Abstract: 【Objective】 To isolate and culture adipose derived mesenchymal stem cells (AD-MSCs) from Holstein cattle in vitro, study their biological properties, and provide a new type of usable seed cells for stem cell clinical research.【Method】 The inguinal adipose tissue of 2-month-old Holstein bovine embryos was collected aseptically,and the cells were digested with type Ⅰ collagenase and cultured.Fluorescent antibody technology and RT-PCR were used to detect the specific markers (CD29,CD44,CD73,CD166,CD34 and CD45) on the cell membrane surface.The growth curves of P4,P10 and P16 generations were calculated using the purified cells and calculate population doubling time.The clone formation rate was calculated using the clone formation assay,the karyotype analysis was performed to determine whether the cells were genetically stable,and the differentiation potential to lipogenesis,osteogenesis and chondrogenesis were verified by adding induction solution to AD-MSCs culture dishes.【Result】 Cells isolated and cultured in vitro grew in an appressed swirling pattern with a long shuttle-shaped cell morphology.The results of fluorescent antibody technique and RT-PCR showed that CD29,CD44,CD73 and CD166 cell membrane surface antigens were expressed,CD34 and CD45 were not expressed,and the isolated cells were identified as AD-MSCs.The cell growth curve was plotted in S-shape,and the cell population multiplication time was 33,35 and 44 h for P4,P10 and P16 generations,respectively,and the clone formation rates were (51.67±1.53)%,(38.00±2.00)%,and (25.00±1.00)%,respectively.The two indicators showed significant differences between the three generations (P＜0.01 or P＜0.05).The karyotype results showed that AD-MSCs were normal diploids (2n=60,XX),and all chromosome morphologies were telomeric with no aberrations.Induced differentiation results showed that in vitro AD-MSCs had lipogenic,osteogenic and chondrogenic differentiation potential,with distinct lipid droplets,calcium nodules and cartilage clusters,respectively,and expressed lipogenic inducible genes lipoprotein lipase (LPL) and peroxisome proliferator-activated receptor (PPAR-γ),osteogenic inducible genes type Ⅰ collagen (COL Ⅰ) and osteochondrogenic bridge protein (OPN),osteogenic chondrogenic gene SRY-like HMG cassette 9 (SOX-9) and chondroprotein glycan antibody (ACAN).【Conclusion】 Primary AD-MSCs could be successfully isolated in vitro using Holstein bovine embryos,and the cell morphology had typical mesenchymal stem cell morphology,with differentiation potential of lipogenic, osteogenic and chondrocytes,and the cells had fast proliferation ability and stability,which could provide germplasm resources for tissue and organ repair.

Key words: adipose mesenchymal stem cells; isolation and culture; induction of differentiation; Holstein cattle