The Generation And Application Of Induced Pluripotent Stem Cells Derived From Periodontal Ligament Cells

The domestic and foreign scholars have been devoted into the study of tissue engineering for tissue generation. Seed cell is one of the three key necessary parts for tissue engineering. Periodontal ligament stem cells (referred to as PDLSCs) are the stem cells that naturally presenting in the periodontal tissues and play a significant role in the regeneration of periodontal tissue. PDLSCs have the potency of differentiate into bone, cementum and periodontal ligament. However, the natural PDLSCs are limited and the isolation process is complicated, it is difficult to the accumulate a large number of PDLSCs as the seed cells for tissue regeneration.Embryonic stem cells (hESCs, referred to as ESCs) have been regarded to own the most differentiation potential among all stem cells. ES cells can differentiate into various types of cells under different conditions. However, due to the limit of ethical issues, ES cells can only be used in library for experiment at present. Thus, scientists have been devoted into creating cells owning stronger differentiation ability. Cell reprogramming is the progress that reversing the differentiated mature somatic cells into the undifferentiated state and the reprogrammed cells can become pluripotent of differentiating into various cell types in the body. Induced pluripotent stem cells (referred to as iPSCs) is one of the hotest researches in the field of stem cells in recent years. In 2006, Yamanaka group from Kyoto University successfully induced mouse fibroblasts into iPSCs. iPSCs from successful induction not only similar to ES cells in cell morphology and differentiation potential, but also avoid the ethical issues surrounding the use of ES cells.Gene reprogramming of induced pluripotent stem cells by reverse transcription factors show the possibility of making one cell into another in clinical treatment. This latest research achievement gives us a whole new idea of solving the problem of bone regeneration even periodontal tissues regeneration. In vitro cultured periodontal ligament cells (PDLCs) can be induced into pluripotent stem cells of undifferentiated state by reprogramming technology, and be used as seed cells for periodontal tissue engineering. This technique will help to break through the bottleneck in this field. PDLCs can grow out from periodontal ligament, which is got from the extraction teeth for orthodontic and wisdom teeth in clinic. Then we can induce PDLCs into iPSCs to use as seed cells in periodontal tissue engineering.In addition, cell sheet technology has a broad application prospect in tissue regeneration. Because of the preservation of extracellular matrix, less transplanted cells and scaffold materials can be used in the transplantation, which is conducive to tissue regeneration and decrease the side effects. the pluripotent stem cells can be made into iPS cell sheet in vitro and applied for the regeneration of periodontal tissues, which provides a new idea for clinical application.ObjectivesThe purpose of this study was to culture human PDLCs from teeth and transfered the exogenous gene (Sox2, KLF4, Oct4 and c-myc) by lentivirus into PDLCs for reprogramming. Biological experiments were made to identified human induced pluripotent stem cells derived from periodontal ligament cells (hPDLC-iPSCs) that successfully generated. After that cell experiments in vitro were designed to evaluate the osteogenic differentiation ability of hPDLC-iPSCs. Finally, we used cell sheet genetrated from hPDLC-iPSCs to repair bone defects, and assessed the application of hPDLC-iPSCs in bone tissue engineering.Materials and methodsThe research content of this study was divided into the following four parts:Part 1:The establishment of iPSCs derived from PDLCs1.1 Primary culture of PDLCsOrthodontic teeth from healthy patients were used in this study with informed consent. Periodontal ligament from the middle third of the teeth was scraped and cut into about 1*1mm for tissue culture method. After culture and amplification, PDLCs from 3rd to 5th were used for subsequent experiments.Identification of PDLCs was done by cell immunofluorescent including targets of cytokeratin, vimentin, fibronectin and type I collagenase.1.2 preparation of CF-1 mouse embryonic fibroblast feederEmbryonic tissue was separated from 13.5 day-pregnanting CF-1 mice and cut into pieces,0.25% trypsin digested for 10 minutes and dispersed into single cell. Amplified the primary MEF cells to 3rd generation and irradiated for 1 hour. The prepared MEF cells were reserved in liquid nitrogen for future use.1.3 retroviral packaging production and collection of virus infection of periodontal ligament cellsRecoveried the frozen 293 T cells and the virus packaging was ready when cell density reached about 80%. Lentiviral vector carried the Oct-4, Sox-2, c-myc and klf-4 gene and virus packaging plasmids were co-transfected 293T cells.48 hours later, fluorescent microscope was used to observe green fluorescence and the supernatants were collected for transfection. lentivirus titer determination of products carrying exogenous gene was prepared for the infection of hPDLCs1.4 cell culture after virus infection and selected iPS cell cloneThe infected cells were cultured on MEF cells and changed with hES cell culture medium.3-4 weeks after infection, clones was picked up for alkaline phosphatase test, after that positive clones were selected for subsequent experiments. Part 2. Identification of hPDLC-iPS2.1 Observation and analysis of cell and nuclear morphologyhPDLC-iPSCs were compared with hES cells and the morphological differences were observed. Changes of cell nuclear morphology after reprogramming of iPSCs were observed.2.2 hPDLC-iPSCs alkaline phosphatase stainingAfter culturing without MEF feeder, iPSCs were fixed with 4% cold paraformaldehyde for 30 minutes, then alkaline phosphatase staining was used to evaluate the activity of alkaline phosphatase in hPDLC-iPSCs for cell type identification.2.3 iPS cell immunofluorescence stainingAfter culturing without MEF feeder, iPSCs were fixed with 4% cold paraformaldehyde for 30 minutes. Cell immunofluorescence was used to detect whether iPSCs express ES cell specific membrane proteins (SSEA3, SSEA4), cytoplasmic proteins (TRA-1-60, TRA-1-81) and nuclear proteins (Oct-4, Nanog).2.4 in vivo teratoma formation2-3* 106 of hPDLC-iPSCs were injected into back and leg of the nude mice. After two months, the nude mice were sacrificed and observed whether there was a formation of teratoma. After taking out, the teratoma was embedded into paraffin and sliced. Hematoxylin eosin staining of the tissue was tested whether hPDLC-iPSCs have the potency to form teratoma and differentiate into ectoderm, mesoderm and entoderm.2.5 embryoid body experimentIPSCs were carefully cultured in suspension to form embryoid bodies. The structure of embryoid body was observed and the embryoid was made to grow with adherence 7 days later.Part 3. Evaluation of the osteogenic ability of hPDLC-iPSCs in vitro3.1 hPDLC-iPSCs, hES cells and osteogenic induction of PDLCThree types of cells, hPDLC-iPSCs, hES cells and PDLC cells were cultured. Primary culture medium was replaced by mineralization-induced medium (medium with beta glycerol sodium phosphate, vitamin C and dexamethasone) after cells reached a density of 80%. The osteogenic ability of the three kinds of cells were observed.3.2 Mineralized nodule formation and alizarin red stainingCells were cultured in mineralization-induced medium for 14 days and fixed in 4% paraformaldehyde for 30 minutes.1% alizarin red was added to cells and keep 37? for 30 minutes after a PBS washing. The mineralized nodules was observed and quantitative comparison was made to evaluate the amount of mineralized nodule formation.3.3 ALP activityCells were cultured in mineralization-induced medium for 7 days or 14 days. Cells were split by triton and the lysates was treated with pNPP, then cells were kept in 37? warm box to avoid light for half an hour. Sodium hydroxide was used to terminate the reaction and OD values were measured at 405 nm. Standard curve was made and the level of ALP was measured.3.4 PCR real-time to detect gene expression levelCells were cultured in mineralization-induced medium for 7 days or 14 days. Trizol was used to split the cells for RNA extraction. Then RNA was reverse transcript into cDNA. Quantitative real-time PCR detected gene expression level of ALP, OCN, Col-I and Runx2 in different cells.Part 4. The application of hPDLC-iPS cell sheet in bone defect restoration4.1 Preparation of hPDLC-iPSCs, hES cells and PDLC cell sheetshPDLC-iPSCs, hES cells and PDLC were cultured in mineralization-induced medium added with vitamin C for 21 days. At the late stage of culture, a white film was visible at the bottom of the culture dish. Then we carefully scraped the film to obtain the cell sheet.4.2 Cell sheet restoration of skull defect in nude miceThree groups were set for the experiment: hPDLC-iPS cell sheet group, hES cell sheet group and PDLC cell sheet group. Nude mice got anesthesia by chloral hydrate, then the incision of head skin was made to expose the skull. A 3.5mm trephine was used to create a bone defect, cell sheet was implanted onto the defect after carefully removing the bone chips. Finally, a skin stitching was made, and all the mice were treated with antibiotics.4.3 Evaluation of the cell sheet applicationAt the time of 4 and 8 weeks after operation, the nude mice were sacrificed. The bone generation was evaluated by ?CT. After that, the HE staining and Masson staining were used to analyze the new bone formation.Results1 After one week of primary culture, cells could be observed growing from the periodontal ligament tissue in microscope. Identification of cell immunofluorescence showed that vimentin, type I collagenase were positive and keratin was negative, which corresponded to the right type of PDLCs. PDLCs were cultured on MEF feeder after the infection of Lentivirus carried Oct-4, Sox-2, c-Myc and klf-4.2. Visible changes of the morphogenesis of PDLCs could be observed after the first week that the fibroblast like shape changed to round clone shape. The AP staining positive clones was picked after 3 weeks. The iPSCs got the typical morphology of hES cells and expressed typical ES cell specific proteins, including cell membrane proteins (SSEA3, SSEA4), cytoplasmic proteins (TRA-1-60, TRA-1-81) and nuclear protein (Oct-4, Nanog). Furthermore, hPDLC-iPS could form teratomas in nude mice that able to differentiate into all three germ layers and form embryoid body in vitro.3. After 14 days of Osteogenic induction, alizarin red staining showed hPDLC-iPSCs showed more mineralized nodule formation than the other two group of cells, and the ALP test confirmed this result. In addition, the expression of ALP, OCN, Col-I and Runx2 in hPDLC-iPSCs was higher than the other two cells. These results showed that the osteogenic ability of hPDLC-iPSCs had a stronger osteogenic ability than that of hES cells and PDLCs.4. Animal experiment set three group, hPDLC-iPSCs, hES cells and PDLC cells were successfully made into cell sheet. ?CT reconstruction analysis found that the hPDLC-iPS cell sheet had a stronger effect of bone defect regeneration than the other two kinds of cell sheet, while the results of HE and Masson staining kept the same with?CT analysis.Conclusions1. hPDLC-iPSCs were successfully constructed. hPDLC-iPSCs could express the specific surface markers of embryonic stem cells, and has the ability of multi differentiation and form into terotoma in nude mice.2. Alizarin red staining, ALP test and real-time PCR results showed that hPDLC-iPSCs had a better osteogenic ablility in vitro than hES cells and hPDLCs.3. Cell sheet method was based on cell extracellular matrix secretion and it is a simple and more effective tissue engineering method in the repairing of bone defects. hPDLC-iPS cell membrane can be used in bone regeneration and proposed a new method for us in periodontal tissue engineering in the future.