The Study Of Transdifferentiation Of Adipose-derived Stem Cells Into Salivary Gland Acinar-like Cells

BackgroundThe irradiation injury always leads to irreversible damage to salivary glands and brings about complications like xerostomia, decays, mucositis and problems associated with speaking and swallowing. Until nowadays, the effects of traditional managements for salivary glands dysfunction are limited and many treatments are always pervaded by numerous side effects. In recent years, with the deep research of stem cells, it is possible for the treatment of many diseases and transplantation of bone marrow- derived stem cells(BMSCs) and adipose-derived stem cells(ADSCs) have been proved to ameliorate salivary glands dysfunction. We found in our previous study that the use of ADSCs and platelet-rich fibrin(PRF) can restore the function of irradiation damaged salivary glands and had better effects than using ADSCs only. However, the related mechanism remains unclear. According to the previous report, we assumed that ADSCs can transdifferentiate into salivary gland acinar-like cells( SGALCs) and PRF can promote the transdifferentiation of ADSCs. In this study, we co-cultured salivary gland cells(SGCs) and ADSCs by using transwell and added exudates of PRF to the co-culture system to confirm our assumption.ObjectiveThis study is to affirm whether ADSCs can transdifferentiate into SGACs and whether PRF can promote the transdifferentiation. It will provide mechanism for the treatment of irradiation injured salivary glands by using ADSCs and PRF. What’s more, it will find a more suitable seed cells for the tissue engineering regeneration of salivary glands.Methods 1. SGCs were obtained from submandibular glands of C3 H mouse with collagenase type Ⅱ treatment under sterile circumstances and then cultured at 37 ℃ in a 5%CO2 atmosphere in a humidified incubator. The cells were identified in the aspects of morphology, zymogen granules under transmission electron microscope, immunofluorescence(IF) staining of α-amylase, cytokeratin8(CK8) and Vimentin, western blot test ofα-amylase and RT-PCR test of m RNA expression of α-amylase, CK8, AQP-5 and vimentin.2. ADSCs were isolated from adipose tissue in the inguinal part of C3 H mouse. Then the cells were cultured in incubator as SGCs and characterized by the following aspects: morphology under inverted phase contrast microscope and HE staining, growth curve, flow cytometry analysis of stem cell-related antigens and multi-differentiation potential analysis by osteogenic and adipogenic induction.3. Arterial blood of rabbit was collected in two 10 ml glass tubes and centrifuged to obtain PRF. Then we freeze-dried the fresh PRF and ground it into powder and added it to serum-free Dulbecco’s Modified Eagle’s Medium(DMEM) in a ratio of 25 mg PRF powder per 1ml DMEM to make PRF leaching solution. The release of vascular endothelial growth factor(VEGF) and platelet-derived growth factor BB(PDGF-BB) was assessed by enzyme-linked immunosorbent assay(ELISA). Finally, we analyzed the effect of PRF leaching solution on the growth and migration of ADSCs.4. Transwell inserts and culture plates were used to establish co-culture system in which SGCs were seeded at the bottom of inserts and ADSCs were seeded at the bottom of culture plates. We set two groups that included control group and experimental group and there was no SGCs in the inserts of control group. After 7days, 14 days and 21 days ofco-culture, the morphology of ADSCs in both control and experimental group were observed and the transdifferentiation ratios were calculated according to α-amylase IF staining. To further identify the transdifferentiation of ADSCs, the IF staining of α-amylase, CK8 and Vimentin, the western blot test ofα-amylase and RT-PCR analysis of α-amylase,AQP-5,CK8 and Vimentin were carried out.5. Different concentrations of PRF leaching solutions(0.5%,1%,3%,5%,7%,10%)were added to the osteogenic induction of ADSCs. Then the expression of osteogenic genes of ALP,COLⅠ,RUNX2 and OCN were assessed after 7 days of induction and the ADSCs were stained by Alizarin red after 21 days of induction. According to the results of above research, three kinds of PRF concentrations(0.5%,1%and 5%) were chosen to add into the co-culture system. After co-cultured for 7 days, the ADSCs in the groups which had or had no PRF leaching solution were carried out with α-amylase IF staining and western blot test and the transdifferentiation ratios were calculated according to the staining. At the same time, the m RNA expression of α-amylase,AQP-5,CK8 and Vimentin in different groups were analyzed by RT-PCR.Results1. The culture of functional SGCs:The SGCs of C3 H mouse cultured in vitro were polygonous, rounded or ovate in morphology and arranged like slabstone when they became confluent. In HE staining, the cytoplasm was red, nucleus was blue and located in the center of the cell. The black particles with high density that called zymogen granules were observed under TEM. The expression of α-amylase, AQP-5 and CK8 were detected by IF staining and RT-PCR analysis. The western blot also confirmed the exist of α-amylase.2. The culture of functional ADSCs:The ADSCs cultured in vitro shaped like spindle and arranged regular when they became confluent. HE staining showed that cytoplasm is red and nucleus is blue. The growth curve shaped like ‘S’ and was same as that of other stem cells. The expression of stem cell-related antigens was positive and the osteogenic and adipogenic induction showed the multi-differentiation potentials of ADSCs.3. PRF can release growth factors continuously and promote proliferation and migration of ADSCs in a limited dose:The fresh PRF clots obtained from the rabbit arteriole blood were freeze-dried and then ground into power. It could release VEGF and PDGF-BB continuously for a long time. The effects of PRF leaching solution on biological bahaviour of ADSCs differed with the concentration. 1% PRF leaching solution had strongest promotion to the growth and osteogenic differentiation of ADSCs and 50%PRF leaching solution had most obvious promotion to the migration of ADSCs.4. ADSCs can transdifferentiate into SMGLCs: The expression α-amylase, AQP-5 and CK8 approved the transdifferentiation of ADSCs co-cultured in the experimental groups. The transdifferentiation ratios of 7,14,21 days co-culture were 22.87 ± 1.66%; 40.56 ± 1.74%; 49.84 ± 1.93%, respectively. The α-amylase bands of experimental groups at three time points in western blot existed at 53 k Da and the gray level increased in order. At the same time, the m RNA expression of α-amylase, AQP-5 and CK8 at three time points increased successively while the expression of Vimentin reduced in order. There was no evidence to indicate the ADSCs in control group had transdifferentiated into SGALCs.5. PRF can promote the transdifferentiation of ADSCs in a limited dose: The concentrations of PRF leaching solutions ranged from 0.5% to 5%could promote the osteogenesis of ADSCs. The groups of 0.5%,1%,3% and 5% PRF leaching solutions showed a high level expressions of osteogenic related genes of ALP,COLⅠ,RUNX2 and OCN. The Alizarin red staining of ADSCs also had the same trend by showing more mineralized nodules in groups of0.5%, 1%,3% and 5% PRF leaching solutions. The transdifferentiation ratios of ADSCs in 0.5%,1%and 5% PRF leaching solution groups in 7days co-culture were 30.39±1.51%,36.20±1.33%, 28.78±0.83%, respectively. When compared the groups with or without PRF leaching solution, there were significant statistical differences between the transdifferentiation ratios and the group with 1%PRF leaching solution had the highest ratio(P<0.05 compared with 0.5% and 5% PRF leaching solution groups). The expression of α-amylase in western blot and m RNAexpression of it in RT-PCR also approved the differences(P<0.05).Conclusion1. we successfully cultured functional SGCs which contained special zymogen granules and expressed α-amylase.2. ADSCs were isolated and cultured successfully. They shaped like spindle and had strong self-renewal capacity. ADSCs expressed high levels of stem cell-related antigens and possessed mlti-lineage potentials including osteogenesis and adipogenesis.3. PRF contained a variety of growth factors such as VEGF and PDGF-BB and could release these growth factors continually for a long time. It could affect the growth, differentiation and migration of ADSCs in a dose-dependent way.4. ADSCs could transdifferentiated into SGALCs in vitro and the transdifferentiation ratios increased with time in a given period.5. PRF could promote the transdifferentiation of ADSCs into SGALCs in a dose-dependent way.6. The combination of ADSCs and PRF may provide enough suitable seed cells for the tissue engineering regeneration of salivary glands.