Lineage Mapping of Extracellular Vesicles: What Cells Do They Come from And Where Do They Go?

Significance: Release of extracellular vesicles (EVs) by various cell types has been shown to mediate the delivery of biologically active payloads from donor cells to recipient cells; however, it remains unclear what cell types these EVs come from. With a focus on fluorescent reporters to monitor the release of EVs, especially those under the control of cell type-specific promoters, we address the translational relevance of genetic tools in cultured cells, normal tissues, and in models of development, injury, cancer, and wound healing. Recent Advances: It is well established that EVs are released by many cell types in the body via fusion and release processes at the plasma membrane. Since there remains debate about what fraction of EVs are released through regulated endosomal trafficking pathways versus nonspecific mechanisms, the development and validation of novel molecular tools are important to address the cellular source of EVs. Critical Issues: There is a need to develop and characterize new cell type-specific reporter mouse models that build upon the examples detailed here to identify the cellular source of EVs with genetic approaches being useful in addressing these critical limitations. Future Directions: Advances in reporter systems will drive a better understanding of EV subsets to identify compartment-specific EV localization to guide the development of more translationally relevant models for the wound healing field.

Xanthogranulomatous pyelonephritis in a patient with polycystic kidney disease without underlying risk factors: a case report.

Xanthogranulomatous pyelonephritis (XGP) is an extremely rare, chronic granulomatous inflammatory condition thought to arise secondary to a combination of obstruction, recurrent bacterial infection and an incomplete immune response although the etiology of XGP is more complex. We would like to report a case of XGP occurring in a patient with polycystic kidney disease (PCKD), which has not been previously documented in etiology. A 29-year-old woman presented to our hospital with right upper quadrant pain for 5 days. She had experienced a low-grade fever, generalized weakness, and myalgia throughout her body for 2 weeks. She had no history of renal stones or recurrent UTIs. Contrast-enhanced CT revealed a well-enhancing large septated cystic mass in the right kidney and numerous cysts in the liver and both kidneys. Open right radical nephrectomy was performed due to the suspicion of renal cell carcinoma, as there was no response to antibiotics over 7 days. Gross specimen demonstrated architectural distortion due to xanthomatous nodules and a dilated pelvico-calyceal system filled with pus and blood. Microscopic examination revealed infiltration of neutrophils and lipid-laden macrophages. The patient is currently being followed up in the outpatient clinic without recurrence of XGP. This is the first reported case of XGP in a patient with underlying PCKD. Physicians should consider PCKD as a potential underlying cause of XGP.

Spontaneous Remission of Minimal Change Disease in a Colon Cancer Patient: A Case Report.

Introduction: Minimal change disease (MCD) is most often primary but may occur secondary to other systemic diseases such as malignancy. In secondary MCD, spontaneous remission of nephrotic syndrome after the treatment of related diseases without steroid therapy is rare. Case Presentation: A 78-year-old man visited the outpatient clinic with foamy urine and generalized edema that had persisted for 2 months. The patient had nephrotic syndrome. Before a kidney biopsy, he underwent several tests to determine the secondary cause of the nephrotic syndrome. The serum CEA was slightly elevated, and colon cancer was detected in the sigmoid colon. MCD was diagnosed from a kidney biopsy. He immediately underwent surgery for colon cancer. Complete remission of the MCD was achieved within 2 weeks after surgery. Conclusion: Here, we report a rare case of a patient with secondary MCD who successfully achieved spontaneous remission after colon cancer surgery.

Defining the activity of pro-reparative extracellular vesicles in wound healing based on miRNA payloads and cell type-specific lineage mapping.

Small extracellular vesicles (EVs) are released by cells and deliver biologically active payloads to coordinate the response of multiple cell types in cutaneous wound healing. Here we used a cutaneous injury model as a donor of pro-reparative EVs to treat recipient diabetic obese mice, a model of impaired wound healing. We established a functional screen for microRNAs (miRNAs) that increased the pro-reparative activity of EVs and identified a down-regulation of miR-425-5p in EVs in vivo and in vitro associated with the regulation of adiponectin. We tested a cell type-specific reporter of a tetraspanin CD9 fusion with GFP to lineage map the release of EVs from macrophages in the wound bed, based on the expression of miR-425-5p in macrophage-derived EVs and the abundance of macrophages in EV donor sites. Analysis of different promoters demonstrated that EV release under the control of a macrophage-specific promoter was most abundant and that these EVs were internalized by dermal fibroblasts. These findings suggested that pro-reparative EVs deliver miRNAs, such as miR-425-5p, that stimulate the expression of adiponectin that has insulin-sensitizing properties. We propose that EVs promote intercellular signaling between cell layers in the skin to resolve inflammation, induce proliferation of basal keratinocytes, and accelerate wound closure.

A heterogenous population of extracellular vesicles mobilize to the alveoli postinjury.

BACKGROUND: Acute lung injury and subsequent resolution following severe injury are coordinated by a complex lung microenvironment that includes extracellular vesicles (EVs). We hypothesized that there is a heterogenous population of EVs recruited to the alveoli postinjury and that we could identify specific immune-relevant mediators expressed on bronchoalveolar lavage (BAL) EVs as candidate biomarkers of injury and injury resolution. METHODS: Mice underwent 30% TBSA burn injury and BAL fluid was collected 4 hours postinjury and compared with sham. Extracellular vesicles were purified and single vesicle flow cytometry (vFC) was performed using fluorescent antibodies to quantify the expression of specific cell surface markers on individual EVs. Next, we evaluated human BAL specimens from injured patients to establish translational relevance of the mouse vFC analysis. Human BAL was collected from intubated patients following trauma or burn injury, EVs were purified, then subjected to vFC analysis. RESULTS: A diverse population of EVs were mobilized to the alveoli after burn injury in mice. Quantitative BAL vFC identified significant increases in macrophage-derived CD44+ EVs (preinjury, 10.8% vs. postinjury, 13%; p < 0.05) and decreases in IL-6 receptor alpha (CD126) EVs (preinjury, 19.3% vs. postinjury, 9.3%, p < 0.05). Bronchoalveolar lavage from injured patients also contained a heterogeneous population of EVs derived from myeloid cells, endothelium, and epithelium sources, with CD44+ EVs being highly detected. CONCLUSION: Injury causes mobilization of a heterogeneous population of EVs to the alveoli in both animal models and injured patients. Defining EV release after injury will be critical in identifying diagnostic and therapeutic targets to limit postinjury acute lung injury.

IgA nephropathy with minimal change disease associated with primary Sjögren's syndrome: A case report.

RATIONALE: Only 1 case of IgA nephropathy (IgAN) with minimal change disease (MCD) associated with primary Sjögren's syndrome (SS) has been reported. We additionally describe IgAN with MCD associated with primary SS. PATIENT CONCERNS: A 80-year-old woman visited our hospital complaining of generalized edema that had started 4 weeks prior. She reported a sense of thirst and dry eye for the last 5 years. DIAGNOSES: Her initial laboratory findings were compatible with nephrotic syndrome; both the antinuclear antibody (1:80) and anti-SS-A (Ro) antibody (200 U/mL) tests were positive. A salivary gland scan revealed markedly decreased uptake for both the parotid and submandibular glands. The Schirmer test was positive. The random urine protein/creatinine ratio was 10 mg/mg. Renal biopsy was compatible with IgAN with superimposed MCD. INTERVENTIONS: Furosemide was intravenously administered with intermittent albumin infusion for her edema control. She was started on prednisone 40mg daily for 6 weeks, which was tapered to 5 mg for another 6 months after starting prednisolone. OUTCOMES: Over the next 6 months, her edema improved and the proteinuria decreased significantly. LESSONS: Physician should suspect IgA with MCD when patient with SS clinically showed nephrotic syndrome, and perform renal biopsy for pathologically diagnosis and appropriate treatment.

Melanin Treatment Effect of Vacuoles-Zinc Oxide Nanoparticles Combined with Ascorbic Acid.

Currently, ascorbic acid (AA) is widely used as a skin whitening material, but, AA, an unstable hydrophilic molecule, cannot penetrate the skin easily, due to the hydrophobic character of the stratum corneum. Therefore, we conjugated AA with hydrated zinc oxide-an inorganic matrix with positive surface charge, to improve the stability of AA. The metal-conjugated-ascorbic acid (ZnAA) was then combined with yeast vacuole through the vacuolar membrane proteins that relate to metal transportation to create an enhanced vacuole that contained ZnAA. The characteristics of vacuole with ZnAA (ZnAA_Vac) were next examined by various tests that included X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FT-IR), Field emission scanning electron microscopy (FE-SEM), and energy-dispersive X-ray (EDX) analysis. Furthermore, the ability of ZnAA_Vac to degrade melanin was confirmed in both melanoma cell line B16F10, and the artificial human skin MelanoDerm. The results showed that ZnAA_Vac possessed a higher depigmenting effect than the wild-type vacuole or ascorbic acid by reducing 75% of melanin color. Interestingly, ZnAA_Vac was found to be harmless, and did not cause any cytotoxicity to the cells. Overall, ZnAA_Vac is expected to provide a robust, harmless, and effective whitening agent for the skin.

Acute kidney injury due to direct infiltration by lymphoplasmacytic lymphoma secreting IgG paraproteins: A case report.

INTRODUCTION: Waldenström's macroglobulinemia is a lymphoplasmacytic lymphoma (LPL) associated with a monoclonal immunoglobulin M protein. Although acute kidney injury (AKI) due to immunoglobulin M paraprotein infiltration into the renal interstitium has been reported, there has been no report of AKI with invasion of the immunoglobulin G paraprotein into the renal interstitium in a patient with LPL. PATIENT CONCERNS: A 65-year-old male was admitted to our hospital with fatigue and decreased renal function. He complained of a 3-kg weight loss in the last 3 months. DIAGNOSIS: The initial blood urea nitrogen and serum creatinine levels were 55.9 and 1.83 mg/dL, respectively. Serum protein electrophoresis revealed a monoclonal component (3.5 g/dL) in the gamma region and immunofixation electrophoresis showed an immunoglobulin G kappa monoclonal protein. Renal pathology revealed that CD3-CD20+ CD138+ lymphoid cells had infiltrated the renal interstitium. A bone marrow biopsy was compatible with LPL. INTERVENTIONS: Intravenous methylprednisolone (1 mg/kg) was administered after confirming the renal pathological findings. OUTCOMES: Serum creatinine decreased to 0.8 mg/dL 14 days after treatment. CONCLUSIONS: Physicians should recognize LPL secreting various immunoglobulins as a possible cause of AKI when renal failure of unknown etiology and serum immunoglobulin paraprotein is present. A kidney biopsy should be performed for definitive diagnosis and appropriate management.

Intrahepatic inflammatory IgA+PD-L1high monocytes in hepatocellular carcinoma development and immunotherapy.

BACKGROUND: IgA neutralizes pathogens to prevent infection at mucosal sites. However, emerging evidence shows that IgA contributes to aggravating inflammation or dismantling antitumor immunity in human diseased liver. The aim of this study was to elucidate the roles of inflammation-induced intrahepatic inflammatory IgA+ monocytes in the development of hepatocellular carcinoma (HCC). METHODS: Patient cohorts including steatohepatitis cohort (n=61) and HCC cohort (n=271) were established. Patients' surgical and biopsy specimens were analyzed using immunohistochemistry. Multicolor flow cytometry was performed with a subset of patient samples. Single-cell RNA-Seq analysis was performed using Gene Expression Omnibus (GEO) datasets. Additionally, we performed in vitro differentiation of macrophages, stimulation with coated IgA, and RNA sequencing. Hepa1-6 cells and C57BL/6N mice were used to obtain HCC syngeneic mouse models. RESULTS: Serum IgA levels were associated (p<0.001) with fibrosis progression and HCC development in patients with chronic liver diseases. Additionally, immunohistochemical staining of inflamed livers or HCC revealed IgA positivity in monocytes, with a correlation between IgA+ cell frequency and IgA serum levels. Compared with IgA- monocytes, intrahepatic IgA+ monocytes expressed higher levels of programmed death-ligand 1 (PD-L1) in inflamed livers and in HCC tumor microenvironment. Single-cell RNA sequencing using NCBI GEO database indicated an upregulation in inflammation-associated genes in the monocytes of patients whose plasma cell IGHA1 expression was greater than or equal to the median value. Bulk RNA sequencing demonstrated that in vitro stimulation of M2-polarized macrophages using coated IgA complex induced PD-L1 upregulation via YAP-mediated signaling. In vivo blockade of IgA signaling decreased the number of tumor-infiltrating IgA+PD-L1high macrophages and increased the number of CD69+CD8+ T cells to enhance antitumor effects in HCC mice models. CONCLUSIONS: Overall, the findings of this study showed that serum IgA levels was correlated with intrahepatic and intratumoral infiltration of inflammatory IgA+PD-L1high monocytes in chronic liver diseases and HCC, providing potential therapeutic targets.

Protective effect of MSC-derived exosomes against cisplatin-induced apoptosis via heat shock protein 70 in auditory explant model.

Therapeutics based on stem cell technology, including stem cell-derived exosomes, have emerged in recent years for the treatment of what were otherwise considered incurable diseases. In this study, we evaluated the efficacy of human MSC-derived exosomes for protection against cisplatin induced ototoxic hearing loss. Incubation of cochlear explants with MSC-derived exosomes prior to addition of cisplatin induced a reduction in cisplatin-induced drug toxicity in auditory hair cells but not when the exosomes were introduced simultaneously with or after cisplatin. The delivery of MSC-derived exosomes to cochlear explants was confirmed by the increasing protein levels of the exosome markers CD63 and HSP70 to reduce apoptosis. These results were consistent with those from a model in which MSC-derived exosomes protect auditory hair cells from cisplatin-induced drug toxicity in an ex vivo cochlear explant model and support future studies into the therapeutic benefits of stem cell-derived exosomes in clinical applications.

Acute interstitial nephritis associated with ingesting a Momordica charantia extract: A case report.

RATIONALE: Momordica charantia is often used to treat type 2 diabetes mellitus in Korea. Drug-induced acute interstitial nephritis (AIN) accounts for 60% to 70% of AIN cases. However, only 1 case of AIN associated with ingesting M charantia has been reported in the English literature. We report an extremely rare case of AIN that occurred after a patient ingested a pure M charantia extract over 7 months. PATIENT CONCERNS: A 60-year-old Korean woman was admitted to our hospital for a renal biopsy. Her renal function had decreased gradually over the last 9 months without symptoms or signs. DIAGNOSIS: Her blood urea nitrogen and serum creatinine levels were 29.7 mg/dL (range: 8.0-20.0 mg/dL) and 1.45 mg/dL (range: 0.51-0.95 mg/dL) on admission. Renal histology indicated AIN; there was immune cell infiltration into the interstitium, tubulitis, and epithelial casts, although the glomeruli were largely intact. INTERVENTIONS: M charantia was discontinued and prednisolone was prescribed. OUTCOMES: The value of serum creatinine has almost been restored to the baseline level after 3 months. CONCLUSION: s: This is the first case report of AIN associated with the ingestion of a pure M charantia extract. Recognition of the possible adverse effects of these agents by physicians is very important for early diagnosis and appropriate management.

Spontaneous rupture of a renal artery pseudoaneurysm in a hemodialysis patient: A case report.

RATIONALE: Renal artery pseudoaneurysm is a rare vascular lesion usually caused by trauma or percutaneous urological procedures. Spontaneous rupture of pseudoaneurysms without predisposing events, especially in hemodialysis patients, has rarely been reported. PATIENT CONCERNS: A 25-year-old man receiving maintenance hemodialysis visited the emergency room because of sudden severe right flank pain. He had no history of trauma or urological procedures except for a left renal biopsy to diagnose Alport syndrome 10 years prior. DIAGNOSIS: Contrast-enhanced computed tomography revealed a right perirenal hematoma with pseudoaneurysms. INTERVENTIONS: On renal angiography, multiple pseudoaneurysms were observed in the right renal artery branches and embolization was performed. OUTCOMES: Post-angiography showed no pseudoaneurysms. His abdominal pain improved, and he was discharged 2 weeks after embolization. LESSONS: When maintenance dialysis patients complain of severe abdominal pain, spontaneous rupture of a renal pseudoaneurysm should be considered as a differential diagnosis, even if the patient has no history of trauma or previous urological procedures.

Preferential Expression of Programmed Death Ligand 1 Protein in Tumor-Associated Macrophages and Its Potential Role in Immunotherapy for Hepatocellular Carcinoma.

A predictive biomarker of immune checkpoint inhibitor (ICI)-based treatments in hepatocellular carcinoma (HCC) has not been clearly demonstrated. In this study, we focused on the infiltration and programmed death ligand 1 (PD-L1) expression of tumor-associated macrophages (TAMs) in the tumor microenvironment of HCC. Immunohistochemistry demonstrated that PD-L1 was preferentially expressed on CD68+ macrophages in the tumor microenvironment of HCC, suggestive of its expression in TAMs rather than in T cells or tumor cells (P < 0.05). A co-culture experiment using activated T cells and M2 macrophages confirmed a significant increase in T cell functionality after the pretreatment of M2 macrophages with anti-PD-L1. Syngeneic mouse model experiments demonstrated that TAMs expressed PD-L1 and tumors treated with anti-PD-L1 showed smaller diameters than those treated with IgG. In these mice, anti-PD-L1 treatment increased activation markers in intratumoral CD8+ T cells and reduced the size of the TAM population. Regarding nivolumab-treated patients, three of eight patients responded to the anti-PD-1 treatment. The percentage of Ki-67-positive CD4+ and CD8+ T cells was higher in responders than non-responders after nivolumab. Overall, PD-L1 expression on TAMs may be targeted by immune-based HCC treatment, and ICI treatment results in the reinvigoration of exhausted CD8+ T cells in HCC.

Alteration of payload in extracellular vesicles by crosstalk with mesenchymal stem cells from different origin.

BACKGROUND: The application of extracellular vesicles (EVs) derived from mesenchymal stem cells (MSCs) requires customized materials to target disease or cell damage. We hypothesized that EVs exert different inflammatory effects on one recipient cell, although stem cells of different origins in humans have similar payloads. RESULTS: Here, the payload of EVs released by crosstalk between MSCs and human middle ear epithelial cells (HMEECs) extracted from adipose tissue, bone marrow and tonsils significantly increased the level of anti-inflammatory factors. EVs derived from the co-culture medium decreased TNF-α, COX-2, IL-1ß, and IL-6 levels to approximately zero within 3 h in HMEECs. Expression of miR-638 and amyloid-ß A4 precursor protein-binding family A member 2 was analyzed using microarrays and gene ontology analysis, respectively. CONCLUSIONS: In conclusion, stem cells of different origins have different payloads through crosstalk with recipient-specific cells. Inducing specific factors in EVs by co-culture with MSCs could be valuable in regenerative medicine.

Nardostachys jatamansi Root Extract Attenuates Tumor Progression in Hepatocellular Carcinoma via Inhibition of ERK/STAT3 Pathways.

BACKGROUND/AIM: Hepatocellular carcinoma (HCC) is a highly prevalent disease and treatment is limited. Therefore, development of new therapeutic agents is urgent. The aim of this study was to investigate the in vitro and in vivo anti-cancer effects of Nardostachys jatamansi root extract (NJRE) against HCC and underlying mechanisms involved in such effects. MATERIALS AND METHODS: Effects of NJRE on viability of HCC cell lines were determined by MTT analysis and annexin/PI apoptosis assays. Expression levels of proteins in MAPK and STAT3 pathways and caspase-3 and PARP after treatment with NJRE in HCC cell lines were determined by western blotting. In a syngeneic model using mouse HCC cells Hepa1-6, inhibition of tumor formation after oral administration of NJRE was determined and expression levels of phospho-ERK and phospho-STAT3 in liver tissues were analyzed by immunohistochemical staining. RESULTS: NJRE reduced the activation of STAT3 by inhibiting the expression of ERK and finally attenuated the proliferation of HCC. CONCLUSION: NJRE has anti-cancer effects against HCC. It has potential to be used in the treatment of human HCC.

Biodistribution of poly clustered superparamagnetic iron oxide nanoparticle labeled mesenchymal stem cells in aminoglycoside induced ototoxic mouse model.

Recently, application of stem cell therapy in regenerative medicine has become an active field of study. Mesenchymal stem cells (MSCs) are known to have a strong ability for homing. MSCs labeled with superparamagnetic iron oxide nanoparticles (SPIONs) exhibit enhanced homing due to magnetic attraction. We have designed a SPION that has a cluster core of iron oxide-based nanoparticles coated with PLGA-Cy5.5. We optimized the nanoparticles for internalization to enable the transport of PCS nanoparticles through endocytosis into MSCs. The migration of magnetized MSCs with SPION by static magnets was seen in vitro. The auditory hair cells do not regenerate once damaged, ototoxic mouse model was generated by administration of kanamycin and furosemide. SPION labeled MSC's were administered through different injection routes in the ototoxic animal model. As result, the intratympanic administration group with magnet had the highest number of cells in the brain followed by the liver, cochlea, and kidney as compared to those in the control groups. The synthesized PCS (poly clustered superparamagnetic iron oxide) nanoparticles, together with MSCs, by magnetic attraction, could synergistically enhance stem cell delivery. The poly clustered superparamagnetic iron oxide nanoparticle labeled in the mesenchymal stem cells have increased the efficacy of homing of the MSC's to the target area by synergetic effect of magnetic attraction and chemotaxis (SDF-1/CXCR4 axis). This technique allows delivery of the stem cells to the areas with limited vasculatures. The nanoparticle in the biomedicine allows drug delivery, thus, the combination of nanomedicince together with the regenerative medicine will provide highly effective therapy.

In vitro Time-lapse Live-Cell Imaging to Explore Cell Migration toward the Organ of Corti.

To study the effects of mesenchymal stem cells (MSCs) on cell regeneration and treatment, this method tracks MSC migration and morphological changes after co-culture with cochlear epithelium. The organ of Corti was immobilized on a plastic coverslip by pressing a portion of the Reissner's membrane generated during the dissection. MSCs confined by a glass cylinder migrated toward cochlear epithelium when the cylinder was removed. Their predominant localization was observed in the modiolus of the organ of Corti, aligned in a direction similarly to that of the nerve fibers. However, some MSCs were localized in the limbus area and showed a horizontally elongated shape. In addition, migration into the hair cell area was increased, and the morphology of the MSCs changed to various forms after kanamycin treatment. In conclusion, the results of this study indicate that the coculture of MSCs with cochlear epithelium will be useful for the development of therapeutics via cell transplantation and for studies of cell regeneration that can examine various conditions and factors.

Paricalcitol Attenuates Contrast-Induced Acute Kidney Injury by Regulating Mitophagy and Senescence.

Contrast-induced acute kidney injury (CI-AKI) is the third most common cause of hospital-acquired renal failure, with an incidence of 11%. However, the disease mechanism remains unclear, and no effective treatment is available. Paricalcitol has been reported to be effective in animal models of kidney injury. We hypothesized that paricalcitol could play a renoprotective role against CI-AKI. Rats were divided into control, paricalcitol, contrast, and paricalcitol-plus-contrast groups. We used a previously published protocol to produce CI-AKI. Paricalcitol (0.3 µg/kg) was administered intraperitoneally before 24 h and 30 min before indomethacin. We used HK-2 cells to evaluate the effects of paricalcitol on mitophagy and senescence. Ioversol triggered renal dysfunction, increasing blood urea nitrogen and serum creatinine. Significant tubular damage, increased 8-OHdG expression, and apoptosis were apparent. Ioversol injection induced high expression levels of the mitophagy markers Pink1, Parkin, and LC3 and the senescence markers ß-galactosidase and p16INK4A. Paricalcitol pretreatment prevented renal dysfunction and reduced tissue damage by reducing both mitophagy and senescence. Cellular morphological changes were found, and expression of LC3B and HMGB1 was increased by ioversol in HK-2 cells. Paricalcitol countered these effects. This study showed that mitochondria might drive injury phenotypes in CI-AKI, and that paricalcitol protects against CI-AKI by decreasing mitochondrial damage.

Improvement of stem cell-derived exosome release efficiency by surface-modified nanoparticles.

BACKGROUND: Mesenchymal stem cells (MSCs) are pluripotent stromal cells that release extracellular vesicles (EVs). EVs contain various growth factors and antioxidants that can positively affect the surrounding cells. Nanoscale MSC-derived EVs, such as exosomes, have been developed as bio-stable nano-type materials. However, some issues, such as low yield and difficulty in quantification, limit their use. We hypothesized that enhancing exosome production using nanoparticles would stimulate the release of intracellular molecules. RESULTS: The aim of this study was to elucidate the molecular mechanisms of exosome generation by comparing the internalization of surface-modified, positively charged nanoparticles and exosome generation from MSCs. We determined that Rab7, a late endosome and auto-phagosome marker, was increased upon exosome expression and was associated with autophagosome formation. CONCLUSIONS: It was concluded that the nanoparticles we developed were transported to the lysosome by clathrin-mediated endocytosis. additionally, entered nanoparticles stimulated that autophagy related factors to release exosome from the MSC. MSC-derived exosomes using nanoparticles may increase exosome yield and enable the discovery of nanoparticle-induced genetic factors.

Endocytic trafficking of polymeric clustered superparamagnetic iron oxide nanoparticles in mesenchymal stem cells.

The technology of directing nanoparticles to specific locations in the body continues to be an area of great interest in a myriad of research fields. In the present study, we have developed nanoparticles and a method that allows the nanoparticles to move to specific sites by simultaneously utilizing the homing ability and magnetism of stem cells. Polymeric clustered SPIO (PCS) nanoparticles are composed of a superparamagnetic iron oxide nanoparticle (SPION) cluster core coated with poly lactic-co-glycolic acid (PLGA) and labeled with the fluorescent dye Cy5.5 for tracking. PCS is designed to be internalized by stem cells via endocytosis and then moved to the desired subcellular location through magnetism. Here, we investigated the interactions between SPIONs and mesenchymal stem cells (MSCs), including their absorption mechanism and subcellular localization. Exposure to the nanoparticles at 40 µg/mL for over 96 h did not affect cell survival or differentiation. We used a variety of endocytosis inhibitors and identified the potential cellular internalization pathway of SPIONs to be clathrin-mediated endocytosis. Antibodies to organelles were used to accumulate lysosomes through early and late endosomes. PCS at 40 µg/mL was internalized and stored without significant deleterious effects on stem cells, indicating that MSCs can act as an effective nanoparticle carrier. These findings also demonstrate the successful localization of the novel particles using magnetic attraction.