The Characterization Of Stem Cells From Human Exfoliated Deciduous Teeth(SHED) After Cartilaginous Differentiation And Osteogenic Induction In Vitro

Backround and objectiveSince the concept of tissue engineering was proposed, seed cells, scaffold materials and training environment have been three important elements, in which seed cells is the basis for tissue repair and regeneration. Stem cells,with their own features, are of great advantage in all the seed cells. Although embryonic stem cells (embryonicstem cells, ESCs) have the potential to differentiate into all types of cells, its development is greatly hindered by the allograft ethical constraints. Therefore, adult stem cells (adultstem cells, ASCs) has become the focus of research for seed cells in tissue engineering at present. What is more, some progress have been made in the renovation of bone and cartilage defects and regeneration of craniofacial tissue.In adult stem cells, stem cells derived from human exfoliated deciduous teeeh (SHED) is of great application potential in regenerative medicine. SHED is close to the mature cells, has little immune rejection, but has plasticity and potential to highly proliferate and differentiate. In the meanwhile, SHED could differentiate into some specific cell types and produce sub-breed of precursor cells in given conditions. In addition, the present study found that SHED had the great potential to be induced to form bone, which is of great significance to the bone defect repair.Although many adult stem cells have been reported to have the potential to differentiate into cartilage cells from the present research, there is no report showing SHED differentiate into cartilage In recent years, many reports show SHED have the potential to differentiate into osteoblasts, but there reseaches are conducted with no specific selection. Therefore, we determine the marker of cartilage cells after induction of SHED in vitro in the first part of this issue. In the second part, using SHED with speicific markers selected by FACS, we analyze the proecess of SHED differentiation into osteoblasts at different time points. The purpose of the present study is to provide references for the follow-up studies on the role of SHED in repair of cartilage tissue and bone tissue in vivo.Partâ… The characterization of SHED after cartilaginous differentiation in vitroMethods1. Isolating the stem cells from human exfoliated deciduous teeth and their culturing According to Miura et al experimental method, pulp tissues from children of 6-10 years old were obtained, digested by enzyme and cultured in the medium. The filtered cell suspension was collected and cultured in the container.2. Observation and identification of SHED2.1 Cell morphological observationThe growth condition, proliferation and morphologic characteristics of both the primary and passage cells were observed, and the cytohistological features of the primary cells were observed by HE staining.2.2 Identification of cell surface antigenPrimary cells were analyzed by Immunocytochemistry staining to detect the expression of STRO-1. 2.3 Induction of SHED into adipocytesThe primary cells were cultured in the adipogenic medium.Oil red O staining was conducted when the formation of lipid droplets occurred.3. Induction of SHED into cartilage cells in vitro3.1 cell morphology after inductionAccording to Johnstone B, et al Experimental Methods, two weeks after induction of SHED, the induced cells were analyzed by HE staining to detect the morphological and histological features.3.2 Toluidine blue and Safranin O staining on cellsAfter induction, SHED cells were stained by toluidine blue and Safranin O to analyze the the coloring of proteoglycans inside and outside the cells.3.3 Typeâ…¡collagen and Aggrecan expressionAfter induction, SHED cells were analyzed by immunochemical staining and immunohistochemical staining to observe the expression of typeâ…¡collagen and Aggrecan.3.4 Detection of typeâ…¡collagen gene expression using RT-PCRThe gene expression of typeâ…¡collagen of SHED after induction is analyzed using RT-PCR method.Results1. Isolation and culture of SHEDIn general, three days after culture, the primary cells became adherent to the plate and presented multiple formes.Then the polymorphism gradually reduced as the passages of cells. SHED showed a fibroblast-like morphology, with cell body enlarging, cell color darkening,and sizes and appearance becoming consistent.2. observation and identification of SHED2.1 HE stainingHE staining results showed that most of the cells presented fusiform shape with smaller size.The nucleus is round and large. A few cells contain more than one nucleolus.2.2 Detection of cell surface antigen using immunocytochemistryThe immune cell staining of primar SHED for STRO-1 expression showed positive expression of STRO-1.2.3 Induction of SHED into adipocytes in vitroRefraction points of high intensity were showed in several cells after 15 days culture in medium with adipogenic induction, Increased transparent and string-beads-like refraction points in highlight were found after 20 days, some points are mixing together and the cells are positive for oil red-O staining.3. SHED induced into cartilage cells in vitro3.1 Cell morphological observationHE staining two weeks after induction:The cell morphology changed from the spindle into a polygon shape,with a small size and deeply stained nuclei.3.2 Toluidine blue and Safranin O staining on cellsBoth of the toluidine blue and Safranin O staining of the cartilage cells differentiated from SHED showed positive results.3.3 Detection of markers of cartilage cellImmunocytochemical staining showed positive expression of collagen typeâ…¡, immunofluorescence staining showed positive expression of Aggrecan.3.4 Detection of typeâ…¡collagen gene expression with RT-PCRAfter 2 weeks of specific induction, typeâ…¡collagen could be detected with a zise at 300-400bpConclusionThe experiment was a success in culturing human primairy deciduous dental pulp stem cells and inducing them into the cartilage cells in vitro. The cell morphology and markers of cartilage cells were charactrized,showing SHED had the potential to differentiate into cartilage cells in vitro. Partâ…¡Osteogenic capacity of SHED in vitroMethods1. Isolate the stem cells from human exfoliated deciduous teeth and their culturing Please refer to the first part of the experiment2. Sorting cells using flow cytometry When the second generation of the deciduous dental pulp stem cells fused into monolayer cells, digest them into single cell suspension.FITC-CD34 and PE-CD117 antibodies are added and the cells are sorted into two subgroups using FACS. A Group:CD34+/CD117+ double positive cells, B Group:the rest of cells.3. Cell morphological observationObserve of A and B group cells growthwith inverted microscope. Histological HE staining is conducted.4. Immunocytochemical staining of SHED to detect its differentiation into osteoblasts 30 days after cell sorting, detect the RUNX-2, OC, BSP expression in A and B group using immunocytochemical staining.5. Detection of osteoblast gene expression using Real time-PCR 40 days after cell sorting,detect RUNX-2, OC, BSP mRNA expression in A and B group using quantitative PCR instrument MX300p Real time-PCR6. Detection of the ability of differention into mineralized nodule 50 days after cell sorting, detect mineralized nodules in.A and B group using Von Kossa's staining and alizarin red stainingResult1.Detection of cell surface antigen and screening using flow cytometry The cells with CD34+/CD117+ in A group, accounts for 40.2% of total cells, while the rest in B group, accounts for 58.5% of total cells.2. Cell growthAfter sorting, the morphology of cells in A and B groups are similar to that before sorting. The cells are spindle-shaped and growing in colony.HE staining shows the cells like fibroblast,with a smaller size and clear outlines. The nuclei is oval or round,which is colored depth, while the cytoplam is slightly stained.3. ImmunocytochemistryThe immune staining shows the osteoblast marker RUNX-2, OC, BSP expression in both A and B groups.4. Detection of RUNX-2, OC, BSP mRNA expression using Q-PCRFluorescence quantitative PCR results showed that RUNX-2, OC, BSP mRNA expression could be detected both in A and B groups, with the expression in group A is higher than that in B.5. Detection of mineralization abilityVon Kossa's staining and alizarin red staining showed positive expression in A group, and negative in B group.ConclusionThis experiment showed that preliminary purification of CD34+/CD117+ double positive SHED could be acheived with FACS.Purified CD34+/CD117+ cells have the potential to differentiate into osteoblast and formthe mineralized nodules in vitro.