Abstract
The bone marrow (BM) microenvironment plays an important role in regulation of thrombopoiesis through release of thrombopoietic growth factor, among which thrombopoietin (TPO) is the most essential regulator. It has been realized that the TPO blood level and platelet count is inversely related, but the precise controlling mechanism is still under investigation. TPO is constitutively produced by the liver but can be induced to generate from BM mesenchymal stromal cells (MSCs) in pathological conditions such as thrombocytopenia. Since serotonin (5-HT) can be released from the active platelets when the body undergoing severe thrombocytopenia or stress, it may function as an essential humoral mediator regulating TPO production under this model. Our previous studies suggested that 5-HT enhances the growth of the colony-forming unit-fibroblast (CFU-F), a progenitor of MSCs (Yang M et al, 2007). However, the exact functions of 5-HT on MSCs and especially MSCs-derived TPO production are still under investigated. In this study, we showed that TPO RNA expression in hMSCs was enhanced in a time-dependent manner in response to the 5-HT treatment (200nM). The maximum reaction peak was observed at 24 hrs with 1.5-fold increase compared to the untreated control. A significant increase TPO protein was found in the supernatants secreted by 5-HT-treated hMSCs. The changes measured by ELISA and cytokinearray and were 4.5-fold (P=0.0372, n=4) and 2.14-fold increased compared to the control sample. Microparticles (MPs) are membrane-derived particles (0.1-1.0μm in size) produced from activated or apoptotic cells. They have been described as an important mediator in regulating a wide range of physiological and pathological response by transforming a spectrum of cytokines, signaling proteins, mRNAs and microRNAs. Recent studies have suggested that BM MSCs is one of the major sources for MPs production and that RNA contained in MSC-derived MPs can be isolated. Therefore, TPO mRNA released from MSCs could also be stored in MPs and released into the circulation or functions in a paracrine manner. In this study, in addition to the evaluation of the effect of 5-HT on naked-TPO release from MSCs, we also investigate its impacts on the RNA expression of TPO contained within the MSC-derived MPs. 5-HT was added to the hMSC culture for 24hrs, 36hrs and 48hrs respectively. It significantly stimulated MPs released from MSCs detected by flow cytometry. The maximal response time was observed at 36hrs. The AnnexinV positive population increased remarkably under 5-HT treatment (P=0.0007, n=4). HtMSC Cell movement and MPs released were visualized under phase contrast microscopy. HtMSC cells with 5-HT treatment were stained with AnnexinV-PE and the plates were inspected under the Tirf microscopy to trace the shedding process of MPs. By using phase contrast microscopy, a notable generation of MPs was observed from approximate 6hrs after the addition of 5HT, while only a little amount of MPs were detected in the untreated control group. Budding MPs were observed in 5HT-treated MSCs under both SEM and TEM. The morphology of these MPs was compatible to those published in other studies. And these results were complementary to our previous data obtained by flow cytometry and Tirf microscopy. Our data proved that hMSCs-derived MPs contained inducible TPO mRNA, which was enhanced under 5-HT treatment with more than 10-fold increase compared to the untreated control (P=0.0025, n=3). We further examined protein level of TPO in MPs using ELISA. The TPO protein level was increased more than 3-fold in 5-HT treated sample (P=0.0052, n=3). In summary, our findings suggested that 5-HT stimulated TPO released from MSCs in both dissociative and MP-bounded form, which indirectly promotes megakaryopoiesis and thrombopoiesis.
Disclosures:
No relevant conflicts of interest to declare.
Segmentation of bone marrow stromal cells in phase contrast microscopy images
