| Stem cell research is one of hotspots in the field of life science research, there are a lotof research achievements applied in clinical practice.This study by Chinses unique localpoultry breed Beijing fatty chicken as the research object. Mesenchymal stem cells fromliver tissue of7d old chicken embryo were separated and incubated in vitro and cultured inoptimized virto environment to25generations, cryopreserved156Beijing fatty chickenMSCs. Immunofluorescence and RT-PCR identified separation passage cells and passagedcell cryopreservation and recovery in order to explore the possibility of this preciousgenetic resources Cells were stored Beijing fatty chicken; cells induced by different cultureconditions, differentiation respectively into adipose cells,bone cells, nerve cells, andmyocardial cells, immunofluorescence and RT-PCR induced differentiation of cells wereidentified.The results prove that:（1）Mesenchymal stem cells from liver tissue of chicken embryo still showedproliferation and differentiation potential;the characteristics of cells meet the standard ofmesenchymal stem cells;which isolated cells were mesenchymal stem cells originated fromliver of Beijing fatty chicken embryo.（2）Chicken fetal liver MSCs cultured in vitro performed vigorous growth under10%FBS,5ng/mL bFGF and DMEM/F12culture system after comparison andoptimization, which performed typical fibroblast appearance. Majority of the MSCsdisplayed long fusiform or irregular triangles form; cells were arranged in swirling orflame-shaped, With the increase of in vitro culture cell number of batches. cell morphologytend to consistent, low generation cell proliferation faster, high generation cell growthslowed significantly, with the number of batches senescent cells increased.（3）Immunofluorescence cytochemistry detected surface markers of differentgenerations chicken fetal liver MSCs CD29and CD44were positive expressed, was notexpressed CK19and CD34. RT-PCR detection and identification on different generations’MSCs surface markers on RNA transcript levels for CD44, CD29, CD71and CD73wereall showed positive expression. Grayscale analysis showed that except from weakenedexpression quantity in CD73as generation increase, the expression levels in other markersdid not differ significantly. Isolated cells were mesenchymal stem cells, which were able to maintain the original characteristics.（4）As the increasing of generation, MSCs from chicken fetal liver showeddownward trend of cell cryopreservation and resuscitation survival rate. Differentgenerations of chicken fetal liver MSCs displayed classical "S" shape, cell multiplicationexperienced incubation period, logarithmic growth period and growth stagnation period.The cultured low generation chicken fetal liver MSCs contained higher number of S-phasecells, and lower number of G0/G1phase cells. As generation increase the G0/G1phase cellsproportion increased and number of S-phase cells significantly decreased. Thawed cellscultured in vitro in line with the growth pattern of the cell culture in vitro.（5）Chicken fetal liver MSCs in different generations of cells under certain cultureconditions are able to differentiate to adipocytes, osteoblasts, nerve cells and myocardialcells, followed by some morphology changes and death of small part of cells. However,cell differentiation capacity diminished as generation increase and differentiated cells fromhigh-generation were aging significantly. In the experiment, differentiated cells werephysical detected for testing the differentiation effects through corresponding stainingmethods, followed by test on PPAR-γ、LPL, Collage typeⅠ、Osteopontin, Nestin、NF andDesmin、MyoD1four cell markers through RT-PCR, each includes two types respectively.Furthermore, analysis was done on changing rule of, so as to prove the high generationcells showed downward trend of expression level，compared to low generation cells. It isindicating that low generation cells are more easily induced to differentiate in vitro, celldifferentiation ability is weakened as generation increase. The experiment proved thatMSCs isolated from7d chicken embryonic liver are pluripotent MSCs.Research showed that MSCs isolated from Beijing fatty chicken embryo liveraccordance with biological characteristics of MSCs. The cells cultured in vitro are able topass on up to25generations at most; cells preserved in liquid nitrogen are able to besubcultured and maintain biological characteristics of MSCs after recovery. Chicken MSCscan be induced and differentiated to adipocytes, osteoblasts, nerve cells and myocardialcells. In this study, isolation and identification methods and experiment processes of thechicken fetal liver MSCs are initially established. |
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