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 (ALP) activity was increased by 130% by NELL1 overexpression at 14 d (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 14 d, runt-related transcription factor 2 (RUNX2) gene level of NELL1-iPSC-MSCs was 2.0-fold that of RFP-iPSC-MSCs. Osteocalcin (OC) level of NELL1-iPSC-MSCs was 3.1-fold that of RFP-iPSC-MSCs (p < 0.05). Collagen type I alpha 1 (COL1A1) gene level of NELL1-iPSC-MSCs was 1.7-fold that of RFP-iPSC-MSCs at 7 d (p < 0.05). Mineral synthesis was increased by 81% in NELL1-iPSC-MSCs at 21 d. 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 to enhance bone engineering.

Engineering human bone grafts with new macroporous calcium phosphate cement scaffolds

“…Previous studies in vitro have shown that macroporous CPC scaffolds (with different formulation) support cell viability and differentiation of human mesenchymal stem cells (MSCs; Vater et al, ; Weir & Xu, ), and grafts of CPC scaffolds seeded with human MSCs promote skeletal regeneration in animal models (Chen et al, ), with good prospects for future developments and clinical applications. To overcome the limitations associated with isolation and poor regeneration potential of adult stem cells, researchers have recently explored the possibility to use human pluripotent stem cells as source of mesenchymal progenitors for bone engineering applications (Idowu, Cama, Deb, & Di Silvio, ; J. Liu, Chen, Zhao, & Xu, ; X. Liu, Wang, et al, ; Sladkova et al, ; Wang et al, ). In particular, human induced pluripotent stem cells (iPSCs) can be derived from every patient, proliferate indefinitely, and give rise to all cells constituting the bone tissue (Takahashi et al, ).…”

Applied Induced Pluripotent Stem Cells in Combination With Biomaterials in Bone Tissue Engineering

“…In another study, they compared osteogenic potential of bone marrow derived iPSCs while cultured on the modified CPC by five biofunctional agents such as RGD, fibronectin, fibronectin‐like engineered polymer protein, ECM Geltrex, and platelet concentrate, and they results were demonstrated that modified CPC have a great osteogenic potential in comparison to the CPC, among the modified CPC, almost all osteogenic markers were increased in the fibronrctin‐CPC, Platelet‐CPC, and RGD‐CPC groups compared to the other without significantly differences [TheinHan et al, ]. This scaffold was also used in another study to evaluation of osteogenic differentiation and mineralization detection by culture of NEL‐like protein‐1 overexpressed iPSCs‐MSCs, and their results were shown that overexpressed stem cells have been shown significantly higher osteogenic gene markers and mineralization assays and the authors suggest this stem cell‐scaffold construction for used in bone tissue engineering applications [Liu et al, ].…”

Stem and progenitor cells: advancing bone tissue engineering

“…Here, the authors reported that polyethersulfone scaffolds seeded with iPSCs induced greater bone regeneration than scaffold alone [62], results which have also been replicated in periodontal bone regeneration [63]. Furthermore, Liu recently reported that NELL1 (which encodes a protein involved in cell growth regulation and differentiation) overexpression greatly enhanced the osteogenic differentiation and mineral synthesis of iPSC-MSCs on Arg-Gly-Asp-grafted calcium phosphate cement scaffold [64]. Ideally, implementing the many arms of tissue engineering, the design of simple and robust biomaterials with an innate ability to induce lineage-specificity of iPSCs is desirable for effective clinical implementation of iPSC-based bone tissue engineering.…”