| Bone marrow mesenchymal stem cells(BMSCs)are an adult plurip-otent progenitor cell found in the bone marrow that supports the growth of hematopoietic stem cells.After isolation,it can be expanded in vitro w-ithout loss of pluripotency and differentiation under specific conditions.It is widely used in regenerative medicine,tissue engineering,avian develo-pmental biology,and production of chimeras in the formation of bone,ca-rtilage,fat,muscle,nerve,and endothelium.At present,most studies on BMSCs focus on mammals such as humans,mice,rabbits and sheep,and there are few reports on the study of chicken BMSCs.Therefore,the iso-lation and purification of chicken BMSCs and the establishment of in vit-ro culture systems are imperative.The aim of this study was to establish an isolation and culture syst-em for chicken BMSCs,which will lay the foundation for the further use of BMSCs to produce transgenic chickens and gene editing at the cellular level.In this experiment,Guangxi Yellow Chicken,1-15 days old,was sel-ected as the test material.First,the femur and tibia of the chicken were a-septically isolated and the two epiphyseal ends of the femur and tibia we-re excised,then all the bone marrow was washed out with D-PBS or basal medium(DMEM/F12),and then the BMSCs were preliminarily analyzed by Ficoll400 density gradient centrifugation.Purification,and finally B-MSCs were repurified by differential digestion of differential adherence during subculture.When the purity of isolated BMSCs reached 90%or m-ore,they were initially identified by morphological observation,prolifera-tion kinetics test,colony formation ability test,histochemical det-ection,RT-PCR analysis,and then passed through in vitro differentiation test and chimera test.It was further identified.Among them,histochem-ical detec-tion mainly uses glycogen staining(PAS),alkaline phosphatase assay(AL P),immunofluorescence detection(IF)of cell surface markers,oil red O staining,etc.RT-PCR analysis test is passed Primers were des-igned to detect whether cells express several marker genes on the surface of BM-SCs and several transcription factors controlling pluripotency;in vitro di-fferentiation assays induced differentiation of adipogenic cells and osteo-blasts into BMSCs,and then The results of differentiation were ver-ified by histochemical detection and RT-PCR analysis;the chimeric assay was performed by injecting EDU-labeled BMSCs into the dorsal aorta of chi-cken embryos incubated for 53 hours,and then continuing the incubation by shell-changing culture on day 7 and The method of detecting the EDU signal in the chicken embryo at 11 days was carried out.Morphological observation showed that the cells isolated in this stu-dy were inoculated to the cell culture dish and adhered at 12-24 h.The cell morphology gradually changed from a circular shape to a spindle sh-ape,an elliptical shape,and a flat shape.During further purification by d-ifferential digestion of differential adherence,the cell morphology tends to be uniform and appears to be typically fibrillar.The results of prolifera-tion kinetics showed that the growth curve of the cells isolated in this stu-dy showed a typical "S" shape during the culture,and the doubling time gradually prolonged with the increase of cell algebra.The colony forming ability test indicated that the cells isolated in this study had the potential to form colonies,and as the amount of cells inoculated increased,the nu-mber of colonies formed increased.The histochemical test results showed that the glycogen staining was positive,the alkaline phosphatase test was negative,the cell surface markers CD29,CD44 and vimentin were posit-ive for immunofluorescence staining,and the hematopoietic stem cell sur-face markers CD34 and CD45 were negative for immunofluorescence st-aining.RT-PCR analysis showed that BMSCs expressed cell surface mar-ker genes CD29,CD44,CD71,and transcription factors Sox2,Myc,N-anog,and PouV which control cell pluripotency.In vitro differentiation a-ssays showed that the cells isolated in this study have the potential for m-ultidirectional differentiation and can differentiate into osteoblasts and a-dipocytes under specific conditions.The results of the chimera test indic-ate that the cells we isolated have the potential to be a powerful tool for preparing chimeras.The BMSCs derived from Guangxi Huangyu chicken isolated in this study have the characteristics of easy isolation,amplification,low immu-nogenicity and long-term production and maintenance of multilineage di-fferentiation in vitro.Hopefully for use in regenerative medicine,cell tr-ansplantation therapy,avian developmental biology,and preparation of genetically modified chickens. |
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