Original Research
Isolation and identification of multidirectional differentiation of human skin fibroblasts
Chuanfei Wei, Wei Wang, Yanming Liu, Jing Duan, Xianjie Lu, Yan Wang, Nan Zhang, Mengpeng Li, Chao Chen, Yang Zhang, Jiabei Tong, Fabin Han
Published 2019-10-01
Cite as Chin J Cell Stem Cell (Electronic Edition), 2019, 09(5): 267-275. DOI: 10.3877/cma.j.issn.2095-1221.2019.05.003
Abstract
ObjectiveTo 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.
MethodsThe 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.
ResultsThe 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.
ConclusionIsolated 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
Contributor Information
Chuanfei Wei
The Institute for Tissue Engineering and Regenerative Medicine, Liaocheng University/The Liaocheng People's Hospital, Liaocheng 252000, China
Wei Wang
The Institute for Tissue Engineering and Regenerative Medicine, Liaocheng University/The Liaocheng People's Hospital, Liaocheng 252000, China
Yanming Liu
The Institute for Tissue Engineering and Regenerative Medicine, Liaocheng University/The Liaocheng People's Hospital, Liaocheng 252000, China
Jing Duan
The Institute for Tissue Engineering and Regenerative Medicine, Liaocheng University/The Liaocheng People's Hospital, Liaocheng 252000, China
Xianjie Lu
The Institute for Tissue Engineering and Regenerative Medicine, Liaocheng University/The Liaocheng People's Hospital, Liaocheng 252000, China
Yan Wang
The Institute for Tissue Engineering and Regenerative Medicine, Liaocheng University/The Liaocheng People's Hospital, Liaocheng 252000, China
Nan Zhang
The Institute for Tissue Engineering and Regenerative Medicine, Liaocheng University/The Liaocheng People's Hospital, Liaocheng 252000, China
Mengpeng Li
The Institute for Tissue Engineering and Regenerative Medicine, Liaocheng University/The Liaocheng People's Hospital, Liaocheng 252000, China
Chao Chen
The Institute for Tissue Engineering and Regenerative Medicine, Liaocheng University/The Liaocheng People's Hospital, Liaocheng 252000, China
Yang Zhang
The Institute for Tissue Engineering and Regenerative Medicine, Liaocheng University/The Liaocheng People's Hospital, Liaocheng 252000, China
Jiabei Tong
Shandong Meijia Saipei Biotechnology Co., Ltd. Jinan 250000, China
Fabin Han
The Institute for Tissue Engineering and Regenerative Medicine, Liaocheng University/The Liaocheng People's Hospital, Liaocheng 252000, China
Institute of Translational Medicine, The Second Hospital of Shandong University, Jinan 250000, China
