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Single-cell spatiotemporal analysis reveals cell fates and functions of transplanted mesenchymal stromal cells during bone repair.

ABSTRACT: Mesenchymal stromal cells (MSCs) transplantation could enhance bone repair. However, the cell fate of transplanted MSCs, in terms of their local distribution and spatial associations with other types of cells were poorly understood. Here, we developed a single-cell 3D spatial correlation (sc3DSC) method to track transplanted MSCs based on deep tissue microscopy of fluorescent nanoparticles (fNPs) and immunofluorescence of key proteins. Locally delivered fNP-labeled MSCs enhanced tibial defect repair, increased the number of stem cells and vascular maturity in mice. fNP-MSCs persisted in the defect throughout repair. But only a small portion of transplanted cells underwent osteogenic differentiation (OSX+); a significant portion has maintained their expression of mesenchymal stem cell and skeletal stem cell markers (SCA-1 and PRRX1). Our results contribute to the optimization of MSC-based therapies. The sc3DSC method may be useful in studying cell-based therapies for the regeneration of other tissue types or disease models.

SUBMITTER: Yang C

PROVIDER: S-EPMC9561611 | biostudies-literature | 2022 Oct

REPOSITORIES: biostudies-literature

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Single-cell spatiotemporal analysis reveals cell fates and functions of transplanted mesenchymal stromal cells during bone repair.

Yang Chengyu C Li Zeshun Z Liu Yang Y Hou Runpeng R Lin Minmin M Fu Linhao L Wu Decheng D Liu Quanying Q Li Kai K Liu Chao C

Stem cell reports 20220922 10

Mesenchymal stromal cells (MSCs) transplantation could enhance bone repair. However, the cell fate of transplanted MSCs, in terms of their local distribution and spatial associations with other types of cells were poorly understood. Here, we developed a single-cell 3D spatial correlation (sc3DSC) method to track transplanted MSCs based on deep tissue microscopy of fluorescent nanoparticles (fNPs) and immunofluorescence of key proteins. Locally delivered fNP-labeled MSCs enhanced tibial defect re ...[more]

PMID: 36150383

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Project description:ObjectivesThe goal of this study was to validate a pathway-specific reporter gene that could be used to noninvasively image the oxidative status of progenitor cells.BackgroundIn cell therapy studies, reporter gene imaging plays a valuable role in the assessment of cell fate in living subjects. After myocardial injury, noxious stimuli in the host tissue confer oxidative stress to transplanted cells that may influence their survival and reparative function.MethodsRat mesenchymal stromal cells (MSCs) were studied for phenotypic evidence of increased oxidative stress under in vitro stress. On the basis of their up-regulation of the pro-oxidant enzyme p67(phox) subunit of nicotinamide adenine dinucleotide phosphate (NAD[P]H oxidase p67(phox)), an oxidative stress sensor was constructed, comprising the firefly luciferase (Fluc) reporter gene driven by the NAD(P)H p67(phox) promoter. MSCs cotransfected with NAD(P)H p67(phox)-Fluc and a cell viability reporter gene (cytomegalovirus-Renilla luciferase) were studied under in vitro and in vivo pro-oxidant conditions.ResultsAfter in vitro validation of the sensor during low-serum culture, transfected MSCs were transplanted into a rat model of myocardial ischemia/reperfusion (IR) and monitored by using bioluminescence imaging. Compared with sham controls (no IR), cardiac Fluc intensity was significantly higher in IR rats (3.5-fold at 6 h, 2.6-fold at 24 h, 5.4-fold at 48 h; p < 0.01), indicating increased cellular oxidative stress. This finding was corroborated by ex vivo luminometry after correcting for Renilla luciferase activity as a measure of viable MSC number (Fluc:Renilla luciferase ratio 0.011 ± 0.003 for sham vs. 0.026 ± 0.004 for IR at 48 h; p < 0.05). Furthermore, in IR animals that received MSCs preconditioned with an antioxidant agent (tempol), Fluc signal was strongly attenuated, substantiating the specificity of the oxidative stress sensor.ConclusionsPathway-specific reporter gene imaging allows assessment of changes in the oxidative status of MSCs after delivery to ischemic myocardium, providing a template to monitor key biological interactions between transplanted cells and their host environment in living subjects.

Project description:BackgroundIntravenous administration of mesenchymal stromal cells (MSCs) has an acknowledged competence of cardiac repair, despite a lack of systematic description of the underlying biological mechanisms. The lung, but not the heart, is the main trapped site for intravenously transplanted MSCs, which leaves a spatial gap between intravenously transplanted MSCs and the injured myocardium. How lung-trapped MSCs after intravenous transplantation rejuvenate the injured myocardium remains unknown.MethodsMSCs were isolated from human placenta tissue, and DF-MSCs or Gluc-MSCs were generated by transduced with firefly luciferase (Fluc)/enhanced green fluorescence protein (eGFP) or Gaussia luciferase (Gluc) lactadherin fusion protein. The therapeutic efficiency of intravenously transplanted MSCs was investigated in a murine model of doxorubicin (Dox)-induced cardiotoxicity. Trans-organ communication from the lung to the heart with the delivery of blood was investigated by testing the release of MSC-derived extracellular vesicles (MSC-EVs), and the potential miRNA inner MSC-EVs were screened out and verified. The potential therapeutic miRNA inner MSC-EVs were then upregulated or downregulated to assess the further therapeutic efficiency RESULTS: Dox-induced cardiotoxicity, characterized by cardiac atrophy, left ventricular dysfunction, and injured myocardium, was alleviated by consecutive doses of MSCs. These cardioprotective effects might be attributed to suppressing GRP78 triggering endoplasmic reticulum (ER) stress-induced apoptosis in cardiomyocytes. Our results confirmed that miR-181a-5p from MSCs-derived EVs (MSC-EVs) inhibited GRP78. Intravenous DF-MSCs were trapped in lung vasculature, secreted a certain number of EVs into serum, which could be confirmed by the detection of eGFP+ EVs. GLuc activity was increased in serum EVs from mice administrated with GLuc-MSCs. MiR-181a-5p, inhibiting GRP78 with high efficacy, was highly expressed in serum EVs and myocardium after injecting consecutive doses of MSCs into mice treated with Dox. Finally, upregulation or downregulation of miR-181a-5p levels in MSC-EVs enhanced or weakened therapeutic effects on Dox-induced cardiotoxicity through modulating ER stress-induced apoptosis.ConclusionsThis study identifies intravenously transplanted MSCs, as an endocrine reservoir, to secrete cardioprotective EVs into blood continuously and gradually to confer the trans-organ communication that relieves Dox-induced cardiotoxicity.

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Single-cell spatiotemporal analysis reveals cell fates and functions of transplanted mesenchymal stromal cells during bone repair.

Publications

Single-cell spatiotemporal analysis reveals cell fates and functions of transplanted mesenchymal stromal cells during bone repair.

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