Abstract: This study utilized Holstein bovine lung resident mesenchymal stem cells (LR-MSCs) and observed their osteogenic differentiation ability in vitro under different conditions, with the hope of providing new seed cells for clinical application and tissue engineering. The Holstein calf (healthy Holstein fetal calf, 0 days old, male) was used to obtain lung tissue via sterile methods and to isolate LR-MSCs through techniques like tissue adherence. The cells were primarily cultured. The growth curves were drew, proliferation timings, and potency for the 3rd, 9th, and 15th generations were measured. The expression of stem cell surface markers (CD29, CD44, CD73, CD90, CD106, CD166, CD34 and CD45) was detected using RT-PCR. Chromosomal karyotype analysis was performed on these genes, and their pluripotency was observed after genetic transformation. The results indicate that LR-MSCs could be obtained through both techniques, exhibiting spindle and spiral shapes in vitro and showing a classic "S" growth curve. The 9th generation had a shorter doubling time than the 15th generation, but longer than the 3rd (P<0.01); colony-forming rates for the 9th generation LR-MSCs were significantly decreased compared to the 3rd generation, but higher for the 15th generation, showing statistical significance (P<0.05). RT-PCR revealed the specific expression of CD29, CD44, CD73, CD90 in LR-MSCs. The preliminary studies found that LR-MSCs could specifically express CD29, CD44, CD73, CD90, CD106, CD166, but not CD34, CD45. Chromosome sequencing showed LR-MSCs to be of a normal diploid karyotype (2n=60, XY). Immunofluorescence staining and RT-PCR analysis proved the multilineage differentiation potential of Holstein bovine LR-MSCs into adipocytes, osteocytes, and chondrocytes, which may offer a new seed cell type for upcoming tissue engineering research.

Key words: lung resident mesenchymal stem cells, primary culture, induced differentiation, assay, multidirectional potential study