title: Effect of NELL1 gene overexpression in iPSC-MSCs seeded on calcium phosphate cement.
abstract: Human induced pluripotent stem cell-derived mesenchymal stem cells (iPSC-MSCs) are a promising source of patient-specific stem cells with great regenerative potential. There has been no report on NEL-like protein 1 (NELL1) gene modification of iPSC-MSCs. The objectives of this study were to genetically modify iPSC-MSCs with NELL1 overexpression for bone tissue engineering, and investigate the osteogenic differentiation of NELL1 gene-modified iPSC-MSCs seeded on Arg-Gly-Asp (RGD)-grafted calcium phosphate cement (CPC) scaffold. Cells were transduced with red fluorescence protein (RFP-iPSC-MSCs) or NELL1 (NELL1-iPSC-MSCs) by a lentiviral vector. Cell proliferation on RGD-grafted CPC scaffold, osteogenic differentiation and bone mineral synthesis were evaluated. RFP-iPSC-MSCs stably expressed high levels of RFP. Both the NELL1 gene and NELL1 protein levels were confirmed higher in NELL1-iPSC-MSCs than in RFP-iPSC-MSCs using RT-PCR and Western blot (P<0.05). Alkaline phosphatase activity was increased by 130% by NELL1 overexpression at 14days (P<0.05), indicating that NELL1 promoted iPSC-MSC osteogenic differentiation. When seeded on RGD-grafted CPC, NELL1-iPSC-MSCs attached and expanded similarly well to RFP-iPSC-MSCs. At 14days, the runt-related transcription factor 2 (RUNX2) gene level of NELL1-iPSC-MSCs was 2.0-fold that of RFP-iPSC-MSCs. The osteocalcin (OC) level of NELL1-iPSC-MSCs was 3.1-fold that of RFP-iPSC-MSCs (P<0.05). The collagen type I alpha 1 (COL1A1) gene level of NELL1-iPSC-MSCs was 1.7-fold that of RFP-iPSC-MSCs at 7days (P<0.05). Mineral synthesis was increased by 81% in NELL1-iPSC-MSCs at 21days. In conclusion, NELL1 overexpression greatly enhanced the osteogenic differentiation and mineral synthesis of iPSC-MSCs on RGD-grafted CPC scaffold for the first time. The novel NELL1-iPSC-MSC seeded RGD-CPC construct is promising for enhancing bone engineering.
authors.affiliation: State Key Laboratory of Oral Diseases, West China Hospital of Stomatology, Sichuan University, Chengdu 610041, People's Republic of China; Biomaterials & Tissue Engineering Division, Department of Endodontics, Prosthodontics and Operative Dentistry, University of Maryland Dental School, Baltimore, MD 21201, USA.
authors.forename: Jun
authors.initials: J
authors.lastname: Liu
authors.affiliation: State Key Laboratory of Oral Diseases, West China Hospital of Stomatology, Sichuan University, Chengdu 610041, People's Republic of China; Biomaterials & Tissue Engineering Division, Department of Endodontics, Prosthodontics and Operative Dentistry, University of Maryland Dental School, Baltimore, MD 21201, USA.
authors.forename: Wenchuan
authors.initials: W
authors.lastname: Chen
authors.affiliation: State Key Laboratory of Oral Diseases, West China Hospital of Stomatology, Sichuan University, Chengdu 610041, People's Republic of China.
authors.forename: Zhihe
authors.initials: Z
authors.lastname: Zhao
authors.affiliation: Biomaterials & Tissue Engineering Division, Department of Endodontics, Prosthodontics and Operative Dentistry, University of Maryland Dental School, Baltimore, MD 21201, USA; Center for Stem Cell Biology & Regenerative Medicine, University of Maryland School of Medicine, Baltimore, MD 21201, USA; Marlene and Stewart Greenebaum Cancer Center, University of Maryland School of Medicine, Baltimore, MD 21201, USA; Department of Mechanical Engineering, University of Maryland, Baltimore County, MD 21250, USA. Electronic address: hxu@umaryland.edu.
authors.forename: Hockin H K
authors.initials: HHK
authors.lastname: Xu
mesh_terms: D001843:Bone Cements / Q000737:chemistry; D002130:Calcium Phosphates / Q000737:chemistry*; D002135:Calcium-Binding Proteins; D002454:Cell Differentiation / Q000502:physiology; D002460:Cell Line; D000090602:Collagen Type I, alpha 1 Chain; D004867:Equipment Design; D019544:Equipment Failure Analysis; D024861:Genetic Enhancement / Q000379:methods; D006801:Humans; D059630:Mesenchymal Stem Cells / Q000166:cytology* / Q000502:physiology*; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000378:metabolism*; D009842:Oligopeptides / Q000737:chemistry; D010006:Osteoblasts / Q000166:cytology* / Q000502:physiology*; D010012:Osteogenesis / Q000502:physiology; D011994:Recombinant Proteins / Q000378:metabolism; D023822:Tissue Engineering / Q000295:instrumentation / Q000379:methods; D054457:Tissue Scaffolds; D015854:Up-Regulation
chemical_list: D001843:Bone Cements; C000716127:COL1A1 protein, human; D002130:Calcium Phosphates; D002135:Calcium-Binding Proteins; D000090602:Collagen Type I, alpha 1 Chain; C104144:NELL1 protein, human; D009419:Nerve Tissue Proteins; D009842:Oligopeptides; D011994:Recombinant Proteins; C047981:arginyl-glycyl-aspartic acid; C020243:calcium phosphate
keywords: Calcium phosphate cement scaffold; Gene therapy; Induced pluripotent stem cells; NELL1; Osteogenic differentiation