Isolation and identification of multidirectional differentiation of human skin fibroblasts

Abstract

Objective To efficiently isolate human skin fibroblast cells with rapid proliferation and to investigate the multi-lineage differentiation potential of fibroblasts into adipogenic, osteogenic, chondrogenic and neurogenic directions. Methods The human primary fibroblast cells were isolated and cultured from the skin tissues. Morphological structures, flow cytometry and vimentin staining were used to identify the fibroblasts. Growth curves analysis, mitochondrial staining and karyotype analysis were used to identify the cell state of different generations. And finally fibroblasts were induced to adipogenic, osteogenic, chondrogenic and neurogenic directions. The difference between cell proliferation rate and the relative amount of mitochondrial was compared by t-test. Results The isolated fibroblasts showed a typical spindle-like morphology. Flow cytometry analysis showed that the isolated cells were highly express fibroblast surface markers CD90 (NCF1, NCF2 accounted for 99.9%, 98.7%, respectively) and CD73 (NCF1, NCF2 accounted for 98.2%, 85.6%, respectively) , rarely express hematopoietic stem cell marker CD34 (NCF1, NCF2 accounted for 1.8%, 2.6%, respectively) . All the fibroblasts positively express vimentin, the positive rate was 100%. The results of growth curves analyse showed that there was no significant difference in cell proliferation rate among multiple passages (t = 1.586, P = 0.1567) . The relative content of mitochondria showed no significant difference (t = 0.664, P = 0.543) between the fifth generation (relative fluorescence intensity: 6876±577.8) and the 10th generation (relative fluorescence intensity: 7371 ± 471.9) of the same cell line. Karyotype analysis showed that the number of chromosomes was 46 and the morphology of chromosomes were similar during passage. The multi-lineage differentiation assay showed that the fibroblasts could express adipogenic, osteogenic, cartilaginous and neurogenic differentiation markers. Conclusion Isolated fibroblasts have adipogenic, osteogenic, chondrogenic, neurogenic multi-directional differentiation potential, which can meet the needs of preclinical studies and provide cell sources for bone trauma, cartilage injury and neurodegenerative diseases. Key words: Skin fibroblasts; Explant culture; Flow cytometry analysis; Multidirectional differentiation