Deciphering the in vivo Dynamic Proteomics of Mesenchymal Stem Cells in Critical Limb Ischemia

Abstract

ObjectiveRegenerative therapy using mesenchymal stem cells (MSC) is a promising therapeutic method for critical limb ischemia (CLI). To understand how the cells are involved in the regenerative process of limb ischemia locally, we proposed a metabolic protein labeling method to label cell proteomes in situ and then decipher the proteome dynamics of MSCs in ischemic hind limb.Methods and ResultsIn this study, we overexpressed mutant methionyl-tRNA synthetase (MetRS), which could utilize azidonorleucine (ANL) instead of methionine (Met) during protein synthesis in MSCs. Fluorescent non-canonical amino-acid tagging (FUNCAT) was performed to detect the utilization of ANL in mutant MSCs. Mice with hindlimb ischemia (HLI) or Sham surgery were treated with MetRSmut MSCs or PBS, followed by i.p. administration of ANL at days 0, 2 6, and 13 after surgery. FUNCAT was also performed in hindlimb tissue sections to demonstrate the incorporation of ANL in transplanted cells in situ. At days 1, 3, 7, and 14 after the surgery, laser doppler imaging were performed to detect the blood reperfusion of ischemic limbs. Ischemic tissues were also collected at these four time points for histological analysis including HE staining and vessel staining, and processed for click reaction based protein enrichment followed by mass spectrometry and bioinformatics analysis. The MetRSmut MSCs showed strong green signal in cell culture and in HLI muscles as well, indicating efficient incorporation of ANL in nascent protein synthesis. By 14 days post-treatment, MSCs significantly increased blood reperfusion and vessel density, while reducing inflammation in HLI model compared to PBS. Proteins enriched by click reaction were distinctive in the HLI group vs. the Sham group. 34, 31, 49, and 26 proteins were significantly up-regulated whereas 28, 32, 62, and 27 proteins were significantly down-regulated in HLI vs. Sham at days 1, 3, 7, and 14, respectively. The differentially expressed proteins were more pronounced in the pathways of apoptosis and energy metabolism.ConclusionIn conclusion, mutant MetRS allows efficient and specific identification of dynamic cell proteomics in situ, which reflect the functions and adaptive changes of MSCs that may be leveraged to understand and improve stem cell therapy in critical limb ischemia.

Bioorthogonal Chemistry and Its Applications

“…FUNCAT was performed to decipher the proteome dynamics of mesenchymal stem cells (MSC) for understanding the local regenerative process of limb ischemia . Mice expressing mutant methionyl-tRNA synthetase (MetRS) with hindlimb ischemia (HLI) or Sham surgery were administrated with azidonorleucine (ANL).…”

“…FUNCAT was performed to decipher the proteome dynamics of mesenchymal stem cells (MSC) for understanding the local regenerative process of limb ischemia. 94 Mice expressing mutant methionyl-tRNA synthetase (MetRS) with hindlimb ischemia (HLI) or Sham surgery were administrated with azidonorleucine (ANL). Ischemic tissues were collected afterward for histological analysis and processed for click reaction-based protein enrichment followed by mass spectrometry and bioinformatics analysis.…”

Monitoring regional astrocyte diversity by cell type‐specific proteomic labeling in vivo

“…Therefore, cell type‐specific tagging by ANL and MetRS L274G might be applicable for studying static or dynamic cell type‐specific proteome changes under various conditions and labeling duration and in several species, either to monitor general net flux changes of the proteome by FUNCAT (Niehues et al, 2015; Shahar & Schuman, 2020) or in combination with proteomic analysis (Alvarez‐Castelao et al, 2017; Azizian et al, 2021; Dunzheng Han et al, 2020; Liu et al, 2017). As either stable expression of MetRS L274G or ANL integration do not interfere with animal health, (learning) behavior, or cellular interaction (Liu et al, 2017; Shahar & Schuman, 2020) and metabolic labeling is also applicable in stem cells and metabolic active tumor cells (Azizian et al, 2021; Du et al, 2021), the application of this method can be likely extended to further unravel the role of astrocytes in nervous system development, aging and diseases progression, for example.…”