An engineered hypoxia-response promoter for human umbilical cord-derived mesenchymal stem cell-based therapeutics

Abstract

Myocardial infarction, characterized by insufficient blood supply to the heart, leads to ischemia and hypoxia of myocardial tissues, causing injury and decreased cardiac function. Despite improvements in pharmaceutical and interventional therapies, it remains a leading cause of death worldwide. Human umbilical cord mesenchymal stem cells (hUC-MSCs) play an important role in the repair of infarcted myocardium by promoting angiogenesis, reducing inflammation, secreting growth factors and cytokines. However, the harsh hypoxic microenvironment of infarcted myocardial tissue poses a threat to the survival and function of transplanted hUC-MSCs. In this study, we modified the candidate gene promoter of hUC-MSCs under hypoxic conditions and created a promoter that can respond quickly under hypoxic conditions. We found that the modified promoter significantly promoted the transcription efficiency as hypoxia time increased. This indicates that the engineered hypoxia-response promoter can effectively drive gene expression in a hypoxic environment. Furthermore, the transcription efficiency of the modified promoter under normoxic conditions is lower than that of common promoters in eukaryotic organisms, suggesting that this effect can improve the efficacy and safety of hUC-MSC-based myocardial infarction treatment by ensuring that cells function effectively in the damaged hypoxic area.

Keywords: Gene promoter modification; Human umbilical cord mesenchymal stem cells (hUC-MSCs); Hypoxia inducible factor (HIF); Hypoxia response element (HRE).

Fig. 1

(A) The diagram shows the experimental design. Firstly, hUC-MSCs were cultured under hypoxia and total RNA was extracted at different time points for sequencing. Then, the genes continuously expressed during the hypoxia treatment were selected. Finally, the appropriate target gene promoter was modified and functionally verified. (B) Representative fluorescence activated cell sorting (FACS) analysis of the apoptosis marker annexin V (conjugated with Allophycocyanin (APC)) in hUC-MSCs after treatment of hypoxia. In the negative control group, DPBS buffer was used instead of the annexin V staining reagent. (C) The proportion of apoptotic cells in hypoxic culture and normoxic culture on different exposure times. (D) To assess cell viability, we performed a CCK8 assay over three days under both hypoxic and normoxic conditions. The graph shows cell viability as measured by the A450 absorbance. Hypoxic conditions led to slightly higher cell viability compared to normoxic conditions during the first two days. (E and F) Additionally, we conducted MTT and Alamar blue assays to evaluate cell viability under the same conditions. During the first two days, cell viability under normoxic conditions was significantly lower than under hypoxic conditions, as indicated by both the Alamar blue assay and the MTT assay. The statistical significance in this figure was expressed by asterisks: **, p < 0.01; ***, p < 0.001; ****, p < 0.0001; ns: no significant difference. The comparison of each group’s P value can be seen in the Supplementary Table 1, Supplementary Table 6 of supplementary materials. Data represent mean ± SEM from n = 3 independent biological replicates

Fig. 2

(A) A heat map of up-regulated genes with a continuous increase in expression from 2 h to 72 h of hypoxia culture and the genes were sorted from high to low according to the log2 value (fold change) of the 72-hour hypoxia group. (B) Volcano map of differentially expressed genes between hypoxic culture and normal culture. Red dots and green dots represent up-regulated genes and down-regulated genes, respectively. (C) Gene ontology (GO) analysis of up-regulated and down-regulated genes in response to hypoxia. The bar graphs display the number of genes associated with various GO terms categorized into three main categories: Molecular Function (green), Biological Process (blue), and Cellular Component (yellow). (D) Gene ontology (GO) enrichment analysis of up-regulated and down-regulated genes in hypoxia 72 h group (GO item: molecular function, biological process, and cellular component)

Fig. 3

(A) The structure diagram of the CA9 series promoters. (B) Dual-luciferase reporter assay results show the relative firefly luciferase (FLuc) activity under normoxic and hypoxic conditions at various time points. The FLuc activity was normalized to Renilla luciferase (RLuc) activity for each sample. The transcription efficiency of CA9 series promoters under normoxic (Left) and hypoxic (Right) conditions for 2 h, 6 h, and 24 h. (C) The comparison of transcription efficiency between CA9-4HRE-TA promoter and some common eukaryotic promoters under hypoxia and normoxia 2 H, 24 H, 48 H, 72 H, and 96 H conditions was similar to Fig C, showing multiple changes in FLuc activity. The statistical significance in this figure was expressed by asterisk: *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001; ns: no significant difference. The comparison of each group’s P value can be seen in Supplementary Table 2, Supplementary Table 3, Supplementary Table 4, Supplementary Table 5, of supplementary materials. Data represent mean ± SEM from at least n = 3 independent biological replicates