Human umbilical cord mesenchymal stem cell-derived exosomes promote murine skin wound healing by neutrophil and macrophage modulations revealed by single-cell RNA sequencing

Abstract

IntroductionFull-thickness skin wound healing remains a serious undertaking for patients. While stem cell-derived exosomes have been proposed as a potential therapeutic approach, the underlying mechanism of action has yet to be fully elucidated. The current study aimed to investigate the impact of exosomes derived from human umbilical cord mesenchymal stem cells (hucMSC-Exosomes) on the single-cell transcriptome of neutrophils and macrophages in the context of wound healing.MethodsUtilizing single-cell RNA sequencing, the transcriptomic diversity of neutrophils and macrophages was analyzed in order to predict the cellular fate of these immune cells under the influence of hucMSC-Exosomes and to identify alterations of ligand-receptor interactions that may influence the wound microenvironment. The validity of the findings obtained from this analysis was subsequently corroborated by immunofluorescence, ELISA, and qRT-PCR. Neutrophil origins were characterized based on RNA velocity profiles.ResultsThe expression of RETNLG and SLC2A3 was associated with migrating neutrophils, while BCL2A1B was linked to proliferating neutrophils. The hucMSC-Exosomes group exhibited significantly higher levels of M1 macrophages (215 vs 76, p < 0.00001), M2 macrophages (1231 vs 670, p < 0.00001), and neutrophils (930 vs 157, p < 0.00001) when compared to control group. Additionally, it was observed that hucMSC-Exosomes elicit alterations in the differentiation trajectories of macrophages towards more anti-inflammatory phenotypes, concomitant with changes in ligand-receptor interactions, thereby facilitating healing.DiscussionThis study has revealed the transcriptomic heterogeneity of neutrophils and macrophages in the context of skin wound repair following hucMSC-Exosomes interventions, providing a deeper understanding of cellular responses to hucMSC-Exosomes, a rising target of wound healing intervention.

Heterogeneity of immune cells and their communications unveiled by transcriptome profiling in acute inflammatory lung injury

“…Perhaps Clec4e expression in macrophages and neutrophils can be used as an indicator to identify the different factors contributing to ARDS. Retnlg has been shown to be associated with neutrophil migration ( 39 , 40 ), whereas in our study, Retnlg present in top50 DEGs of multiple cell clusters in the pulmonary ALI, was not found in the DEGs of neutrophils. S100 Calcium Binding Protein A9 (S100a9) usually present as a heterodimer in vivo , mainly released by immune cells, such as neutrophils and macrophages ( 41 ), and is an early-warning protein for inflammation, initiated by Toll-like Receptor 4 (TLR4) in response to LPS stimulation ( 42 ).…”

HucMSC-Exo Induced N2 Polarization of Neutrophils: Implications for Angiogenesis and Tissue Restoration in Wound Healing

“…Previously, we characterized the heterogeneity of neutrophils and macrophages during HucMSC-Exo-mediated tissue repair through single-cell sequencing. 16 Herein, we found that the pro-healing effect of HucMSC-Exo heavily depended on infiltrated neutrophils rather than macrophages. Adding complexity to this, HucMSC-Exo guided the phenotypic switching of neutrophils at the wound site, directly reprogramming naïve neutrophils (N0) into an anti-inflammatory proangiogenetic status (N2).…”

“…Our prior recognition of the participation of myeloid cells, specifically macrophages and neutrophils, in the wound healing process led us to investigate their role in HucMSC-Exo-guided wound healing. 16 We deployed two strategies to selectively deplete either circulating monocytes (via anti-F4/80 treatment) or neutrophils (via anti-Ly6G) in recipient mice ( Figure 2A ). Intraperitoneal injection of the anti-Ly6G antibody resulted in the effective depletion of neutrophils without affecting circulating monocytes.…”

“… 8 , 10 , 36 Our prior findings provided evidence that HucMSC-Exo significantly ups the count of infiltrated neutrophils at the wound site in mice (930 vs 157, p < 0.00001). 16 These cells are paramount in early inflammation, serving as a frontline defense against microbes. However, emerging experimental evidences suggest that neutrophils possess alternative functions beyond their classic bactericidal roles.…”

HucMSCs Delay Muscle Atrophy After Peripheral Nerve Injury Through Exosomes by Repressing Muscle-Specific Ubiquitin Ligases

“…These changes are accompanied by shifts in ligand-receptor interactions, ultimately promoting a conducive environment for healing. 52 HucMSC-EXOs have significant effects on the regulation of microglia activation in dorsal root ganglia (DRGs) 53 and suppress macrophages in peripheral nerves. 54 Exosomes from alternative stem cell sources, such as bone marrow mesenchymal stem cells, 55 gingival mesenchymal stem cells, 56 and Schwann cells, 57 among others, affect dorsal root ganglion (DRG) neurons, and promote peripheral nerves regeneration.…”