Chinese Journal of Tissue Engineering Research ›› 2013, Vol. 17 ›› Issue (32): 5757-5764.doi: 10.3969/j.issn.2095-4344.2013.32.003
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Induction ways of bone marrow mesenchymal stem cells differentiating into nerve cells
Chen Zeng-sheng1, Chu Qiang2, Liu Yan-feng3, Song Xuan4, Li Ping5
- 1 Department of Laboratory, Qingdao Municipal Hospital, Qingdao 266071, Shandong Province, China
2 Department of Ultrasound, Qingdao Municipal Hospital, Qingdao 266071, Shandong Province, China
3 Department of Laboratory, Sifang District Hospital, Qingdao 266033, Shandong Province, China
4 Department of Blood Transfusion, Qingdao Municipal Hospital, Qingdao 266071, Shandong Province, China
5 Department of Blood Transfusion, Affiliated Hospital, Medical School of Qingdao University, Qingdao 266000, Shandong Province, China
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Received:2013-05-11Revised:2013-06-22Online:2013-08-06Published:2013-08-06 -
Contact:Li Ping, Master, Technician-in-charge, Department of Blood Transfusion, Affiliated Hospital, Medical School of Qingdao University, Qingdao 266000, Shandong Province, China lpxkck@126.com -
About author:Chen Zeng-sheng★, Master, Technician, Department of Laboratory, Qingdao Municipal Hospital, Qingdao 266071, Shandong Province, China Redapple_02@126.com
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Chen Zeng-sheng, Chu Qiang, Liu Yan-feng, Song Xuan, Li Ping. Induction ways of bone marrow mesenchymal stem cells differentiating into nerve cells[J]. Chinese Journal of Tissue Engineering Research, 2013, 17(32): 5757-5764.
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Morphology of cultured bone marrow mesenchymal stem cells Lots of erythrocytes covered the entire bottom of culture flask with whole bone marrow culture after inoculation. A large number of blood cells were removed after low glucose Dulbecco’s modified Eagle’s medium was changed at the first time, scattered adherent cells could be seen in the bottom of the bottle, with fusiform or polygonal appearance, and uneven part was a whirlpool-like growth. The formation of multiple fibroblast-like cell clusters could be seen after 6 or 8 days, few round cells could be seen around the cell clusters. Fibroblast-like growth was pooled into a piece after 12 or 16 days. Adherent cells were long spindle-shaped after passage for 2-3 days. Primary cultured bone marrow mesenchymal stem cells had a multiple differentiation potential and maintained a higher purity over 90% after repeated passages[22-23, 25-26] (Figure 1)."
Surface marker expression of bone marrow mesenchymal stem cells Flow cytometry results showed that bone marrow mesenchymal stem cells strongly expressed hyaluronan receptor CD44, with the positive rate of over 99%, but did not express the hematopoietic stem cell marker CD34 (< 1%) (Figure 2)."
Co-culture and chemical induction methods for inducing differentiation into nerve cells Most of the cultured nerve cells grew adherent, and the cell body was full and fusiform, withshining brilliantly cytoplasm. Small protrusions were visible in the cell body for 2 or 3 days. Glial cell body was flat and had slightly larger size, and one or four short rod protrusions were around the cell body. The nerve cell bodies grew larger, and rounded protrusions around the cell body were longer and increased to form a unipolar, bipolar or multipolar projection for 6 days. Nerve cells inoculated in the 6-well plates of Transwell co-culture system, bone marrow mesenchymal stem cells were found to have pyknosis smaller than flat cells. Formerly, most of the cell bodies had rounded protrusion formation, and cells gradually became long stellate for 3 days. The stellate cells gradually increased, and could cross each other to form a connection with a neuron-like morphology (Figure 3) for 4 or 5 days. Neural-like cells accounted for 62.4% of the total cells. Immunofluorescence results showed Neuron-specific enolase positive rate was (60.47± 1.89)%, suggesting the performance characteristics of neurons (Figure 3). Most of the cells were still flat and wide long spindle-shaped similar as morphology characteristics of bone marrow mesenchymal stem cells, and did not form a neuron-like morphology in the control group after cultured for 8 days. Neuron-specific enolase was also negative in the control group."
Chemically induced culture results Cells showed adherent growth after 1-3 days in the chemically induced group, and no obvious morphological changes were observed. The cell body significantly shrank and a circle or a triangle extended at 4 days. Some cells formed longer dendritic-like branches, and mutual extending connection; some cells were similar to neurons, and the long axons appeared to render the nerve cell-like morphology (Figure 4) at 5-6 days. Nerve cells accounted for 50.4% of the total cells, and the positive rate for neuron-specific enolase was (40.91± 1.63)%."
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