR4-activating peptide), p = 0.0162 (vs no stimulation), and 25.1±3.8 (0.5 U/ml thrombin), p = 0.0162 (vs no stimulation) (Figure 4B). These observations indicated that OP9 cells differentiated into MKs and platelets in vitro.


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)

Flow cytometric analyses for megakaryocytes and platelets differentiated from OP9 cells. A, Representative flowcytometry histogram of CD41 and CD42b expression on megakaryocyte-sized cells. Dot-line shows data using isotype control. B, Representative flowcytometry histogram of CD41 and CD42b expression on platelet-sized cells. Dot-line shows data using isotype control. C, DNA ploidy analysis in OP9-derived CD41+ cells on day 8.
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Related In: Results  -  Collection

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

pone-0058123-g001: Flow cytometric analyses for megakaryocytes and platelets differentiated from OP9 cells. A, Representative flowcytometry histogram of CD41 and CD42b expression on megakaryocyte-sized cells. Dot-line shows data using isotype control. B, Representative flowcytometry histogram of CD41 and CD42b expression on platelet-sized cells. Dot-line shows data using isotype control. C, DNA ploidy analysis in OP9-derived CD41+ cells on day 8.
Mentions: By flow cytometric analysis with MK lineage specific markers, approximately 95% and 60% of these OP9-derived MK-sized cells on day 8 expressed CD41, a surface marker throughout MK differentiation, and CD42b, a surface marker for the late stage of MK differentiation, respectively (Figure 1A). Approximately 70% and 60% of OP9-derived platelet-sized cells on day 12 expressed CD41 and CD42b, respectively (Figure 1B). Regarding the number of the OP9-derived MKs and platelets, approximately 4×104 MKs and 1×105 platelets were generated from 1×106 OP9 cells before the MK induction. OP9 cells before the MK induction (day 0) did not express CD41 and CD42b (Figure 1A, 1B). DNA ploidy of OP9-derived CD41+ cells ranged from 2N to 32N (Figure 1C). Immunohistochemical analyses showed that VWF and P-selectin, cytoplasmic proteins of MK lineage cells, were positive in OP9-derived CD41+ cells (Figure 2A, 2B). Under electron microscopic observation, OP9-derived MK-sized CD41+ cells had typical organelles for MKs, such as granules, demarcation membrane system, and lobulated nuclei, and OP9-derived platelet-sized CD41+ cells showed typical features for platelets, such as granules, mitochondria, and open canalicular system (Figure 2C, 2D). The present observations were similar to what described in MKs derived from mBMMNCs (Figure S3). Also, the present observations were similar to what described in MKs and platelets derived from mouse ES cells, mouse pre-adipocyte cell line 3T3-L1, human bone marrow CD34-positive cells, and human adipose tissues [19], [20]. OP9-derived MK-sized CD41+ cells were examined for proplatelet formation under scanning electron microscopic observation. We observed proplatelets forming OP9-derived MK-sized CD41+ cells (Figure 3A). Moreover, proplatelet-forming OP9-derived MK showing CD41 and alpha-tubulin was observed (Figure 3B). To examine whether OP9-derived platelets are functional, fibrinogen-binding assay was performed on OP9-derived platelet-sized cells on day 12. Binding of Alexa Fluor 488-labeled fibrinogen to OP9-derived platelet-sized cells was increased upon stimulation when assessed by mean fluorescence (mean±S.D.): 9.7±0.9 (no stimulation), 25.7±0.3 (10 µM ADP), p<0.0001 (vs no stimulation), 28.6±0.4 (1.5 mM PAR4-activating peptide), p<0.0001 (vs no stimulation), and 29.2±1.6 (0.5 U/ml thrombin), p<0.0001 (vs no stimulation) (Figure 4A). The representative data of dot-plots in this assay are shown in Figure S4. Moreover, P-selectin surface exposure, a marker for platelet activation, on OP9-derived platelet-sized cells on day 12 was examined in the presence or absence of stimulation, and mean fluorescence (mean±S.D.) was 7.8±1.1 (no stimulation), 17.9±1.5 (10 µM ADP), p = 0.0159 (vs no stimulation), 19.1±1.8 (1.5 mM PAR4-activating peptide), p = 0.0162 (vs no stimulation), and 25.1±3.8 (0.5 U/ml thrombin), p = 0.0162 (vs no stimulation) (Figure 4B). These observations indicated that OP9 cells differentiated into MKs and platelets in vitro.

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