Visualization and Identification of Bioorthogonally Labeled Exosome Proteins Following Systemic Administration in Mice

Exosomes transport biologically active cargo (e.g., proteins and microRNA) between cells, including many of the paracrine factors that mediate the beneficial effects associated with stem-cell therapy. Stem cell derived exosomes, in particular mesenchymal stem cells (MSCs), have been shown previously to largely replicate the therapeutic activity associated with the cells themselves, which suggests that exosomes may be a useful cell-free alternative for the treatment of cardiovascular disorders. However, the mechanisms that govern how exosomes home to damaged cells and tissues or the uptake and distribution of exosomal cargo are poorly characterized, because techniques for distinguishing between exosomal proteins and proteins in the targeted tissues are lacking. Here, we report the development of an in vivo model that enabled the visualization, tracking, and quantification of proteins from systemically administered MSC exosomes. The model uses bioorthogonal chemistry and cell-selective metabolic labeling to incorporate the non-canonical amino acid azidonorleucine (ANL) into the MSC proteome. ANL incorporation is facilitated via expression of a mutant (L274G) methionyl-tRNA-synthetase (MetRS∗) and subsequent incubation with ANL-supplemented media; after which ANL can be covalently linked to alkyne-conjugated reagents (e.g., dyes and resins) via click chemistry. Our results demonstrate that when the exosomes produced by ANL-treated, MetRS∗-expressing MSCs were systemically administered to mice, the ANL-labeled exosomal proteins could be accurately and reliably identified, isolated, and quantified from a variety of mouse organs, and that myocardial infarction (MI) both increased the abundance of exosomal proteins and redistributed a number of them from the membrane fraction of intact hearts to the cytosol of cells in infarcted hearts. Additionally, we found that Desmoglein-1c is enriched in MSC exosomes and taken up by ischemic myocardium. Collectively, our results indicate that this newly developed bioorthogonal system can provide crucial insights into exosome homing, as well as the uptake and biodistribution of exosomal proteins.


INTRODUCTION
The therapeutic benefits of stem cells are mediated primarily via the secretion of paracrine factors (Bagno et al., 2018), and recent evidence suggests that the exosomes produced by mesenchymal stem cells (MSCs) largely replicate the improvements associated with administration of the cells themselves in large-mammal models of myocardial injury (Charles et al., 2020). Thus, while exosomes could be a useful cell-free alternative to MSC therapy, little is known of how exosomes are directed toward injured tissue, the distribution of the exosomal cargo in target organs and cells, or the physiological mechanisms that are induced by exosome uptake. This information is crucial for identifying the appropriate dose, timing, and route of administration for exosome therapy but remains difficult to decipher, as techniques for tracking exosomes and the exosome cargo are limited. Radiolabeled exosomes have been used to determine the half-life of exosomes in the circulation and to identify some of the major sites of exosome uptake (Morishita et al., 2015), but they cannot track the presence of exosome proteins in target organs and are dependent on half-life of the radiolabel. Thus, methods for accurately and reliably identifying both the source and targets of the exosomal cargo in living organisms will dramatically advance exosome research (Mathieu et al., 2019).
Cell-selective metabolic labeling generates proteins containing non-canonical amino acids with functional groups that can be linked to affinity reagents or fluorescent dyes for subsequent identification, isolation, and imaging. It was previously shown that the mutant E. coli methionyl-tRNA synthetase (MetRS L274G or MetRS * ) selectively appends the azide-bearing non-canonical amino acid azidonorleucine (ANL) to tRNA. Thus, when cells that express MetRS * are incubated with ANL, they produce proteins that contain ANL in lieu of endogenous methionine (Ngo et al., 2009) and can be covalently linked to alkyne-tagged reagents (e.g., dyes and resins) via click chemical reactions, such as copper-catalyzed azide-alkyne cycloaddition. These noncanonical amino acids are not produced in vivo and as such are excluded during normal protein synthesis.
For this study, we used non-canonical amino-acid labeling to develop an in vivo model that enables us to visualize, track, and measure the biodistribution of proteins from systemically administered MSC exosomes. MSCs were transfected with a lentiviral vector coding for MetRS * and incubated with ANLsupplemented media; then, exosomes were isolated from the MSC-conditioned media and intravenously administered to mice. Our results demonstrate that the ANL-labeled exosomal proteins can be accurately and reliably identified, isolated, and quantified from a variety of mouse organs, and that surgically induced myocardial infarction (MI) both increased the abundance of labeled exosomal proteins and redistributed the localization from the membrane to the cytosol within organs following systemic administration.

MSC Culture and Characterization
Mouse (C57BL/6) bone marrow MSCs were purchased from Cyagen Biosciences (Sunnyvale, CA, United States, Catalog Number: MUBMX-01001), cultured in Dulbecco modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS), and passaged 5-8 times before use. MSC identity was a confirmed via flow-cytometry analyses of MSC surface marker expression and assessments of osteogenic and adipose differentiation potential. Differentiation assays were performed by following the manufacturer's protocols (Cyagen Biosciences, United States). Briefly, the cells were cultured on 0.1% gelatincoated six-well plates (2 × 10 4 cells/cm 2 ) in complete medium until confluent and then treated with osteogenic differentiation medium for 3 weeks or with adipocyte induction medium and maintenance medium for 2-3 weeks; osteogenic and adipose differentiation were analyzed by Alizarin staining and by Oil Red O staining (for lipid droplets), respectively.

qRT-PCR
Quantitative reverse transcription PCR (qRT-PCR) was performed as previously described (Tang et al., 2009). Briefly, total MSC RNA was reverse transcribed with random hexamer primer, and 25 ng of the reverse-transcribed product was used for RT-PCR; results with the random primer were consistent with those obtained previously with a primer common to the 3 untranslated region (UTR) of Dsg1-a,b, and -c. Values were normalized to equivalent assessments of glyceraldehyde phosphate dehydrogenase (GAPDH) mRNA abundance.

Western Blotting
For protein extraction, 1 × 10 7 cells or 100 mg of frozen tissue were homogenized in 1 mL RIPA lysis buffer containing protease inhibitors (Sigma, 4693132001) and phosphatase inhibitors (Sigma,4906837001). Samples were incubated with agitation for 30 min at 4 • C and centrifuged at 13,000 rpm for 10 min at 4 • C; then, the protein concentration in the supernatant was determined via bicinchoninic acid (BCA) assay (Pierce). For immunoblotting, proteins in the supernatant were denatured by heating at 95 • C for 10 min, separated by SDS-PAGE, and then transferred onto a polyvinylidene difluoride (PVDF) membrane (Bio-Rad). The membrane was incubated in 5% nonfat milk blocking buffer (tris-buffered saline [TBS]) for 1 h, incubated with primary antibody in TBS-containing 3% bovine serum albumin (BSA) overnight at 4 • C, washed three times with TBS (0.5% Tween 20), incubated with secondary antibody, washed with TBS Tween 20, and then developed with Enhanced Chemiluminescence Detection Reagents (ECL, Thermo Fisher). Protein signals were imaged with a Bio-Rad ChemiDoc System.

Lentivirus Transduction and ANL Labeling of Nascent Cellular Proteins
For lentivirus transduction, MSCs were seeded at ∼80% confluence, and the lentivirus (MOI = 4) was applied with polybrene (8 µg/mL); transduction efficiency was evaluated 72 h later by monitoring mCherry fluorescence with a fluorescence microscope or via fluorescence-activated cell-sorting (FACS); control assessments were conducted with MSCs that had been transduced with Lenti.CAG.H2B-Dendra2.W. For ANL labeling, cells were washed twice in PBS and then suspended in methionine-, glutamine-, and cystine-depleted DMEM (Gibco, Cat. #21013024) containing 1 mM ANL (IRIS Biotech, Cat. #159610-92-1); the DMEM had been made complete with glutamine and cystine and supplemented with exosome-free FBS (Gibco, Cat. #A270801). Cells were cultured for 72 h before use in subsequent experiments.

Bio-Orthogonal Non-canonical Amino Acid Tagging (BONCAT)
MetRS * -transduced MSCs were incubated in 1 mM ANL for 24 h and lysed in RIPA buffer supplemented with protease inhibitors (Sigma, United States); then, total protein was collected, and the Click-iT reaction was performed with a Click-iT Protein Reaction Buffer Kit (Invitrogen, Waltham, MA, United States) as directed by the manufacturer's instructions. Briefly, up to 200 µg of ANL-labeled protein was reacted with 100 µL of Click-iT reaction buffer containing alkyne-Cy7 and copper-sulfate catalyst in a rotator for 20 min. Methanol and chloroform were added to remove residual reaction components and precipitate the proteins; then, the precipitated proteins were solubilized in Laemmli buffer and loaded for gel electrophoresis. Gels were immediately imaged with a ChemiDoc MP Imaging System (Bio-Rad, California, United States) under both stain-free mode and Cy7 mode.

Fluorescent Non-canonical Amino-Acid Tagging
For fluorescent non-canonical amino-acid tagging (FUNCAT) of MetRS * -transduced MSCs in vitro, MSCs were cultured in 1 mM ANL (Jena Bioscience, Jena, Germany) for 24 h, gently rinsed with PBS, fixed in 4% formaldehyde/PBS and permeabilized with 0.5% Triton X-100/PBS; then, the Click-iT reaction was performed with Click-iT reaction cocktails containing Alexa Fluor 488 alkyne (Click-iT Alexa Fluor 488 Protein Synthesis HCS Assay kit, Life Technologies, Waltham, MA, United States) as directed by the manufacturer's protocol. Cells were stained with Hoechst 33342 prior to imaging. For FUNCAT of MetRS * exosomal proteins in vivo, the general methodology was adapted from previous studies (Ngo et al., 2013). Briefly, mice were euthanized 2 h after exosome administration; then, the hearts were explanted, perfused, fixed by incubation in 4% paraformaldehyde at room temperature for 4 h, cryoprotected in 30% sucrose/PBS solution overnight, frozen in OCT compound, cut into 5-µM-thick sections, and stored at −20 • C. Frozen sections were incubated at room temperature for 30 min, washed once with PBS, permeabilized in 0.25% Triton/PBS for 35 min, washed in PBS (3 washes, 5 min each), blocked in commercial blocking buffer (Thermo Scientific, TA-125-PBQ) for 7 min, and washed in PBS again (3 washes, 5 min each); then, the ANL-labeled proteins were conjugated to biotin-alkyne by incubating the sections at 4 • C overnight in a 1:10,000 dilutio of biotin-alkyne in 2× Catalyst solution from the Click-iT Enrichment kit (Invitrogen, Cat. #C10416). Sections were thoroughly washed in PBS (5 washes, 5 min each) to remove residual biotin, sequentially incubated with primary biotin antibodies (1:1000 dilution) at 4 • C overnight and with secondary antibodies at 4 • C overnight, washed PBS (three times), incubated with Sudan Black for 30 min, and then washed again in PBS (three times).

Isolation and Characterization of MSC Exosomes
MetRS * MSCs were plated at ∼50% confluence in complete DMEM for 24 h, after which they were cultured in methionine-depleted DMEM supplemented with 1 mM ANL for 72 h. Conditioned media was collected from the MSCs, spun at 1,000 G to remove cell debris, passed through a 0.22-micron filter, and ultracentrifuged at 120,000 G for 2 h; then, the supernatant was removed and replaced with cold PBS, and the exosomes were ultracentrifuged at 120,000 G for 2 h. Exosome pellets were resuspended in 10 mL of cold PBS and stored at −80 • C until use in subsequent experiments. The size and quantity of freshly isolated exosomes was estimated via nanoparticletracking analyses (NanoSight), and morphological assessments were conducted via electron microscopy as previously described (Cheng et al., 2019).

Mouse MI Model and Intravenous Exosome Delivery
All animal experiments in this report were approved by the Institutional Animal Care and Use Committee (IACUC) of the University of Alabama at Birmingham and performed in compliance with the National Institutes of Health (NIH) publication Guide for the Care and Use of Laboratory Animals. Twelve-to 16-week-old C57BL/6J mice (Jackson Laboratory, Bar Harbor, ME, United States) were used for all experiments unless otherwise specified. For surgically induced MI, mice were intubated and connected to a ventilator, and anesthesia was initiated and maintained with inhaled 2% isoflurane USP (Fluriso TM , VetOne). The heart was exposed via left thoracotomy, and MI was induced by permanently ligating the left-anterior descending (LAD) coronary artery, as previously described (Qin et al., 2006); sham-operated animals underwent the identical surgical procedure, except LAD artery ligation was omitted. After chest closure, buprenorphine hydrochloride (0.1 mg/kg, Buprenex, Reckitt Benckiser Pharmaceuticals Inc.) and carprofen (5 mg/kg, Rimadyl, Zoetis) were administered via intraperitoneal injection every 12 h for 3 days. Exosomes (10 × 10 9 in 200 µL PBS) were delivered via tail-vein injection 24 h after MI or Sham surgery, and mice were euthanized via CO 2 inhalation.

Isolation of ANL-Labeled Proteins From
Whole Organs (Click-Catalyzed Alkyne-Agarose Capture) Animals were euthanized via CO 2 inhalation and perfusion with 10 mL cold PBS; then, the heart, brain, lung, kidney, liver, and spleen were isolated, cut into ∼20-mg sections, rinsed twice in PBS, and flash frozen with liquid nitrogen. Membrane and cytosolic total protein fractions were isolated with a Mem-PER Plus Membrane Protein Extraction kit (Thermo Scientific, Cat. #89842), precipitated via methanolchloroform extraction, and stored at −80 • C. For ANL-labeling and MetRS * protein capture, fractions were resolubilized in Click-iT buffer solution and processed as directed by the Click-iT Protein Enrichment Kit protocol (Invitrogen, Cat. #C10416). Captured peptides were stored at −80 • C until identification via liquid chromatography mass spectrometry (LC-MS). To collect protein samples from non-administered exosomes, ∼1 × 10 9 exosomes were suspended in 100 µL PBS and prepared in parallel with organ samples for LC-MS identification.

Sample Preparation and Data Acquisition by Liquid Chromatography Mass Spectrometry
Dried peptides were reconstituted in 16 µL of 0.1% formic acid (FA); then, 8 µL of each sample was injected onto a 1,260 Infinity nano high-performance liquid chromatography (nHPLC) stack (Agilent Technologies, Santa Clara, CA, United States), and peptides were separated on a 75-micron (internal diameter) by 15-cm pulled tip C-18 column (Jupiter C-18 300 Å, five micron, Phenomenex) that was attached to a Thermo Orbitrap Velos Pro hybrid mass spectrometer equipped with a nanoelectrospray source (Thermo Fisher Scientific, Waltham, MA, United States). All data were collected in CID mode, and the nHPLC was configured with binary mobile phases consisting of solvent A (0.1%FA in ddH2O) and solvent B (0.1% FA in 15% ddH 2 O / 85% ACN) programmed as follows; 10 min at 2% solvent B; 90 min at 5-40% solvent B; 5 min at 70% solvent B; and 10 min at 0% solvent B. After each parent ion scan (300-1,200 m/z; 60 k resolution), fragmentation data (MS2) was collected for the 15 most-intense ions. For data dependent scans, charge state screening and dynamic exclusion were enabled with a repeat count of two, repeat duration of 30 s, and exclusion duration of 90 s.

MS Data Conversion and Searches
XCalibur RAW files were collected in profile mode, centroided, and converted to MzXML with ReAdWv.3.5.1; then, the mgf files were created by using MzXML2Search (included in TPP v. 3.5) for all scans. Data was searched via SEQUEST, which was set for two maximum missed cleavages, a precursor mass window of 20 ppm, trypsin digestion, and variable modifications C and M at 57.0293 and 15.9949, respectively. Searches were performed with a specific subset of the UniRef100 database.

Peptide Filtering, Grouping, and Quantification
The lists of peptide identifications (IDs) generated via SEQUEST (Thermo Fisher Scientific, Waltham, MA, United States) were filtered with Scaffold (Protein Sciences, Portland, Oregon, United States). Scaffold generates and retains only highconfidence IDs while producing normalized spectral counts across all samples, and the spectral counts can subsequently be used for relative quantification. Only peptides with charge states ≥2+, minimum lengths of six amino acids, and nonzero quantities for all six mass tags were analyzed. For large datasets, Scaffold incorporates the two most common methods for statistical validation: false discovery rate (FDR), and protein probability; FDR was set at <1% cutoff, individual peptide probabilities were ≥0.8, protein probabilities were ≥0.99, and at least two peptides were assigned per protein. Relative quantification was performed via spectral counting and spectral count abundances were normalized between samples.

Bioinformatics and Statistics Analysis
For protein abundance data, the Pearson coefficient was calculated to evaluate correlations in protein expression between samples. Hierarchical clustering of samples and protein profiles was based on the complete linkage method, and differences in protein expression between the MI and Sham groups was evaluated with the Mann Whitney U-test. Differentially expressed proteins (DEPs) were defined as those with nominal p-value < 5% and fold-change >1.5, and were visualized via heatmaps. For proteins that were detected in ≥60% of one group and absent in the other group, we used the method developed by Kojima et al. (2012), with a more stringent cutoff (>60% in only one group); proteins meeting this criteria were defined as "all-or-nothing" hits and included in the list of DEPs.

MetRS * Expression in MSCs Facilitates ANL-Labeling of Nascent MSC Proteins
Murine MSCs were obtained from a commercial vendor, and the characteristic pattern of MSC marker expression (i.e., positive for Sca-1 and CD44; negative for CD11b, CD45, and CD34) was verified via flow-cytometry (Supplementary Figure 1A). Phase-contrast microscopy confirmed that the cells were uniformly spindle-shaped, and robust multipotency was verified by differentiating the cells into adipocytes and osteocytes (Supplementary Figures 1B,C). To facilitate non-canonical amino acid tagging, MSCs were transfected with our newly generated MetRS * lentiviral vector (Lenti-CAG-MetRS L274G -mCherry). The vector also coded for mCherry, which enabled stably transfected cells ( MetRS * MSCs) to be selected via flow cytometry for mCherry fluorescence, and proteins produced by the MetRS * MSCs were labeled with ANL by incubating the selected cells in 1 µM ANL-supplemented media. Control assessments were conducted with MSCs that had been transfected with an equivalent vector that lacked the MetRS L274G -mCherry cassette ( CTL MSCs).
Widespread mCherry expression was observed after just a single transfection (Figure 1A), and there were no apparent morphological differences between MetRS * MSCs and CTL MSCs after 72 h of incubation in the ANL-supplemented media. To confirm that ANL was incorporated into the MetRS * MSC proteome, BONCAT was conducted with lysates from ANLtreated MetRS * MSCs and CTL MSCs: proteins in the lysate were separated on a gel and covalently stained with alkyne-conjugated Cy7. Overall protein levels in the two MSC populations were equivalent; however, the alkyne-Cy7 stain was only observed in proteins from MetRS * MSC lysates, which spanned a wide spectrum of masses ( Figure 1B). The results from our BONCAT assessments were also corroborated in intact cells via FUNCAT: the cells were permeabilized, and then click reactions were conducted with a cocktail containing Alexa Fluor 488 alkyne. The fluorescent signal (i.e., ANL-incorporated protein) was highly abundant in the cytosol of MetRS * MSCs but completely absent in CTL MSC cytosol ( Figure 1C).

ANL-Alkyne Click Reactions Detect ANL-Labeled Proteins From MetRS * MSC Exosomes With High Sensitivity and Specificity
Assessments via nanoparticle-tracking analysis (Figure 2A) and transmission electron microscopy ( Figure 2B) confirmed that treatment with ANL-supplemented media (1 µM for 72 h) did not substantially alter the production, size, and ultrastructure of exosomes produced by MetRS * MSCs. Furthermore, when exosomes were collected from MetRS * MSCs (i.e., MetRS * MSC-Exos) and CTL MSCs ( CTL MSC-Exos) that had been grown in media supplemented with or without ANL, BONCAT assessments of their protein cargo indicated that the total amount of protein in all four groups was similar, but alkyne-Cy7 fluorescence was only detected for proteins in exosomes from ANL-treated MetRS * MSC-Exos ( Figure 2C). Thus, ANL labeling and subsequent detection was both sensitive and specific for proteins in MetRS * MSC-Exos.

ANL-Alkyne Click Reactions Detect ANL-Labeled Proteins From Systemically Administered MetRS * MSC-Exos in the Organs of Mice
To confirm that our method for detecting ANL-labeled MetRS * MSC-Exo proteins could effectively monitor the trafficking of proteins from systemically administered MetRS * MSC-Exos in vivo, exosomes were isolated from ANL-or vehicletreated MetRS * MSCs and intravenously delivered into mice (20 µg/mouse); 2 h later, cardiac tissues were harvested, sectioned, and analyzed via FUNCAT. The fluorescent signal was strongly observed in both the vasculature and cardiomyocytes of mice that received the ANL-treated MetRS * MSC-Exos but was completely absent in sections from mice that were administered vehicle-treated MetRS * MSC-Exos (Figure 3). Having confirmed the fidelity of click-catalyzed ANL-staining in our in vivo model, we investigated whether ANL-labeled exosome proteins could be isolated from the organs of mice after MetRS * MSC-Exo administration. Proteins were precipitated from whole mouse organs (heart, kidney, spleen, lung, liver, and brain), and then MetRS * MSC-Exo proteins (i.e., proteins containing the ANL-label) were captured from the precipitate with an agarose resin containing alkyne functional groups that, when catalyzed via click reaction, formed covalent links with the ANL label. Because the MetRS * MSC-Exo proteins comprised only a very small fraction of the total amount of protein in each organ, we confirmed the specificity and accuracy of click-catalyzed alkyne-agarose capture by comparing the data for each individual MetRS * MSC-Exo protein to data for the same protein in the corresponding organs from MetRS * MSC-Exo-treated animals when the click catalysis reaction was omitted and from animals that were not treated with MetRS * MSC-Exos.
LC-MS analysis identified 205 MetRS * MSC-Exo proteins that were isolated from the hearts of MetRS * MSC-Exo-treated mice exclusively via click-catalyzed alkyne-agarose capture, compared to 29 proteins that were captured only when the click reaction was omitted, and 48 that were isolated both with and without click catalysis ( Figure 4A); the abundance of each individual MetRS * MSC-Exo protein varied widely among organs ( Figure 4B). Notably, most (but not all) of the exosome-marker proteins that were present in MetRS * MSC-Exos before administration (Supplementary Table 2) were also isolated from at least one of the organs of MetRS * MSC-Exotreated mice, but they were not equally distributed across all organs ( Figure 4C).

MI Increases Cardiac Uptake and Alters the Subcellular Distribution of ANL-Labeled MetRS * MSC-Exo Proteins
To determine whether tissue injury changes the biodistribution of MSC exosomes, ANL-labeled MetRS * MSC-Exos were administered to mice via tail-vein injection 24 h after surgically induced MI (the MI group) or sham surgery (the Sham group). Cardiac tissue was collected 12 h after MetRS * MSC-Exo administration, and MetRS * MSC-Exo proteins were isolated via click-catalyzed alkyne agarose capture. The number of MetRS * MSC-Exo proteins was dramatically greater in hearts from MI than from Sham animals (Figure 5A), and of those that were present in the hearts of both groups, nearly all were more abundant in MI hearts ( Figure 5B). These observations are consistent with the MI-induced uptake of MSC exosomes in cardiac cells. Furthermore, when the membrane and cytosol fractions were evaluated separately, a number of MetRS * MSC-Exo proteins that were associated with the membranes of cells in the hearts of Sham animals were found primarily in the cytosol fraction of MI hearts (Figure 6).

Desmoglein-1c (DSG-1c) Is Expressed in MSCs, Enriched in MSC Exosomes, and Taken up by Ischemic Myocardium
The results from our click-catalyzed alkyne-agarose capture assessments indicated that desmoglein 1 (DSG-1) was more  P50462  P21550  Q60932  Q6P8J7  A1BN54  Q8K0E8  Q8VHX6  P43274  P43277  Q9D0M3  P38647  P19536  Q01339  P11276  Q91VR2  Q62234  Q91VD9  Q9JI91  P09528  P05064  P09411  P50752  Q9JKS4  P48787  P35486  Q9CRB9  Q99LC5  Q9D855  P51667  P20029  Q52KI8  P07724  P52503  P31001  P17751  Q8BWT1  P04247  abundant in the heart than in other organs of both MI and Sham animals, and that cardiac DSG-1 levels increased in response to MI; however, to our knowledge, DSG-1 expression had not been previously reported in either MSCs or cardiomyocytes. Thus, we evaluated DSG-1 expression in MSCs via RT-PCR and in both MSCs and MSC exosomes via Western blot: our results confirmed that the DSG-1c isoform was expressed in MSCs ( Figure 7A) and enriched in MSC exosomes ( Figure 7B). To determine whether we could identify MSC-derived DSG-1 in the infarcted myocardium of MetRS * MSC-Exo-treated mice via FUNCAT, myocardial sections were click-stained with biotinalkyne, sequentially stained with primary biotin antibodies and fluorescent secondary antibodies, and then counterstained for the presence of DSG-1 and troponin-T (TnT). When visualized via fluorescence, a number of TnT-expressing cells were positive for both the biotin-alkyne and DSG-1 labels (Figure 7C), which confirmed that the cardiomyocytes had taken up MetRS * MSC-Exo-derived DSG-1.

DISCUSSION
Because the benefits of stem-cell therapy for myocardial repair are primarily mediated via paracrine mechanisms, the factors produced by these cells, many of which are transported by exosomes, could serve as a potent, cell-free alternative for the treatment of patients with cardiovascular disease (Liu et al., 2018). Results from a number of preclinical studies indicate that MSCs and the exosomes produced by them are equally beneficial (Xiao et al., 2018); however, the mechanisms that direct the homing of exosomes to injured tissues, as well as the uptake and biodistribution of their cargo, remain largely uncharacterized, because techniques for distinguishing between exosomal proteins and proteins from the targeted tissues are either unavailable or inefficient. Here, we demonstrate that when MSCs are transfected with a vector coding for MetRS * and cultured in ANL-supplemented medium, the proteins produced by the transfected MSCs are selectively labeled with ANL, which can be covalently linked (via click chemistry) to alkyne-tagged reagents for subsequent visualization, identification, and isolation. Thus, when exosomes produced by the MetRS * MSCs were systemically administered to mice, the protein components of their cargo could be accurately and reliably tracked, visualized, identified, and quantified in tissues from a variety of organs, including both intact and infarcted hearts. Notably, as bioorthogonal chemistry can occur in living systems without disrupting native biochemical processes (Prescher et al., 2004;Prescher and Bertozzi, 2005;Sletten and Bertozzi, 2011;Ngo et al., 2013;Han et al., 2020a), this approach can be applied to other cell populations and even extended to wholly in vivo investigations by generating MetRS *transgenic mice (Alvarez-Castelao et al., 2017;Han et al., 2020a).
The overall pattern of MetRS * MSC-Exo protein distribution differed substantially between organs; thus, the mechanisms that govern exosome homing and the processing of exosomal proteins after uptake are likely cell-and tissue-type specific. Furthermore, our observation that a number of canonical exosome marker proteins (e.g., TSG101, Syntenin-1, ALIX, CD9, and CD81) were not present in any of the organs analyzed suggests that they are not suitable for tracking exosome uptake; whether their absence is attributable to rapid degradation in the targeted tissues or another process will be investigated in future studies. We also observed that surgically induced MI led to an increase in the abundance of exosomal proteins in cardiac tissue, which is consistent with the therapeutic mechanism of systemically delivered exosomes (Xiong et al., 2021) but has not been explicitly demonstrated previously. MI was also associated with the redistribution of numerous exosomal proteins from the membrane fraction in intact hearts to the cytosol of cells in infarcted hearts. This observation was not anticipated but likely evolves from increases in membrane permeability, because the loss of cardiomyocyte membrane integrity is a hallmark of MI, and some proposed treatment strategies target membrane dysfunction (Han et al., 2020b). Our results also indicated that the desmosome protein DSG-1c was expressed in MSCs, enriched in MSC exosomes, and taken up by cardiomyocytes in response to MI and MetRS * MSC-Exo administration. DSG-1 expression has not been previously reported in cardiomyocytes; however, cardiomyocytes express DSG-2, which is critical for cardiac integrity and electric conduction. Notably, human DSG-2 mutations are associated with cardiac arrhythmia (Schinner et al., 2020), and genetic deletion of DSG-2 leads to arrhythmogenic cardiomyopathy and heart failure in mice (Pilichou et al., 2009); thus, whether the exosome-mediated transfer of DSG-1 to cardiomyocytes contributes to the benefit of MSC therapy warrants continued investigation, particularly since MSC exosomes appear to reduce the arrhythmogenicity of human cardiomyocytes (Sattayaprasert et al., 2020).
A limitation of this study is that N-terminal processing by methionyl aminopeptidases results in cleavage of the first residue during protein maturation, preventing identification of these proteins. Methionine is the first amino acid translated during protein synthesis in eukaryotes and the average methionine content of mammalian proteins is ∼2.13% (Dieterich et al., 2006). Nevertheless, N-terminal posttranslational processing and acetylation occurs in ∼30% of all mammalian proteins (Meinnel et al., 2005) and only ∼1.02% of the open reading frames of the human database (predicted to be <25 residues and incomplete sequences) do not contain a single methionine residue (Dieterich et al., 2006).
In conclusion, when exosomes were collected from MetRS * MSCs that had been incubated with ANL and then systemically administered to mice, the ANL-labeled exosomal proteins could be accurately and reliably identified, isolated, and quantified from a variety of mouse organs, including both intact and infarcted hearts, via ANLalkyne click chemistry. Thus, this bioorthogonal technique can provide crucial insights into the mechanisms that govern exosome homing and the uptake of exosomal proteins.

DATA AVAILABILITY STATEMENT
The datasets presented in this study can be found in online repositories. The name of the repository and accession numbers can be found below: European Bioinformatics Institute (EBI) PRoteomics IDEntifications Database (PRIDE) Archive, https://www.ebi.ac.uk/pride/archive/, PXD024482, PXD024483, PXD024484, and PXD024485.

ETHICS STATEMENT
The animal study was reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) of the University of Alabama at Birmingham.

AUTHOR CONTRIBUTIONS
EZ and GQ conceptualized the study, interpreted the data, and wrote the manuscript. EZ, YL, CH, CF, LW, WC, YD, DH, SX, YW, and JM performed the experiments and analyzed the data. BA and JM made intellectual contributions and assisted in data interpretation. All authors contributed to the article and approved the submitted version.

FUNDING
This work was supported by the National Institute of Health (R01 Grant Nos. HL113541, HL130052, HL131110, and HL138990 to GQ), American Diabetes Association (Grant No. 1-15-BS-148 to GQ), and American Heart Association (Grant Nos. 19TPA34910227 to GQ, 18POST34070088 to SX, and 18PRE34080358 to EZ).