Detection of ADSCs and identification of differentiation potential
Extracted adipocytes were isolated and cultured in accordance with improved methods by Zuk et al. At different time points 4h, 48h, and 2d (Fig. 1 A), the growth condition was observed under a microscope, and it can be seen that the morphology changed into a spindle-shaped distribution with time.In order to identify the cell type, CD44-FITC antibody was used for specific labeling and fluorescence staining. Red fluorescence was visible under the microscope, and the nuclei of DAPI counterstained were blue (Fig. 1 B), suggesting that the surface antigen CD44 of ADSCs was positively expressed. It was confirmed that the cells obtained from the culture were ADSCs.. To further verify the differentiation potential, Alisin blue was specifically stained after induced differentiation, showing a large number of blue matrix particles (Fig. 1 E), indicating that ADSCs have good chondrogenic differentiation potential.
Effect of Wnt pathway activation on adipose stem cell proliferation
CCK-8 was used to detect the increase of fat stem cells at different times. The results showed that the agonist LiCl stimulated the Wnt pathway in the experimental group, and adipose-derived stem cells entered exponential growth on the second day, but the adipose-derived stem cells of the blank control group began to enter the exponential growth state on the third day (Fig. 1 C). During the observation period, the proliferation rate of the agonist LiCl stimulation group was significantly higher than that of the blank control group. On the seventh day of the experiment, the experimental group and the blank control group stimulated by LiCl were detected and recorded. The expression of proliferating cell nuclear antigen (PCNA) protein was significantly higher in the experimental group than in the control group (Fig. 1 D and Additional file 1).
Cartilage Indexes and β-catenin Expression in Different Stages of ADSCs Chondrogenic Induction
During the cartilage-induced differentiation of ADSCs, the mRNA and protein expression levels of Sox9, Collagen 2a, and Aggrecan chondrogenic differentiation indicators were measured on days 0, 7, 14, and 21. The results showed that Sox9 showed a higher expression on day 7, and slowly increased on day 14 and 21. At the same time, Collagen 2a and Aggrecan also showed high expression on the 7th day, and were significantly expressed on day 14 and 21 (Fig. 2 A). Western blot showed the same results (Fig. 2 B and Additional file 2). Quantitative detection of the content of GAG in the culture medium showed that GAG began to express a small amount on day 7, gradually increased on day 14, and was obviously expressed on day 21 (Fig. 2 C).
Expression of β-catenin protein, an important factor in the Wnt pathway, at the beginning, the expression level was large, but with the progress of chondrogenic induction differentiation, the expression level gradually decreased, and the protein was low on day 7 and 14. However, by day 21, β-catenin expression rebounded and its content was higher than the initial level (Additional file 3).
In the late stage of chondrogenic induction of ADSCs, The mRNA expression levels of the cartilage differentiation markers Collagen 2a, Collagen 10 and RUNX2 were measured on day 21, 28, and 35, The results show: the mRNA expression of Colagen 10 and RUNX 2 increased with the time of cartilage-induced differentiation, and the expression of Collagen 2a gradually decreased (Fig. 2 D). Western blot results showed: Sox9 protein expression gradually decreased with cartilage-induced differentiation time, but β-catenin protein expression gradually increased (Fig. 2 E & F).
Role of Wnt pathway in ADSCs during chondrogenic differentiation
The Wnt pathway is regulated by the agonist LiCl and the inhibitor DKK-1. In the early stage of induced differentiation, on day 7, the expression of Sox9 in the LiCl-excited group was higher than that in the cartilage-induced differentiated group, whereas the DKK-1 inhibited group was the opposite. The changes of the corresponding cartilage indicators Aggrecan and Collagen 2a protein expression were not significant compared with the cartilage-induced differentiation group (Fig. 3 A and Additional file). Compared with the blank control group, β-catenin protein was weakened in the cartilage-induced differentiation group, and various indicators of cell proliferation such as CyclinD protein and PCNA protein were weakened. Compared with the cartilage-induced differentiation group, the ratio of β-catenin protein in LiCl agonized group was significantly higher than that in the latter group, and CyclinD protein and PCNA protein were also significantly increased. However, the DKK-1 inhibition group had the opposite result (Fig. 3 B and Additional file).
In the late stage of induced differentiation, LiCl group activated β-catenin protein expression, while DKK-1 inhibited group suppressed expression. Western blot analysis showed that in the LiCl agonist group, both Collagen 2a and Sox9 increased, especially, Collagen 2a increasing significantly, and the DKK-1 inhibitory group significantly inhibiting expression (Fig. 3 C Additional file). The cartilage indicators Aggrecan, Collagen 2a, and Sox9 mRNA analysis showed that the cartilage indicators Aggrecan and Collagen 2a in the LiCl-excited group were significantly increased, but the DKK-1-inhibited group was significantly inhibited (Fig. 3 D). Alixin blue staining (Fig. 3 E) and quantitative analysis (Fig. 3 F) show that chondroitin sulfate in the LiCl agonizing group was significantly higher than that in the chondrogenic differentiation group, while chondroitin sulfate in the DKK-1 inhibitory group was lower than that in the chondrogenic differentiation group. GAG quantitative analysis results also show the same trend (Fig. 3 G).
In the late stage of induced differentiation, quantitative and qualitative analysis by Western blot showed that LiCl group obviously continued to activate β-catenin protein expression, and DKK-1 inhibited expression. Compared with the cartilage-induced differentiation group, the expression of Sox9 protein in the LiCl agonist group was significantly reduced, while the DKK-1 inhibitory group was slightly decreased (Fig. 3 H & I). Cartilage indicators show that compared with the cartilage-induced differentiation group, the expression of Collagen 2a is reduced, and the expression of Collagen 10 and RUNX 2 is increased in the LiCl-excited group. In the DKK-1 inhibitory group, the expression of Collagen 2a increased, and the expression of Collagen 10 and RUNX 2 decreased (Fig. 3 J & K).
Detection of Sox9 protein expression after ADSCs lentivirus transfection
ADSCs were transfected with a lentivirus containing the full sequence Sox9 gene. Quantitative and qualitative analysis of Western blot revealed that Sox9 was abundantly expressed (Fig. 4 A & B), indicating that genes were successfully integrated into ADSCs and expressed.
Cartilage Indexes and β-catenin Expression at Different Stages of Chondrogenic Induction and Differentiation of ADSCs After Lentivirus Transfection
During transfection-induced differentiation, Sox9 protein expression was significant in the lentiviral transfection group. Compared with the blank control group, the difference between the day 7 and 14 was particularly obvious, and β-catenin protein was lower than that of the blank control group in each period (Fig. 4 C Additional file 5). On days 0, 7, 14, and 21, the expression of the chondrogenic differentiation markers Collagen 2a and Aggrecan mRNA were detected, respectively. The PCR results showed that compared with the blank control group, the mRNA expression of Collagen 2a and Aggrecan in the Sox9 lentivirus transfection group appeared to be higher (Fig. 4 D & E). On the day 7 and 14, the quantitative results of GAG content showed that the Sox9 lentivirus transfection group was significantly higher, but the results of the two groups were not significantly different when monitoring on day 21 (Fig. 4 F).
Effects of Sox9 overexpression on Wnt pathway in cartilage-induced differentiation
On day 21 of transfection-induced differentiation, immunofluorescence tests show that compared with the blank control group, the expression of Collagen 2a protein was significantly higher in the Sox9 lentivirus transfection group (Fig. 4 G). Western blot was used to detect the expression of Wnt signaling pathway-related proteins. The expression of β-catenin and GSK-3β protein increased in the Sox9 lentivirus transfection group, but the total β-catenin protein was significantly reduced (Fig. 4 H & I).