In Vitro Culture Of Chicken Primordial Germ Cells And The Affection Mechanism Of A Key Growth Factor

Primordial germ cells（PGCs）are precursor cells of gonocyte.As a kind of pluripotent stem cell,PGCs have a similar morphology and multilineage potential with the embryonic stem（ES）cell,which can be cultured and subcultured in vitro and maintain its undifferentiated state and pluripotency under certain conditions.At present,PGCs are often cultured with feeder cells and serum,which causes difficulties and confusion for the later research.This study was aim to establish a serum-free and no-feeder layer culture system,and to study the effect of key growth factors on proliferation of PGCs in vitro and its mechanism on this basic.The main contents of this paper are as follows.1.Establishment of no-feeder layer culture system of primordial germ cells of Yuehuang chicken.In this experiment,PGCs were cultured with a variety of essential factors under no-feeder layer system according to its growth status.The results showed that the diameter of PGCs was 6-8μm.The growth curve of PGCs showed"S"shape and its population doubling time was（3.75±0.15）d,which had a good multiplication capacity and expressed the specific genes SSEA-1 and DAZL of PGCs.PGCs were labeled with PKH26 and injected into the dorsal aorta of recipient chicken embryo that hatched to the 17-20HH until day 5.5,the results showed that PGCs could migrate and localize in the gonads,this proved that PGCs could migrate and differentiate in vivo.The results provided important technical support for application of PGCs in PGCs-based modern preservation technology,transgenic technology and developmental biology of pultry.2.Comparison of characteristic of PGCs with different culture system of PGCs in vitro.Under no-feeder layer culture system,PGCs were suspension growth,and there was no adhesion between cells,which had significantly differences from the common feeder layer culture system.Comparing with the cells size,colonies and adhesion factors,the results showed that PGCs cultured with feeder layer were larger and grew into colony instead of adhering to the feeder layer.PGCs cultured with no-feeder layer were smaller and didn’t adhere to each other,which grew as scattered single cell and sank to the bottom of the culture plate.There was significant difference in the response to BMP4 between these two groups of cells.Multiplication capacity(OD₄₅₀)of cells added with BMP4 under no-feeder layer system was significantly increased,while the proliferation of the cells under feeder layer system was not obvious,but the adhesion ability of its single cell was increased and the colony area increased significantly.There was no significant difference in the expression of BMPR1A and BMPR2 between the two culture conditions by detecting BMP4 receptor.Suggesting that the signal pathways of BMP4 and the signal pathways of cell adhesion and adhesion-related both effected PGCs,and the two signal paths had interaction.So the comparison experiment of this two signal pathways is continued.3.Influence mechanism of Signal pathways of BMP4 for the proliferation of PGCs.BMP4 promoted the proliferation of PGCs according to the experiment above,but the relevant signal pathways is not clear yet.This experiment studied the relation of the related factors and adhesion factors with the signal pathways of BMP4.There were experimental groups:culture system with feeder layer（PGC group,PGCs+Noggin group）,culture system with feeder-free layer（PGCs+BMP4 group、PGCs+Noggin+BMP4 group）.The signal pathways Smad1,Smad4 and Smad5 of BMP4,the downstream target gene Id2、BCL2 and the adhesion gene integrinβ1 of BMP4 were detected by qRT-PCR.pSmad1/5/8、Id2、BCL2、Integrinβ1 protein were detected by western blotting.The results showed that there was a significant reaction of the BMP4 signaling pathway-related genes in the feeder-free culture system,which could also be reversed by Noggin.But the reaction was not obvious in the feeder culture system.The results showed that PGCs,which grew as single cell,could be significantly increased,that might be due to the adhesion function of integrinβ1 was inhibited and its function to promote proliferation was enhanced.This study established a feeder-free layer culture system of chicken PGCs.The relation of BMP4 and the adhesion factors was revealed by comparing the growth status of PGCs and the influence on PGCs by BMP4,which laid a foundation for further study of the molecular mechanism of PGCs.