Abstract
Three-dimensional (3D) tissue construction from individual cells is an important process in regenerative medicine to enhance cell functions. In transplantation of tissue-engineered constructs, a limited oxygen/nutrient supply due to insufficient vascular network formation causes cell death. Thus, it is necessary to develop a system for inducing vascular networks into 3D tissue constructs under hypoxic conditions. In our previous study (Ono, A., et al., 2017), we developed a hypoxia-inducible transgene expression system in which a target gene can be expressed in response to hypoxic stress using hypoxia-responsive promoter RTP801 as a trigger, tTA transactivator as an amplifier, and oxygen-dependent degradation sequence as a noise canceler. In this study, to improve oxygen and nutritional limitation within engineered 3D tissue constructs, a hypoxia-responsive gene expression system for vascular endothelial growth factor (VEGF) was introduced into cells. We demonstrated that genetically engineered cells could regulate VEGF expression autonomously in an oxygen concentration-dependent manner. Using the genetically engineered cells, 3D tissue constructs were fabricated using a magnetic force-based tissue engineering technique (Ito, A., et al., 2005). The tissue constructs were transplanted into mice to evaluate the feasibility of the hypoxia-responsive VEGF gene expression system in vivo. The results indicated that the VEGF gene expression system is promising for the induction of vascular networks into 3D tissue constructs.
Highlights
In recent years, regenerative medicine has attracted attention for the purpose of functional recovery using functionalized cells as a solution for defects and dysfunction of tissues and organs
In the hypoxiaresponsive vascular endothelial growth factor (VEGF) gene-expression system, an artificial transcription activator TA-oxygen-dependent degradation (ODD) comprising tetracyclineresponsive transactivator incorporated ODD domain is expressed under control of RTP801 promoter, and the expression of VEGF and EGFP genes is driven by TAODD
Mouse myoblast C2C12 cells were grown in low-glucose Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum (FBS), 0.1 mg/ml streptomycin sulfate, 100 U/ml potassium penicillin G, 3.7 mg/ml sodium hydrogen carbonate (NaHCO3) and 20 mM 2-[4-(2-hydroxyethyl)1-piperazinyl] ethanesulfonic acid (HEPES). 293FT cells were maintained on collagen coated dish in high-glucose DMEM supplemented with 10% FBS, 0.1 mg/ml streptomycin sulfate, 100 U/ml potassium penicillin G, 3.7 mg/ml NaHCO3 and 0.1 mM non-essential amino acid (NEAA)
Summary
Regenerative medicine has attracted attention for the purpose of functional recovery using functionalized cells as a solution for defects and dysfunction of tissues and organs. In transplantation of tissueengineered constructs, lack of oxygen/nutrition supply within the constructs due to “hypovascularity” causes a decrease in cell viability of the constructs To address this problem, approaches such as artificial blood vessels and co-culture with vascular endothelial cells have been examined (Arai et al, 2018; Okudaira, et al, 2016). In the hypoxiaresponsive VEGF gene-expression system, an artificial transcription activator TA-ODD comprising tetracyclineresponsive transactivator incorporated ODD domain is expressed under control of RTP801 promoter, and the expression of VEGF and EGFP genes is driven by TAODD This system allows cells to control VEGF gene expression autonomously in response to hypoxia. The tissue constructs were transplanted into mice to evaluate the feasibility of the hypoxia-responsive VEGF gene expression system in vivo
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