The Potential Role Of Non-adherent Bone Marrow-derived Stem Cells As New Cell Source In Rescuing Of Bone Marrow Acute Radiation Sickness And The Mechanisms Involved

Acute radiation sickness occurs after whole-body or significant partial-body irradiation greater than 1Gy delivered at a relatively high-dose rate within several seconds or days. After being exposed to X-rays or r rays between 1 Gy and 10 Gy, hematopoietic tissue damage especially bone marrow damage is the basic injury which determines further development of the disease. Bone marrow damage mainly shows inhibition or failure of hematopoietic function, pancytopenia, and injury of small blood vessel wall, causing widespread life-threatening hemorrhage and infection. Thus, reducing damage of hemotopoietic organs and promoting hematopoietic reconstruction is the key treatment to ARS.Mesenchymal stem cells (MSCs) are important constituents of bone marrow hematopoietic inductive microenvironment (HIM). They could promote recovery of hematopoiesis not only because of their ability to secrete hematopoietic growth factors and to differentiate into various stromal cel1 types to repair the HIM, but also because of their organ-specific differentiation potential and low immunogenicity, through which they could promote recovery of multiple organ damage resulted from ARS and reduce incidence of GVHD after cotransplanted with HSC in stem cell therapy. Due to these characteristics, it will be prospective to apply MSCs to tissue engineering and gene therapy. Recently, researchers have demonstrated that except adherent MSCs, their was a kind of new stem cells in non-adherent bone marrow, named non-adherent bone marrow-derived stem cells(NA-BMSCs), which was more primitive than MSCs and it could be differentiated into MSCs and various stromal cells under certain condition. Moreover, it is reported that NA-BMSCs is the common stem cells of both hematopoietic stem cells and mesenchymal stem cells. Their detail characteristics and therapeutic potential in tissue regeneration need further research.In view of the above reasons, we isolated NA-BMSCs from human fetal bone marrow using repeatedly-transferred culture, observed the similarities and differences of the chatacteristics of NA-MSCs and MSCs, and then we paid much attention to the therapeutic potential of NA-BMSCs in bone marrow acute radiation disease and the mechanisms involved.PARTⅠIsolation and identification of non-adherent bone marrow-derived stem cellsObjective:To investigate whether NA-BMSCs exist in NA-BMCs from human fetal, and to compare their characteristics with MSCs. Methods:Bone marrow cells of human fetal bone were isolated and cultured, non-adherent parts of bone marrow cells were repeatedly-transferred into new dishes every 24 hours for 4 times, total adherent cells of the first dish was MSCs, and those in the last dish was NA-MSCs. Two kinds of cells in passage 7 were used. The growth curve, cell cycle and many surface markers such as CD29, CD105, CD45, etc were identified using MTT and flow cytometry, respectively. To detect their differentiation potential, 2 conditioned medium were used to induce the two cells into adipocyte and osteoblast cells and then Oil red staining and VonKossa staining were used to determine the formation of lipid drops and calcium . Results:In the process of repeatedly-transferred culture, adherent MSCs occurred from NA-BMSCs and they could proliferate to CFU-F continuously.After adherent, the morphology of NA-BMSCs was similar to MSCs, and it also has great proliferation potential and differentiation potential after generation. It was found that NA-MSCs expressed CD106, CD29, and MSCs-specific surface marker CD105, CD73, but not antigens of hematopoietic cells CD14, CD34 and CD45, and almost not antigens related to graft-versus-host disease (GVHD), such as HLA-DR. However, the proliferation potential and the osteogenic potential of NA-MSCs was higher than those of MSCs. Compared with MSCs, NA-MSCs in passage 13 also maintained high proliferation rate. Conclusion: NA-BMSCs exist in NA-BMCs of human fetal;and NA-MSCs could differentiate into MSCs. In other words, there is a new stromal stem cell in NA-BMCs, which is more primitive than MSCs.PARTⅡTherapeutic potential of non-adherent bone marrow-derived stem cells in bone marrow acute radiation sickness and the mechanisms involvedObjective: To investigate the therapeutic potential of NA-BMSCs in bone marrow acute radiation disease, as well as the mechanisms involved. Methods: After being exposed to 8.5Gy total body irradiation, BALB/c mice were randomly assigned into transplantation group and control group. Mice in transplantation group were given 3×106 CFDA-SE labeled NA-BMSCs-derived NA-MSCs, and those in control group were given 0.3ml normal saline. Then, the survival rate, peripheral white blood cells at different time, pathologic change and angiogenesis of bone marrow were observed.Results: The survival rate of mice in transplantation group (14/21)was much higher than that of the control group(2/21). Compared with the control group, the white blood cells in transplantation group decreased more slowly while recovered more rapid--- the nadir appeared at D14 after transplantation while it recovered within 30d. The bone marrow of mice in transplantation group regenerated more actively and had more hematopoietic islands than control group on day 21. In addition, bone marrow angiogenesis of transplantation group (31.7±3.61)were more obvious than that of the control group(11.2±2.33). Conclusion: Human fetal NA-BMSCs could promote bone marrow angiogenesis in acute radiation disease in mouse model, through which they play an important role in tissue regeneration in ARS.