3.1. BASCs culture, morphology, and growth observation
The BASCs were obtained from 24 days-old broiler adipose tissue, began to adhere to the T-25 flask surface, appeared after a day of seeding, and gradually found a spindle shape (Fig. 1 A, B, and C). Cells formed colonies and, at P0, reached confluency within four days, although in the following passages, after almost seven days, suffused the flask surface. Cells were passaged up to P7 to observe the phenotype, morphological characteristics, and proliferation capacity of BASCs during expansion in vitro (Fig. 1 F).
3.2. The self-Renewable capability of BASCs
CFU-assay is a popular method to evaluate the proliferation ability and colony-forming potential of isolated MSCs. For this purpose, the BASCs at P2 and P5 were seeded at a low density of 175/cm2, 350/cm2, and 520/cm2 cells per well of six well plates. After nine days, the colonies were counted by OpenCFU-3.9.0 software (Geissmann, 2013) and were 107, 136, 213 at P2 and 22, 62, and 84 at P5, respectively (Fig. 2).
3.3. Growth Kinetics
The growth curve of BASCs in the different passages of 2, 4, and 8 were drawn to assay the growth kinetics of broilers mesenchymal stem cells and examine the impact of BASCs age on growth tendency (Fig. 3). At P2 and P4 cells had similar proliferation trends, whereas, at P8 they significantly lost their potential. At P2, BASCs illustrate the growth phase after five days, and until day 11, they kept their upgoing trend; however, the cells' population decreased. At P4, in days 1-4, the cells were in the Lag phase and entered the growth phase till day 9th. On the other hand, P8 shows a dramatic decline in cell viability, and they were in the lag phase till day 5th and, after that, had modest proliferation till day 9th. The average population doubling time of passages 2, 4, and 8 were 37.03, 60.92, and 101.42h, respectively.
3.4. Curcumin effect on Cell Viability and proliferation
Curcumin may have a toxicity effect on some dosages and finding an appropriate dosage that shows the best result is necessary. To evaluate the curcumin impact on BASCs, the cells were treated with different dosages of curcumin (1, 5, 10, 15,20 µM). After 1, 4, and 7 days of treatment, the viability and proliferation of the cells were estimated by MTT assay. In figure 4, the control group is BASCs treated by the basal medium. The viability of this group was considered 100 percent, and other groups were evaluated based on it. As shown, on the first day, curcumin climbed cell proliferation. All the groups had a significant difference from the control group (p<0.01), although the group of 15 µM had a lower difference (p<0.05). Likewise, on day 4, all groups had significant differences from the control group (p<0.01). And finally, on the 7th day, a similar trend was observed, except for a dosage of 20 µM, which did not have a significant difference.
3.5. Multi-linage differentiation potential of BASCs
Cells were differentiated under osteogenic and adipogenic conditions to assess the curcumin impact on BASCs potential.
Alizarin red staining: This qualitative test shows cells' calcification deposition. As is seen in Figure 5, cells illustrate an increase of matrix mineralization at day 11, against day 6, especially in the dosage of 5 µM. In this experiment, the stem cells cultured by BM were considered a control group and compared with different dosages of curcumin.
Calcium Deposition: Precipitation of calcium as a quantitative parameter of osteogenic differentiation was measured for 11 days. On day 6 of curcumin treatment, a significant difference was not observed. However, on the 11th day, cells had differentiated into osteogenic cells, and two dosages of 1 and 5 µM, significantly, were upper than the other groups (p<0.01) (Fig. 6).
ALP activity: To further assess the osteogenic differentiation potential of BASCs, the activity of ALP enzyme, as a critical marker of osteogenesis, was estimated on the 6th and 11th day of curcumin treatment. The result was similar to calcium deposition, and again on day 11th, two dosages of 1 and 5 µM had the most ALP activation, which was significantly more than other concentrate and control group (p<0.01) (Fig. 7). The results in these experiments demonstrate it, curcumin at 1µM and 5µM can induce osteogenic differentiation.
Oil Red O staining: ORO staining, like Alizarin red staining, is a qualitative method, whereas it is applied to recognize lipid vacuole accommodation. Staining of broilers MSCs by ORO illustrates the potential of curcumin to induce adipogenic differentiation. Increased curcumin concentration leads to less lipid vacuole accommodation (Fig. 8).
Gene expression: To evaluate the curcumin effect on differentiated cells, osteogenic and adipogenic gene expression was measured on days 6th and 11th of the experiment in two concentrations of 1 and 5 µM. On the 6th day of treatment, ALP did not show a significant difference (p<0.05), although RUNX2 in 5 µM and COL1 in both 1 and 5 µM had substantial differences with the control group. On the other hand, the expression of PPARγ and FABP4 as the adipogenic marker in the control group was higher than two concentrations of curcumin (p<0.01) (Fig. 9). On day 11th of differentiation, all three osteogenic gene markers illustrate a significant difference with the control group (p<0.01). Whereas, in both PPARγ and FABP4, the expression of gene markers at 1 µM was lower than the control group (not significant) and at 5 µM was significantly lower than the control (P value of PPARγ was (p<0.05), and FABP4 was (p<0.01)) (Fig. 10).