The Impact of GFP Reporter Gene Transduction and Expression on Metabolomics of Placental Mesenchymal Stem Cells Determined by UHPLC-Q/TOF-MS

Abstract

Introduction Green fluorescent protein (GFP) is widely used as a reporter gene in regenerative medicine research to label and track stem cells. Here, we examined whether expressing GFP gene may impact the metabolism of human placental mesenchymal stem cells (hPMSCs). Methods The GFP gene was transduced into hPMSCs using lentiviral-based infection to establish GFP+hPMSCs. A sensitive 13C/12C-dansyl labeling LC-MS method targeting the amine/phenol submetabolome was used for in-depth cell metabolome profiling. Results A total of 1151 peak pairs or metabolites were detected from 12 LC-MS runs. Principal component analysis and partial least squares discriminant analysis showed poor separation, and the volcano plots demonstrated that most of the metabolites were not significantly changed when hPMSCs were tagged with GFP. Overall, 739 metabolites were positively or putatively identified. Only 11 metabolites showed significant changes. Metabolic pathway analyses indicated that three of the identified metabolites were involved in nine pathways. However, these metabolites are unlikely to have a large impact on the metabolic pathways due to their nonessential roles and limited hits in pathway analysis. Conclusion This study indicated that the expression of ectopic GFP reporter gene did not significantly alter the metabolomics pathways covered by the amine/phenol submetabolome.

Mesenchymal Stem Cell Therapy for Spinal Cord Contusion: A Comparative Study on Small and Large Animal Models

“…It is considered that EGFP gene functionally is neutral when integrated and provides convenient cell labeling without a significant impact on basic cellular functions [52], even within MSCs [53]. We demonstrated that within our isolated MSCs, transduction of AD-MSCs, BM-MSCs, and DP-MSCs cells by LV-EGFP did not lead to significant changes in the expression of the investigated markers, which is consistent with other works [54].…”

DNAJA1 Dysregulates Metabolism Promoting an Antiapoptotic Phenotype in Pancreatic Ductal Adenocarcinoma

“…A number of prior studies have failed to identify any metabolic signature associated with the overexpression of a protein in a cell line. 42,43 A tree diagram (Figure 1F) derived from the score plot (Figure 1E DNAJA1 expression, indicating metabolome perturbations accumulated over time. However, the tree diagram indicates that the metabolome of WT-Bx cells did not change over time.…”

Metabolomics studies of cell–cell interactions using single cell mass spectrometry combined with fluorescence microscopy

“…Our experimental results agree well with the previous report that GFP labelling has a negligible influence on cell metabolism. 51 Therefore, it is valid to use HCT116-GFP cells to represent HCT116 cells for the co-culture studies.…”

“…Stable HCT116-GFP cells, generated from HCT116 cells by lentiviral vector transduction, were purchased (Cellomics Technology, Halethorpe, MD, USA). Because GFP (green fluorescent protein) labeling has no significant influence on cell metabolism and functions, 51 HCT116-GFP cells are also defined as drug-sensitive cells in the current study. To maintain the ability of expressing the green fluorescent protein, HCT116-GFP cells were cultured in a complete growth medium containing 1 μg mL −1 puromycin.…”