No abstract

Poor survival of grafted cells is the major impediment of successful cell‐based therapies for bone regeneration. Implanted cells undergo rapid death in an ischemic environment largely because of hypoxia and metabolic stress from glucose deficiency. Understanding the intracellular metabolic processes and finding genes that can improve cell survival in these inhospitable conditions are necessary to enhance the success of cell therapies. Thus, the purpose of this study was to investigate changes of metabolic profile in glucose‐deprived human bone marrow stromal/stem cells (hBMSCs) through metabolomics analysis and discover genes that could promote cell survival and osteogenic differentiation in a glucose‐deprived microenvironment. Metabolomics analysis was performed to determine metabolic changes in a glucose stress metabolic model. In the absence of glucose, expression levels of all metabolites involved in glycolysis were significantly decreased than those in a glucose‐supplemented state. In glucose‐deprived osteogenic differentiation, reliance on tricarboxylic acid cycle (TCA)‐predicted oxidative phosphorylation instead of glycolysis as the main mechanism for energy production in osteogenic induction. By comparing differentially expressed genes between glucose‐deprived and glucose‐supplemented hBMSCs, NR2F1 (Nuclear Receptor Subfamily 2 Group F Member 1) gene was discovered to be associated with enhanced survival and osteogenic differentiation in cells under metabolic stress. Small, interfering RNA (siRNA) for NR2F1 reduced cell viability and osteogenic differentiation of hBMSCs under glucose‐supplemented conditions whereas NR2F1 overexpression enhanced osteogenic differentiation and cell survival of hBMSCs in glucose‐deprived osteogenic conditions via the protein kinase B (AKT)/extracellular signal‐regulated kinase (ERK) pathway. NR2F1‐transfected hBMSCs significantly enhanced new bone formation in a critical size long‐bone defect of rats compared with control vector‐transfected hBMSCs. In conclusion, the results of this study provide an understanding of the metabolic profile of implanted cells in an ischemic microenvironment and demonstrate that NR2F1 treatment may overcome this deprivation by enhancing AKT and ERK regulation. These findings can be utilized in regenerative medicine for bone regeneration. © 2022 American Society for Bone and Mineral Research (ASBMR).

Adipose‐derived stromal/stem cells (ASCs) have been extensively studied as cell sources for regenerative medicine for bone due to their excellent proliferative capacity and the ability to obtain a large number of cells with minimal donor morbidity. On the other hand, the differentiation potential of ASCs is generally lower than that of bone marrow‐derived stromal/stem cells and varies greatly depending on donors. In this study, we mined a marker that can predict the osteogenic potential of ASC clones and also investigated the usefulness of the molecule as the enhancer of osteogenic differentiation of ASCs as well as its mechanism of action. Through RNA‐seq gene analysis, we discovered that GSTT1 (Glutathione S‐transferase theta‐1) was the most distinguished gene marker between highly osteogenic and poorly osteogenic ASC clones. Knockdown of GSTT1 in high osteogenic ASCs by siGSTT1 treatment reduced mineralized matrix formation. On the other hand, GSTT1 overexpression by GSTT1 transfection or GSTT1 recombinant protein treatment enhanced osteogenic differentiation of low osteogenic ASCs. Metabolomic analysis confirmed significant changes of metabolites related to bone differentiation in ASCs transfected with GSTT1. A high total antioxidant capacity, low levels of cellular reactive oxygen species and increased GSH/GSSG ratios were also detected in GSTT1‐transfected ASCs. When the in vivo effect of GSTT1‐transfected ASCs on bone regeneration was investigated with segmental long‐bone defect model in rats, bone regeneration was significantly better after implantation of GSTT1‐transfected ASCs compared to that of control vector‐transfected ASCs. In conclusion, GSTT1 can be a useful marker to screen the highly osteogenic ASC clones and also a therapeutic factor to enhance the osteogenic differentiation of poorly osteogenic ASC clones.This article is protected by copyright. All rights reserved.