Changing Of Stemness During Serial Passage Of HDPSCs In Vitro

Dental pulp stem cells(DPSCs), a group of undifferentiated adult stem cells, are originated from neural crest cells--derived mesenchyme, and can be extracted from dental pulp tissue. In certain induction environment, DPSCs have the capacities to differentiate into bone, cartilage, nerve, hepatic and cardiomyocyte like cells. Compared with BMMSCs, DPSCs show higher differentiated state, easy material obtainment, injury-limited, extensive sources and significant capabilities in differentiation and self-renewal, and are now considered as ideal seeding cells in tissue engineering. However, due to the limited number of cells obtained from dental pulp tissue and the urgent needs of tissue engineering,it is necessary to expand h DPSC in vitro. Meanwhile, quality control of stem cells in serial passage was an important part to control the quantity of h DPSC. Investigation showed that serial passage promote MSCs different degrees of aging.Effects of serial passage on h DPSCs biological activity remains unclear. Objective: To investigate the proliferation, differentiation and self-renewal capacities of h DPSC before and after in vitro continued culture, evaluate the effects of in vitro culture on stem cell properties of DPSCs, and provide theory and experiment basis for stemness mainteinence and application of tissue engineering.Methods:1.Separation, cultivation and identification of h DPSCsHDPSCs were obtained from dental pulp of healthy and intact teeth from individuals undergoing extraction by enzymatic digestion and selected by using limited dilution method. FCM was employed to identify single-colony-derived DPSCs, which showed that h DPSCs highly expressed mesenchymal stem cell markers including CD29, CD90, CD105, CD146 and Stro-1, but did not express hematopoietic markers including CD45 and endothelial marker CD34. The data indicated that the cells obtained were mesenchymal origin. Alizarin red S, ALP and Oil red O positive staining proved that cells can be induced towards odontoblastic and adipogenic differentiation. All the identification demonstrated that we successfully obtain h DPSCs.2.Effects of serial passage on morphology, cell phenotype and senescence of h DPSCHDPSC were serially passage in two-dimensional culture in vitro and stem cell capacity in passages 4, 8, 12, 16 and 20 were evaluated. The results showed that(1)P4 DPSC exhibited a homogeneous, fibroblastoid with a high nucleocytoplasmic ratio morphology, but changed to a wide、flat shape during serial passage.(2) Surface marker expression measurement by FCM showed that, no difference of the expression of CD29 and CD90 were found between groups(P>0.05). There were gradually reduction expression of CD105 and CD146 in HDPSC during continuous passage(P<0.05).(3) β-galactosidase staining revealed that P4 h DPSC showed no senescence phenomenon, the amount of senescent cells proportionately increased in P16 and P20(P<0.05).3.Effects of serial passage on proliferation, differentiation and colony forming unit-fibroblast of h DPSC(1) HDPSCs proliferation viability was detected by MTT method and FCM for cell cycle analysis. Results revealed that the rapid proliferation in P4 h DPSC was observed. Proliferation in P8, P12, P16, P20 gradually reduced(P<0.05). Phase of cell cycle analysis by FCM showed that cells in S stage was consistent to MTT.(2) Differentiation induction of h DPSC into osteoblasts showed that the longer the culture times were, the lighter alizarin red stain and the number of mineral node decreasing were. Subsequently, cells were analyzed by Real-Time PCR to evaluate the expression of the DSPP and RUX2, which showed decreased expressions during serial passage(P<0.05).The amount of PPAR-γ gradually reduced with increasing h DPSC generations, detected by oil red O staining and Real-Time PCR respectively.(3) The amount of CFU-F determined by crystal violet stain showed reduction appearance, indicating colony forming and self-renewal capacity were weakened with increasing h DPSC generations.(4) Methods RT-PCR method was used to detect growth factors. The results showed that h DPSC negatively expressed EGF, FGF-4, and IL-6, and positively expressed IGF-2, TGF-β, HGF, FGF-2, VEGF, FGF and TGF-β, which were downregulated after serial passage.4.Influences of serial passage on stemness related genes(1) Real-Time PCR examination of Oct4 spliced variants Oct4 A and stemness genes(Sox2、Klf4、c-Myc and Nanog) were positive. Oct4 A and c-Myc showed no significant difference between selected passages. Sox2, Klf4 and Nanog were downregulated after continuous passage, but Oct4 B was undetectable in h DPSC.(2)Characterization was further investigated by detecting Oct4 A proteins by immunofluorescence. P4 and P8 h DPSCs showed high nuclear Oct4 A protein presence.While nuclear and cytoplasmic staining evidenced in IF were observed in P12 h DPSCs.With the increasing of generation, P16 and P20 h DPSCs showed high cytoplamic Oct4 A protein presence. Evidence indicated that Oct4 A translocation may have a relation to h DPSCs stemness maintenance.In conclusion, this study preliminary proved the downregulation of the proliferation, odontoblastic capacity and self-renewal capacity in h DPSCs during culture in vitro. Further research identified stemness genes including Sox2 、 Klf4 and Nanog were downregulated after serial passage. Although Oct4 A expression was not changed significantly at m RNA level, Oct4 A translocation appearance indicated that stemness genes such as Klf4、Nanog and Oct4 A may play a crucial role in maintaining stemness of h DPSCs. The follow-up study will focus on the functions of stemness genes including Sox2,Klf4, Nanog and Oct4 A translocation so as to seek out essential mechanisms for h DPSCs stemness maintenance. And controlling these machanisms may lay a theoretical foundation into the application and dissemination in the field of tissue engineering.