Experiment of the differentiation of adipose-derived stem cells into fibroblasts
10.16098/j.issn.0529-1356.2020.04.007
- Author:
Xiao-Jun JU
1
;
Li-Bing RAO
1
;
Li-Ming YI
1
;
Sha XIANG
1
;
You-Ya NIU
2
;
Ying-Jun ZHANG
3
Author Information
1. Department of Human Anatomy, Hunan University of Medicine
2. Department of Cell and Genetics, Hunan University of Medicine
3. Department of Imaging Diagnosis, Hunan University of Medicine
- Publication Type:Journal Article
- Keywords:
Adipose-derived stem cell;
Flow cytometry;
Induced differentiation;
Rat;
Scanning electron microscopy;
Tissue Engineering
- From:
Acta Anatomica Sinica
2020;51(4):513-519
- CountryChina
- Language:Chinese
-
Abstract:
Objective To explore the possibility of adipose-derived stem cells (ADSCs) to differentiate into fibroblasts, and to provide an effective way for the effective solution of skin tissue engineering seed cells. Methods Primary ADSCs were obtained from inguinal fat of ten healthy adult SD rats,weighing 280-320 g,and cultured in vitro and purified. When primary ADSCs expansion to the 3rd passages,the following experiments were performed alkaline phosphatase test on the 16th day after osteogenesis induction,staining of alizarin red mineralized nodules on day 23 after osteogenesis induction, oil red O staining on day 12 after adipogenic induction; Flow cytometry detection of cell surface markers; Addition of conditioned medium to induce differentiation into fibroblasts,Photograph the changes of cell morphology on the 2nd, 4th, 6th, and 8th days after induction,MTT to detect cell viability at various time points;Scanning electron microscopy on day 6 and day 8 after induction;Immunocytochemical staining on day 8 after induction,detect the expression of vimentin, the main marker of fibroblasts. Results Primary ADSCs grew in long spindles, showed strong positive expression of alkaline phosphatase (ALP) after osteogenesis induction,and alizarin red staining showed red mineralized nodules;Aggregation of intracellular red-stained lipid droplets after adipogenic induction were found;Flow cytometry showed positive expression of mesenchymal stem cell-related marker CD90,and hematopoietic stem cell marker CD45 was negative. Morphology of ADSCs started to change on day 2 after induction into fibroblasts. On the 4th day after induction, the cells were in the shape of water droplets or short rods. On the 6th day after induction, the cells were protruded polygonal or triangular. Cells crowded and covered the bottom of the bottle on day 8 after induction,becoming slender fibrous. MTT test showed that the cell viability was significantly lower on the second day after induction than in the control group. There were no significant differences in cell viability on the 4th, 6th, and 8th days after induction compared with the control group. Scanning electron microscopy showed that the cells were triangular on the 6th day after induction, and the surface had more cilia. On the 8th day after induction, the cells were slender and fibrous, with small protrusions, and the surface cilia were dense. Vimentin was positively expressed in most cells on the 8th day after induction. Conclusion ADSCs can have the morphological characteristics of fibroblasts after induced differentiation in vitro; that can express fibroblasts marker protein.
