title: Application of eGFP to label human periodontal ligament stem cells in periodontal tissue engineering.
abstract: OBJECTIVE
  To establish human periodontal ligament stem cells (hPDLSC) with high and stable expression of enhanced green fluorescent protein (eGFP) and to obtain an ideal vector expression system that suitable for gene therapy in periodontal tissue engineering.
  
  
  METHODS
  hPDLSCs were transfected with eGFP for 48h via different MOI (25, 50, 100, 200 and 400) by lentiviral vector, the transfection efficiency was evaluated by fluorescent microscopy and flow cytometry, and transfected hPDLSCs proliferation was evaluated by MTT. Pluripotent, differentiation capacity and ALP expression status were determined further. Osteoblast-associated genes expressions for osteogenesis were evaluated by quantitative-PCR. In addition, rat molar periodontal fenestration defect model was used for evaluating periodontal tissue engineering.
  
  
  RESULTS
  The transfection efficiency after 48h were 44.7%, 60.9%, 71.7%, 85.8%, and 86.9% respectively. There was no significant effect of transfection (at different MOI levels of 25, 50, 100, and 200) on the proliferation of hPDLSCs (designated as eGFP-hPDLSCs) compared with hPDLSCs (P>0.05). However, proliferation of eGFP hPDLSCs at MOI 400 became slower (P<0.05). Both eGFP hPDLSCs and hPDLSCs were able to differentiate into osteocytes and adipocytes under certain conditioned media. At 7 days, expression levels of COL-1, RUNX2 in hPDLSCS were higher than those in eGFP hPDLSCs (P<0.05); expression levels of ALP and OPN in eGFP hPDLSCs were similar to those in hPDLSCs (P>0.05). Newly regenerated bone formation was observed in the defect model used.
  
  
  CONCLUSIONS
  Among the transfection conditions, 48h transfection at MOI 200 is optimal for labelling hPDLSCs with eGFP in a lentiviral vector. There is no change in capability of the eGFP hPDLSCs osteogenesis. The lentiviral vector with eGFP is an appropriate expression vector system and hPDLSCs are ideal seeding cells for gene therapy in periodontal tissue engineering.
authors.affiliation: School of Stomatology, Shandong University, No. 44-1 Wenhua Xi Road, Jinan, Shandong, PR China. wenyongsdu@yahoo.com.cn
authors.forename: Yong
authors.initials: Y
authors.lastname: Wen
authors.forename: Jing
authors.initials: J
authors.lastname: Lan
authors.forename: Haiyun
authors.initials: H
authors.lastname: Huang
authors.forename: Meijiao
authors.initials: M
authors.lastname: Yu
authors.forename: Jun
authors.initials: J
authors.lastname: Cui
authors.forename: Jin
authors.initials: J
authors.lastname: Liang
authors.forename: Baoqi
authors.initials: B
authors.lastname: Jiang
authors.forename: Xin
authors.initials: X
authors.lastname: Xu
mesh_terms: D017667:Adipocytes / Q000502:physiology; D050156:Adipogenesis / Q000502:physiology; D000469:Alkaline Phosphatase / Q000032:analysis; D016301:Alveolar Bone Loss / Q000601:surgery; D000818:Animals; D001861:Bone Regeneration / Q000502:physiology; D018929:Cell Culture Techniques; D002454:Cell Differentiation / Q000502:physiology; D049109:Cell Proliferation; D024042:Collagen Type I / Q000032:analysis; D050659:Core Binding Factor Alpha 1 Subunit / Q000032:analysis; D004195:Disease Models, Animal; D005456:Fluorescent Dyes; D015316:Genetic Therapy; D005822:Genetic Vectors / Q000235:genetics; D049452:Green Fluorescent Proteins; D006801:Humans; D016086:Lentivirus / Q000235:genetics; D010006:Osteoblasts / Q000166:cytology; D010011:Osteocytes / Q000502:physiology; D010012:Osteogenesis / Q000502:physiology; D053244:Osteoprotegerin; D010513:Periodontal Ligament / Q000166:cytology*; D039904:Pluripotent Stem Cells / Q000166:cytology; D051381:Rats; D017207:Rats, Sprague-Dawley; D013234:Stem Cells / Q000166:cytology*; D023822:Tissue Engineering; D014162:Transfection
chemical_list: D024042:Collagen Type I; D050659:Core Binding Factor Alpha 1 Subunit; D005456:Fluorescent Dyes; D053244:Osteoprotegerin; C493709:RUNX2 protein, human; C506030:TNFRSF11B protein, human; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D000469:Alkaline Phosphatase