Investigation On Human Umbilical Cord Blood-derived Stromal Cells For Promoting Megakaryocytopoiesis And Thrombopoiesis

Dysfunction of hematogenesis is a main complication under the condition of chemotherapy, radiotherapy, unexpected leakage of radioactive material and nuclear warfare. Thrombocytopenia caused by the injury of megakaryocyte is to be a formidable puzzle to solve till recently. Many risks, such as bleeding caused by the anticoagulated antibody, might be caused by the application of thrombopoietin (TPO), which is used as a tool to deal with the thrombocytopenia. So, the methods to restore the quantity and function of platelets efficiently and securely are remained to explore.Hematopoietic inductive microenvironment (HIM), the internal environment for sustaining and regulating the growth and development of hematopoietic cell, acts as an important role in the proliferation, differentiation and maturation of megakaryocytes. So, it is worth finding some methods to cope with the injury of megakaryocytes start with the reconstruction of HIM.Bone marrow stromal cells(BMSCs) is the main component of hematopoietic inductive microenvironment, BMSCs was researched systematically since it was cultured in vitro by Dexter in 1977. Infusion of the suspension containing BMSCs expanded in vitro and hemapoietic stem cells is thought to be an effective method to restore the normal function of hematopoiesis previously. But, it can't be applied extensively in clinical practices for some reasons, such as the dysfunction of HIM in autotransplantation and histocompatibility in allograft. Based on some advantages, such as easy-to-obtain, convenient to collect and low immunogenicity, cord blood is now used as a new source of haemopoietic stem cells. Our group also has studied the human umbilical cord blood-derived stromal cells (hUCBDSCs) and the cord blood associated HIM for many years. After the successful acquirement of hUCBDSCs in vitro in our preliminary experiments, bio-characteristics and hematopoietic supporting-capacity of these cells were observed. Interestingly, it was found that many stromal cell specific features was possessed by hUCBDSCs. Moreover, as compared with the alternative expansion system, which human bone marrow stromal cells (hBMSCs) were used as the trophoblastic cells, the colony forming unit-megakaryocte rate was much higher in the co-culture system with hUCBDSCs to be trophoblastic cells. It is hinted that hUCBDSCs might act as a special role for the proliferation of megakaryocytes. Thus, some therapeutic improvements on the megakaryocytic injury might be made if the further understanding of this phenomenon could be reached.Accordingly, based on the construction of megakaryocyte/hUCBDSCs co-culture model and nude mice HIM injury model, the mechanisms of hUCBDSCs to promote the proliferation of megakaryocytes and to reconstruct the HIM in vivo, especially, to restore the quantity of platelet were explored in this study, which were mainly focused on the following three aspects: TPO pathway, SDF-1 pathway and gap junction communication. Some experimental data might be obtained for the improvement of megakaryocytic injury therapy.Methods:1. To observe the influence of hUCBDSCs on the proliferation of megakaryocytic cell line HEL.Groups: HEL suspended culture group; HEL/hBMSCs co-culture group; HEL/ hUCBDSCs co-culture group. The spatial relationship between the HEL cells and stromal cells was observed under the inverted microscope and scanning electron microscope, then, CCK8 assay was used to determine the proliferation of HEL cells. After this, the cell cycle distribution and the rate of apoptosis/necrosis of these cells were assessed by flow cytometry. At last, the ultrastructure of HEL cells was revealed under the transmission electron microscope.2. To observe the reconstruction of HIM, especially the recovery of megakaryocytes caused by the implantation of hUCBDSCs into nude mice.To observe the changes of hemogram, bone marrow biopsy and CFU-F in nude mice at different phases after irradiation in order to explore the proper irradiation dose to apply in this study. At last, 5.0 Gy was used as the experimental dose in the following experiments.Groups: Isotonic Na chloride group; hBMSCs group; hUCBDSCs group. After ionizing radiation, the reconstruction of HIM and the recovery of megakaryocytes in ` different groups were determined in the nude mice with transplantation.3. Possible mechanisms for hUCBDSCs to promote the proliferation of megakaryocytes.3.1 Possible role performed by the TPO pathway.The concentration of TPO in the culture supernatant of hBMSCs and hUCBDSCs was detected by ELISA assay. The expression of C-mp1 at mRNA and protein levels in HEL cells was determined by RT-PCR, confocal microscopy and flow cytometry under the different culture conditions.3.2 Possible role performed by the SDF-1 pathway.The concentration of SDF-1 in the culture supernatant of hBMSCs and hUCBDSCs was detected by ELISA assay. The expression of CXCR4 at mRNA and protein levels in HEL cells was determined by RT-PCR, laser confocal microscopy and flow cytometry under the different culture conditions.3.3 Possible role performed by the gap conjunction communication.The gap conjunction communication between HEL cells and hUCBDSCs was assessed by fluorescence recovery after photobleaching assay (FRAP). The expression of Connexin-43 (Cx43) at mRNA and protein levels in HEL cells and hUCBDSCs under co-culture condition was determined by RT-PCR and laser confocal microscopy, respectively.Results:1. It was revealed under the inverted microscopy that many HEL cells attached on the hUCBDSCs. Under the inverted microscopy, some of them adhered to the hUCBDSCs surface by pseudopodia, the others inserted into the meshes formed by several fused hUCBDSCs. The quantity of attached-HEL cells increased significantly as the culturing time prolonged. As compared with the other groups, HEL cells in the HEL/hUCBDSCs co-culture group grew faster. But, the rate of apoptosis/necrosis in different groups was the same. It is indicated that some special roles might be performed by hUCBDSCs in the proliferation of HEL cells.2. Different degrees of myelosuppression and CFU-F decreasing could be found in the mice, which were administrated with different dose of radiation. The 5.0 Gy was selected as the proper dose in the following studies. It was revealed by the in vivo assay that the two kinds of stromal cells, especially hUCBDSCs, possessed the capacity of promoting the proliferation of platelets. After processed a transient retention at liver, spleen and lung, hUCSDCs homing quickly to bone marrow to reconstruct the impaired HIM.3. The mechanisms of hUCBDSCs to promote the proliferation of megakaryocytes3.1 It was revealed by ELISA assay that the concentration of TPO secreted by hUCBDSCs was higher than that of the hBMSCs did, even though the passage was done. But, the appearance of secretion peak in hUCBDSCs group was late. The expression of C-mpl protein was enhanced as determined by flow cytometry and laser confocal microscopy. But, there was no significant differences of the C-mpl mRNA level between different groups.3.2 The concentration of SDF-1 in different groups was the same at the early stage of culturing. But, 6 days after seeding, it increased significantly in the hUCBDSCs group, even though the passage was done. The expression of CXCR4 protein was weaken as determined by flow cytometry and laser confocal microscopy. Red punctiform fluorescence was detected in endochylema by laser confocal microscopy after HEL cell line co-cultured with hUCBDSCs. But, there was no significant differences of the CXCR4 mRNA level between different groups.3.3 Between the hUCBDSCs and hUCBDSCs, it was revealed by FRAP assay that the fluorescence intensity was recovered quickly in the photobleached cells. But, this phenomenon was not found between the hUCBDSCs and HEL cells. Moreover, under the co-culture condition, the expression level of Cx43 was much weaker in HEL cells than that in hUCBDSCs. The spatial distribution of this protein was also quite different in these two cells. Under laser confocal microscopy, fluorescence displayed as a homogeneous distribution in HEL cells but a plaquelike distribution in hUCBDSCs.Conclusion:1. The proliferation of megakaryocytes can be enhanced by the hUCBDSCs in vitro.2. The impaired HIM, especially the decreasing and aberrant platelets, can be restored after the hUCBDSCs has been implanted into the nude mice.3. High level of TPO is secreted by hUCBDSC. Meanwhile, the expression of C-mpl protein, the receptor of TPO on megakaryocyte also can be upregulated by hUCBDSC. This might be one of the reasons for the hUCBDSC to promote the proliferation, differentiation and maturation of megakaryocytes.4. High level of SDF-1 is secreted by hUCBDSCs to promote the proliferation and migration of megakaryocyte. With migration, the expression of CXCR4 protein on megakaryocyte was decreased and formed"internalized vesic", that regulate the migration of megakaryocyte.5. There is no obvious gap junction communication can be found between the hUCBDSCs and HEL cells. It is indicated that this kind of interaction might not be the main cause of the hUCBDSCs to promote the proliferation of megakaryocytes.