Allogeneic bone marrow mesenchymal stem cell-derived exosomes alleviate human hypoxic AKI-on-a-Chip within a tight treatment window.

BACKGROUND: Acute hypoxic proximal tubule (PT) injury and subsequent maladaptive repair present high mortality and increased risk of acute kidney injury (AKI) - chronic kidney disease (CKD) transition. Human bone marrow mesenchymal stem cell-derived exosomes (hBMMSC-Exos) as potential cell therapeutics can be translated into clinics if drawbacks on safety and efficacy are clarified. Here, we determined the real-time effective dose and treatment window of allogeneic hBMMSC-Exos, evaluated their performance on the structural and functional integrity of 3D microfluidic acute hypoxic PT injury platform. METHODS: hBMMSC-Exos were isolated and characterized. Real-time impedance-based cell proliferation analysis (RTCA) determined the effective dose and treatment window for acute hypoxic PT injury. A 2-lane 3D gravity-driven microfluidic platform was set to mimic PT in vitro. ZO-1, acetylated α-tubulin immunolabelling, and permeability index assessed structural; cell proliferation by WST-1 measured functional integrity of PT. RESULTS: hBMMSC-Exos induced PT proliferation with ED50 of 172,582 µg/ml at the 26th hour. Hypoxia significantly decreased ZO-1, increased permeability index, and decreased cell proliferation rate on 24-48 h in the microfluidic platform. hBMMSC-Exos reinforced polarity by a 1.72-fold increase in ZO-1, restored permeability by 20/45-fold against 20/155 kDa dextran and increased epithelial proliferation 3-fold compared to control. CONCLUSIONS: The real-time potency assay and 3D gravity-driven microfluidic acute hypoxic PT injury platform precisely demonstrated the therapeutic performance window of allogeneic hBMMSC-Exos on ischemic AKI based on structural and functional cellular data. The novel standardized, non-invasive two-step system validates the cell-based personalized theragnostic tool in a real-time physiological microenvironment prior to safe and efficient clinical usage in nephrology.

Adrenomedullin has no effect on segmental bone defect healing but increases bone mineral density in rat model.

OBJECTIVE: This study aimed to investigate the effect of adrenomedullin on the healing of the segmental bone defect in a rat model. METHODS: Thirty-six Wistar rats were randomly divided into 6 groups based on follow-up periods and administered a dose of adrenomedullin hormone. In each group, bilaterally, a 2-mm bone defect was created at the diaphysis of the radius. Sodium chloride solution was administered to sham groups 3 times a week for 4 and 8 weeks intraperitoneally. Adrenomedullin was administered to the study groups 3 times a week: 15 µg-4 weeks, 15 µg-8 weeks, 30 µg-4 weeks, and 30 µg-8 weeks, respectively. After euthanasia, the segmental defects were evaluated by histomorphometric [new bone area (NBA)] and microtomographic [bone volume (BV), bone surface (BS), and bone mineral density (BMD)] analyses. RESULTS: Although the 4- and 8-week 15 µg administered study groups had higher NBA values than the other study and control groups, the histomorphometric analysis did not reveal any statistical difference between the control and study groups regarding NBA (P > .05). In microtomographic analysis, BV was higher in the 15 µg 4-week group than 30 µg 4-week group (296.9 vs. 208.5, P=.003), and BS was lower in the 30 µg 4-week group than in the 4-week control group (695.5 vs. 1334.7, P=.005), but overall, no significant difference was found between the control and study groups (P > .05). Despite these minor differences in histomorphometric and microtomographic criteria indicating new bone formation, the BMD values of the 15 µg 8-week study group showed a significant increase compared with the control group (P=.001, respectively). CONCLUSION: Adrenomedullin positively affected BMD at 15 µg, but this study could not show healing in the segmental defect site at different dose regimens. Further studies are needed to assess its effects on bone tissue trauma.

Comparative analysis of magnetically activated cell sorting and ultracentrifugation methods for exosome isolation.

Mesenchymal stem cell-derived exosomes regulate cell migration, proliferation, differentiation, and synthesis of the extracellular matrix, giving great potential for the treatment of different diseases. The ultracentrifugation method is the gold standard method for exosome isolation due to the simple protocol, and high yield, but presents low purity and requires specialized equipment. Amelioration of technical optimization is required for quick and reliable confinement of exosomes to translate them to the clinic as cell therapeutics In this study, we hypothesized that magnetically activated cell sorting may provide, an effective, reliable, and rapid tool for exosome isolation when compared to ultracentrifugation. We, therefore, aimed to compare the efficiency of magnetically activated cell sorting and ultracentrifugation for human mesenchymal stem cell-derived exosome isolation from culture media by protein quantification, surface biomarker, size, number, and morphological analysis. Magnetically activated cell sorting provided a higher purity and amount of exosomes that carry visible magnetic beads when compared to ultracentrifugation. The particle number of the magnetically activated cell sorting group was higher than the ultracentrifugation. In conclusion, magnetically activated cell sorting presents a quick, and reliable method to collect and present human mesenchymal stem cell exosomes to clinics at high purity for potential cellular therapeutic approaches. The novel isolation and purification method may be extended to different clinical protocols using different autogenic or allogeneic cell sources.

Voltage gated calcium channel antibody-related neurological diseases.

Voltage gated calcium channel (VGCC) antibodies are generally associated with Lambert-Eaton myasthenic syndrome. However the presence of this antibody has been associated with paraneoplastic as well as non-paraneoplastic cerebellar degeneration. Most patients with VGCC-antibody-positivity have small cell lung cancer (SCLC). Lambert-Eaton myasthenic syndrome (LEMS) is an autoimmune disease of the presynaptic part of the neuromuscular junction. Its classical clinical triad is proximal muscle weakness, areflexia and autonomic dysfunction. Fifty to sixty percent of LEMS patients have a neoplasia, usually SCLC. The co-occurrence of SCLC and LEMS causes more severe and progressive disease and shorter survival than non-paraneoplastic LEMS. Treatment includes 3,4 diaminopyridine for symptomatic purposes and immunotherapy with prednisolone, azathioprine or intravenous immunoglobulin in patients unresponsive to 3,4 diaminopyridine. Paraneoplastic cerebellar degeneration (PCD) is a syndrome characterized with severe, subacute pancerebellar dysfunction. Serum is positive for VGCC antibody in 41%-44% of patients, usually with the co-occurrence of SCLC. Clinical and electrophysiological features of LEMS are also present in 20%-40% of these patients. Unfortunately, PCD symptoms do not improve with immunotherapy. The role of VGCC antibody in the immunopathogenesis of LEMS is well known whereas its role in PCD is still unclear. All patients presenting with LEMS or PCD must be investigated for SCLC.