BACKGROUND： Previous studies have reported a neurotrophin-like motif in the N-terminal receptor binding region of the thrombopoietin （TPO） molecule, and have described localization of TPO and TPO receptor in the br...BACKGROUND： Previous studies have reported a neurotrophin-like motif in the N-terminal receptor binding region of the thrombopoietin （TPO） molecule, and have described localization of TPO and TPO receptor in the brain. Therefore, it is believed that TPO may be involved in regulation of neurogenesis. OBJECTIVE： To validate the effect of TPO on trans-differentiation, or differentiation from hematopoietic stem cells （HSCs） to neural stem cells （NSCs）. DESIGN, TIME AND SETTING： Comparative studies were performed from March 2004 to April 2007 at the Department of Experimental Medicine, Northern Hospital, and the Department of Immunology, Fourth Military Medical University of Chinese PLA. MATERIALS： Human fetal liver （FL） was obtained from fetuses after water-balloon abortion. Gestational age ranged from 16 to 20 weeks. The study was approved by the Institutional Review Board and Ethics Committee of the Northern Hospital. TPO was kindly provided by Genentech Inc （USA）. Iscove＇s Modified Dulbecco＇s Medium （IMDM） and neurobasalTM medium were purchased from Invitrogen （USA）. MACS CD34 multisort kit was purchased from Miltenyi Biotec （Germany）. METHODS： CD34^＋ cells were isolated from human FL mononuclear cells using MACS CD34 multisort kit and cultured at 1 × 10^5/mL in IMDM, containing TPO for 60 days with weekly changes of half of the medium. After culturing for 30 and 60 days, the TPO-induced cells were resuspended in neurobasalTM medium containing 10% fetal brain extracts and plated in an 8-well BIOCOAT poly-D-Lysine Culture Slide and cultured for another 7 days. MAIN OUTCOME MEASURES： Cell number, viability, phenotype and expression of hemopoiesis-related and neurogenesis-related proteins were examined by trypan blue exclusion with hemocytometer, immunoblot, immunocytochemistry and flow cytometry. RESULTS： After 60 days of induction with TPO, the cell number increased by 4.6-fold compared to the initial culture. Although the proportion of the cells expressing the hemopoietic stem cell associated antigen （CD34） decreased steadily, both proportions of the cultured FL-derived CD34^＋cells expressing CD41a and CD61 remained unchanged, which still accounted for 10%. Noticeably, the proportions of the cells expressing nestin and epidermal growth factor receptor increased significantly （both 〉 50%）, whereas the expression of more mature neural or glial proteins [microtubule-associated protein-2 （MAP2）, glial fibrillary acidic protein （GFAP）, oligodendrocyte marker 04 （04）] markers on the cultured fetal liver derived-CD34^＋ cells were at lower levels. After another 7 days incubation in neurobasalTM medium, these TPO-induced cells formed neurospheres, which were labeled with nestin, and differentiated into cells with morphological characteristics of neurons, astrocytes and oligodendrocytes, which were labeled with MAP-2, GFAE and 04, respectively. CONCLUSION： TPO can induce FL-derived HSCs to differentiate or trans-differentiate into NSCs and its progenitors.展开更多
基金National Key Basic Research Program of China, No. 2001CB510004the National Natural Science Foundation of China,No.30030130
文摘BACKGROUND： Previous studies have reported a neurotrophin-like motif in the N-terminal receptor binding region of the thrombopoietin （TPO） molecule, and have described localization of TPO and TPO receptor in the brain. Therefore, it is believed that TPO may be involved in regulation of neurogenesis. OBJECTIVE： To validate the effect of TPO on trans-differentiation, or differentiation from hematopoietic stem cells （HSCs） to neural stem cells （NSCs）. DESIGN, TIME AND SETTING： Comparative studies were performed from March 2004 to April 2007 at the Department of Experimental Medicine, Northern Hospital, and the Department of Immunology, Fourth Military Medical University of Chinese PLA. MATERIALS： Human fetal liver （FL） was obtained from fetuses after water-balloon abortion. Gestational age ranged from 16 to 20 weeks. The study was approved by the Institutional Review Board and Ethics Committee of the Northern Hospital. TPO was kindly provided by Genentech Inc （USA）. Iscove＇s Modified Dulbecco＇s Medium （IMDM） and neurobasalTM medium were purchased from Invitrogen （USA）. MACS CD34 multisort kit was purchased from Miltenyi Biotec （Germany）. METHODS： CD34^＋ cells were isolated from human FL mononuclear cells using MACS CD34 multisort kit and cultured at 1 × 10^5/mL in IMDM, containing TPO for 60 days with weekly changes of half of the medium. After culturing for 30 and 60 days, the TPO-induced cells were resuspended in neurobasalTM medium containing 10% fetal brain extracts and plated in an 8-well BIOCOAT poly-D-Lysine Culture Slide and cultured for another 7 days. MAIN OUTCOME MEASURES： Cell number, viability, phenotype and expression of hemopoiesis-related and neurogenesis-related proteins were examined by trypan blue exclusion with hemocytometer, immunoblot, immunocytochemistry and flow cytometry. RESULTS： After 60 days of induction with TPO, the cell number increased by 4.6-fold compared to the initial culture. Although the proportion of the cells expressing the hemopoietic stem cell associated antigen （CD34） decreased steadily, both proportions of the cultured FL-derived CD34^＋cells expressing CD41a and CD61 remained unchanged, which still accounted for 10%. Noticeably, the proportions of the cells expressing nestin and epidermal growth factor receptor increased significantly （both 〉 50%）, whereas the expression of more mature neural or glial proteins [microtubule-associated protein-2 （MAP2）, glial fibrillary acidic protein （GFAP）, oligodendrocyte marker 04 （04）] markers on the cultured fetal liver derived-CD34^＋ cells were at lower levels. After another 7 days incubation in neurobasalTM medium, these TPO-induced cells formed neurospheres, which were labeled with nestin, and differentiated into cells with morphological characteristics of neurons, astrocytes and oligodendrocytes, which were labeled with MAP-2, GFAE and 04, respectively. CONCLUSION： TPO can induce FL-derived HSCs to differentiate or trans-differentiate into NSCs and its progenitors.
