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Thrombopoietic-mesenchymal interaction that may facilitate both endochondral ossification and platelet maturation via CCN2.

Sumiyoshi K, Kubota S, Furuta RA, Yasui K, Aoyama E, Kawaki H, Kawata K, Ohgawara T, Yamashiro T, Takigawa M - J Cell Commun Signal (2009)

Bottom Line: In this study, we initially pursued the possible origin of the CCN2 in platelets.As suspected, CCN2 production by HCS-2/8 was significantly enhanced by the conditioned medium.We further confirmed that human platelets were able to absorb/uptake exogenous CCN2 in vitro.

View Article: PubMed Central - PubMed

ABSTRACT
CCN2 plays a central role in the development and growth of mesenchymal tissue and promotes the regeneration of bone and cartilage in vivo. Of note, abundant CCN2 is contained in platelets, which is thought to play an important role in the tissue regeneration process. In this study, we initially pursued the possible origin of the CCN2 in platelets. First, we examined if the CCN2 in platelets was produced by megakaryocyte progenitors during differentiation. Unexpectedly, neither megakaryocytic CMK cells nor megakaryocytes that had differentiated from human haemopoietic stem cells in culture showed any detectable CCN2 gene expression or protein production. Together with the fact that no appreciable CCN2 was detected in megakaryocytes in vivo, these results suggest that megakaryocytes themselves do not produce CCN2. Next, we suspected that mesenchymal cells situated around megakaryocytes in the bone marrow were stimulated by the latter to produce CCN2, which was then taken up by platelets. To evaluate this hypothesis, we cultured human chondrocytic HCS-2/8 cells with medium conditioned by differentiating megakaryocyte cultures, and then monitored the production of CCN2 by the cells. As suspected, CCN2 production by HCS-2/8 was significantly enhanced by the conditioned medium. We further confirmed that human platelets were able to absorb/uptake exogenous CCN2 in vitro. These findings indicate that megakaryocytes secrete some unknown soluble factor(s) during differentiation, which factor stimulates the mesenchymal cells to produce CCN2 for uptake by the platelets. We also consider that, during bone growth, such thrombopoietic-mesenchymal interaction may contribute to the hypertrophic chondrocyte-specific accumulation of CCN2 that conducts endochondral ossification.

No MeSH data available.


Related in: MedlinePlus

Analysis of CCN2 gene expression and protein production during the course of megakaryocytic differentiation. Human cord blood CD34+ cells were caused by thrombopoietin to differentiate in vitro toward the megakaryocyte lineage. The cells were sampled at the indicated time points and then analyzed by FACS for cell morphology and demonstration of cell-surface antigens (a), by RT-PCR for CCN2 expression (b), and by ELISA for CCN2 production (c). Abbreviations SSC, FSC, PE, and FITC in panel A denote side scatter, forward scatter, propidium iodide, and fluorescein isothiocyanate, respectively. Total RNA from HCS-2/8 cells was utilized as a positive control in panel b. Similar experiments with peripheral blood-derived haemopoietic stem cells yielded comparable results (data not shown)
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Fig2: Analysis of CCN2 gene expression and protein production during the course of megakaryocytic differentiation. Human cord blood CD34+ cells were caused by thrombopoietin to differentiate in vitro toward the megakaryocyte lineage. The cells were sampled at the indicated time points and then analyzed by FACS for cell morphology and demonstration of cell-surface antigens (a), by RT-PCR for CCN2 expression (b), and by ELISA for CCN2 production (c). Abbreviations SSC, FSC, PE, and FITC in panel A denote side scatter, forward scatter, propidium iodide, and fluorescein isothiocyanate, respectively. Total RNA from HCS-2/8 cells was utilized as a positive control in panel b. Similar experiments with peripheral blood-derived haemopoietic stem cells yielded comparable results (data not shown)

Mentions: Since CMK is an established immortal cell line, it may not precisely retain the complete megakaryocytic phenotype. Therefore, we subsequently isolated CD34-positive haemopoietic stem cells from human cord blood leukocytes by cell sorting; and cytodifferentiation towards megakaryocyte progenitors was induced by incubation with recombinant thrombopoietin. According to the result of flow cytometric analysis, those cells initially showed morphological changes such as increased cell size, which was represented by the forward scatter values, 3 days after initiating the induction of differentiation. Thereafter, demonstration of CD41 molecules was clearly observed 10 days after the start of the cell cultures (Fig. 2a). These findings indicate that the isolated cells were mostly composed of haemopoietic stem cells that could differentiate into megakaryocyte progenitors in vitro. Under the same culture conditions, total RNA was isolated and subjected to PCR analysis for the detection of CCN2 mRNA. However, no CCN2 mRNA was detectable in the cells in the differentiating cultures at either 0, 3 or 10 days after the beginning of the cultures; whereas β-actin mRNA was definitely detected at all times (Fig. 2b). Consistent with this result, no CCN2 protein was detected in the cell culture supernatant, either (Fig. 2c). Therefore, we concluded that, during the differentiation in vitro, CCN2 was not produced by megakaryocyte progenitors. In addition, since terminal differentiation to mature megakaryocytes producing platelets was not inducible in vitro, we performed immunohistochemical staining to detect CCN2 in mature megakaryocytes in mouse bone marrow. Consequently, we could detect no positive signal representing CCN2 production (data not shown; Cicha et al. 2004).Fig. 2


Thrombopoietic-mesenchymal interaction that may facilitate both endochondral ossification and platelet maturation via CCN2.

Sumiyoshi K, Kubota S, Furuta RA, Yasui K, Aoyama E, Kawaki H, Kawata K, Ohgawara T, Yamashiro T, Takigawa M - J Cell Commun Signal (2009)

Analysis of CCN2 gene expression and protein production during the course of megakaryocytic differentiation. Human cord blood CD34+ cells were caused by thrombopoietin to differentiate in vitro toward the megakaryocyte lineage. The cells were sampled at the indicated time points and then analyzed by FACS for cell morphology and demonstration of cell-surface antigens (a), by RT-PCR for CCN2 expression (b), and by ELISA for CCN2 production (c). Abbreviations SSC, FSC, PE, and FITC in panel A denote side scatter, forward scatter, propidium iodide, and fluorescein isothiocyanate, respectively. Total RNA from HCS-2/8 cells was utilized as a positive control in panel b. Similar experiments with peripheral blood-derived haemopoietic stem cells yielded comparable results (data not shown)
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2821475&req=5

Fig2: Analysis of CCN2 gene expression and protein production during the course of megakaryocytic differentiation. Human cord blood CD34+ cells were caused by thrombopoietin to differentiate in vitro toward the megakaryocyte lineage. The cells were sampled at the indicated time points and then analyzed by FACS for cell morphology and demonstration of cell-surface antigens (a), by RT-PCR for CCN2 expression (b), and by ELISA for CCN2 production (c). Abbreviations SSC, FSC, PE, and FITC in panel A denote side scatter, forward scatter, propidium iodide, and fluorescein isothiocyanate, respectively. Total RNA from HCS-2/8 cells was utilized as a positive control in panel b. Similar experiments with peripheral blood-derived haemopoietic stem cells yielded comparable results (data not shown)
Mentions: Since CMK is an established immortal cell line, it may not precisely retain the complete megakaryocytic phenotype. Therefore, we subsequently isolated CD34-positive haemopoietic stem cells from human cord blood leukocytes by cell sorting; and cytodifferentiation towards megakaryocyte progenitors was induced by incubation with recombinant thrombopoietin. According to the result of flow cytometric analysis, those cells initially showed morphological changes such as increased cell size, which was represented by the forward scatter values, 3 days after initiating the induction of differentiation. Thereafter, demonstration of CD41 molecules was clearly observed 10 days after the start of the cell cultures (Fig. 2a). These findings indicate that the isolated cells were mostly composed of haemopoietic stem cells that could differentiate into megakaryocyte progenitors in vitro. Under the same culture conditions, total RNA was isolated and subjected to PCR analysis for the detection of CCN2 mRNA. However, no CCN2 mRNA was detectable in the cells in the differentiating cultures at either 0, 3 or 10 days after the beginning of the cultures; whereas β-actin mRNA was definitely detected at all times (Fig. 2b). Consistent with this result, no CCN2 protein was detected in the cell culture supernatant, either (Fig. 2c). Therefore, we concluded that, during the differentiation in vitro, CCN2 was not produced by megakaryocyte progenitors. In addition, since terminal differentiation to mature megakaryocytes producing platelets was not inducible in vitro, we performed immunohistochemical staining to detect CCN2 in mature megakaryocytes in mouse bone marrow. Consequently, we could detect no positive signal representing CCN2 production (data not shown; Cicha et al. 2004).Fig. 2

Bottom Line: In this study, we initially pursued the possible origin of the CCN2 in platelets.As suspected, CCN2 production by HCS-2/8 was significantly enhanced by the conditioned medium.We further confirmed that human platelets were able to absorb/uptake exogenous CCN2 in vitro.

View Article: PubMed Central - PubMed

ABSTRACT
CCN2 plays a central role in the development and growth of mesenchymal tissue and promotes the regeneration of bone and cartilage in vivo. Of note, abundant CCN2 is contained in platelets, which is thought to play an important role in the tissue regeneration process. In this study, we initially pursued the possible origin of the CCN2 in platelets. First, we examined if the CCN2 in platelets was produced by megakaryocyte progenitors during differentiation. Unexpectedly, neither megakaryocytic CMK cells nor megakaryocytes that had differentiated from human haemopoietic stem cells in culture showed any detectable CCN2 gene expression or protein production. Together with the fact that no appreciable CCN2 was detected in megakaryocytes in vivo, these results suggest that megakaryocytes themselves do not produce CCN2. Next, we suspected that mesenchymal cells situated around megakaryocytes in the bone marrow were stimulated by the latter to produce CCN2, which was then taken up by platelets. To evaluate this hypothesis, we cultured human chondrocytic HCS-2/8 cells with medium conditioned by differentiating megakaryocyte cultures, and then monitored the production of CCN2 by the cells. As suspected, CCN2 production by HCS-2/8 was significantly enhanced by the conditioned medium. We further confirmed that human platelets were able to absorb/uptake exogenous CCN2 in vitro. These findings indicate that megakaryocytes secrete some unknown soluble factor(s) during differentiation, which factor stimulates the mesenchymal cells to produce CCN2 for uptake by the platelets. We also consider that, during bone growth, such thrombopoietic-mesenchymal interaction may contribute to the hypertrophic chondrocyte-specific accumulation of CCN2 that conducts endochondral ossification.

No MeSH data available.


Related in: MedlinePlus