Treatment of Chronic Kidney Disease with Extracellular Vesicles from Mesenchymal Stem Cells and CD133+ Expanded Cells: A Comparative Preclinical Analysis.

Chronic kidney disease (CKD) is characterized by structural abnormalities and the progressive loss of kidney function. Extracellular vesicles (EVs) from human umbilical cord tissue (hUCT)-derived mesenchymal stem cells (MSCs) and expanded human umbilical cord blood (hUCB)-derived CD133+ cells (eCD133+) maintain the characteristics of the parent cells, providing a new form of cell-free treatment. We evaluated the effects of EVs from hUCT-derived MSCs and hUCB-derived CD133+ cells on rats with CDK induced by an adenine-enriched diet. EVs were isolated by ultracentrifugation and characterized by nanoparticle tracking analysis (NTA) and electron microscopy. The animals were randomized and divided into the MSC-EV group, eEPC-EV group and control group. Infusions occurred on the seventh and 14th days after CKD induction. Evaluations of kidney function were carried out by biochemical and histological analyses. Intense labeling of the α-SMA protein was observed when comparing the control with MSC-EVs. In both groups treated with EVs, a significant increase in serum albumin was observed, and the increase in cystatin C was inhibited. The results indicated improvements in renal function in CKD, demonstrating the therapeutic potential of EVs derived from MSCs and eCD133+ cells and suggesting the possibility that in the future, more than one type of EV will be used concurrently.

Canine dental pulp and umbilical cord-derived mesenchymal stem cells as alternative sources for cell therapy in dogs.

The use of regenerative medicine for pets has been growing in recent years, and an increasing number of studies have contributed to the widespread use of cell therapies in clinical veterinary medicine. Mesenchymal stem cells (MSCs) can be isolated from different sources such as dental pulp and umbilical cord. Aiming safety and reproducibility of cell therapy in clinical practice by using sources easily obtained that are usually discarded, this study isolated, characterized, and evaluated the proliferation and colony formation potential of canine dental pulp-derived mesenchymal stem cells (cDPSCs) and canine umbilical cord tissue (cUCSCs). Three samples from each source were collected, isolated, and cultured. MSCs were differentiated into three lineages and quantified by spectrophotometry. For immunophenotypic characterization, antibodies were used to analyze the expression of cell surface markers, and 7-AAD and Annexin-V were used to analyze cell viability and apoptosis, respectively. For the clonogenic assay, cells were cultured, the colonies were stained, and counted. For the proliferation assay, the cells were plated in flasks for three days and added EdU nucleoside. cDPSCs and cUCSCs showed plastic adherence and fibroblastic morphology after cultivation. Both sources showed differentiation potential and showed CD29 and CD44 positivity and CD14, CD45, CD34 and HLA-DR negativity, and low mortality and apoptosis rates. There was no difference in proliferation rates between sources. Overall, although cUCSCs had a higher number of colony-forming units than cDPSCs, both sources presented MSCs characteristics and can be used safely as alternative sources in cell therapy.

Cytotoxicity of fluconazole on canine dental pulp-derived stem cells.

OBJECTIVE: In order to use fluconazole as an antifungal in cell cultures, we evaluated its possible cytotoxic effects and its influence on the proliferation and viability of canine dental pulp-derived stem cells (cDPSCs). METHODS: Samples from permanent canine teeth were placed in a sterile tube with IMDM, penicillin-streptomycin, sodium heparin, and different concentrations of fluconazole. Dental pulp was digested (collagenase type II) and expanded in vitro. After 12 days of culture, enzymatic dissociation of the cDPSCs was performed to quantify, differentiate, and characterize the cells. Cytotoxicity was evaluated based on cell viability in response to fluconazole treatment using the 7-AAD dye. RESULTS: Characterization of the cDPSCs revealed that fluconazole had no influence on the immunophenotypic characteristics and differentiation of these cells. Cell proliferation assay revealed that fluconazole did not significantly interfere with the replication capacity of the cDPSCs. Cytotoxicity analysis revealed a loss of cell viability as the fluconazole concentration increased. Although there was an increase in cell mortality, the number of dead cells remained low. Though the higher concentration of fluconazole (240 µg/mL) resulted in a higher number of non-viable cells, it remained safe for use. CONCLUSION: To prevent fungal contamination that causes a loss of samples during expansion of cDPSCs and to maintain minimal cell toxicity, we suggest adding 120 µg/mL of fluconazole to the teeth collection medium and cDPSCs culture.

Lung Tissue Damage Associated with Allergic Asthma in BALB/c Mice Could Be Controlled with a Single Injection of Mesenchymal Stem Cells from Human Bone Marrow up to 14 d After Transplantation.

Mesenchymal stem cell (MSC) research has demonstrated the potential of these cells to modulate lung inflammatory processes and tissue repair; however, the underlying mechanisms and treatment durability remain unknown. Here, we investigated the therapeutic potential of human bone marrow-derived MSCs in the inflammatory process and pulmonary remodeling of asthmatic BALB/c mice up to 14 d after transplantation. Our study used ovalbumin to induce allergic asthma in male BALB/c mice. MSCs were injected intratracheally in the asthma groups. Bronchoalveolar lavage fluid (BALF) was collected, and cytology was performed to measure the total protein, hydrogen peroxide (H2O2), and proinflammatory (IL-5, IL-13, and IL-17A) and anti-inflammatory (IL-10) interleukin (IL) levels. The lungs were removed for the histopathological evaluation. On day zero, the eosinophil and lymphochte percentages, total protein concentrations, and IL-13 and IL-17A levels in the BALF were significantly increased in the asthma group, proving the efficacy of the experimental model of allergic asthma. On day 7, the MSC-treated group exhibited significant reductions in the eosinophil, lymphocyte, total protein, H2O2, IL-5, IL-13, and IL-17A levels in the BALF, while the IL-10 levels were significantly increased. On day 14, the total cell numbers and lymphocyte, total protein, IL-13, and IL-17A levels in the BALF in the MSC-treated group were significantly decreased. A significant decrease in airway remodeling was observed on days 7 and 14 in almost all bronchioles, which showed reduced inflammatory infiltration, collagen deposition, muscle and epithelial thickening, and mucus production. These results demonstrate that treatment with a single injection of MSCs reduces the pathophysiological events occurring in an experimental model of allergic asthma by controlling the inflammatory process up to 14 d after transplantation.

Comparison of the Efficacy of Surgical Decompression Alone and Combined With Canine Adipose Tissue-Derived Stem Cell Transplantation in Dogs With Acute Thoracolumbar Disk Disease and Spinal Cord Injury.

Paraparesis and paraplegia are common conditions in dogs, most often caused by a disc herniation in the thoracolumbar spinal segments (T3-L3), which is a neurological emergency. Surgical decompression should be performed as soon as possible when spinal compression is revealed by myelography, computed tomography, or magnetic resonance imaging. Mesenchymal stem-cell therapy is a promising adjunct treatment for spinal cord injury. This study sought to compare the effects of surgical decompression alone and combined with an allogeneic transplantation of canine adipose tissue-derived mesenchymal stem cells (cAd-MSCs) in the treatment of dogs with acute paraplegia. Twenty-two adult dogs of different breeds with acute paraplegia resulting from a Hansen type I disc herniation in the thoracolumbar region (T3-L3) were evaluated using computed tomography. All dogs had grade IV or V lesions and underwent surgery within 7 days after symptom onset. They were randomly assigned into two groups, 11 dogs in each. The dogs in Group I underwent hemilaminectomy, and those in Group II underwent hemilaminectomy and cAd-MSC epidural transplantation. In both groups, all dogs with grade IV lesions recovered locomotion. The median locomotion recovery period was 7 days for Group II and 21 days for Group I, and this difference was statistically significant (p < 0.05). Moreover, the median length of hospitalization after the surgery was statistically different between the two groups (Group I, 4 days; Group II, 3 days; p < 0.05). There were no statistically significant between-group differences regarding the number of animals with grade IV or V lesions that recovered locomotion and nociception. In conclusion, compared with surgical decompression alone, the use of epidural cAd-MSC transplantation with surgical decompression may contribute to faster locomotor recovery in dogs with acute paraplegia and reduce the length of post-surgery hospitalization.

Management of Airway Remodeling in a Mouse Model of Allergic Airways Inflammation Using Extracellular Vesicles from Human Bone Marrow-Derived Mesenchymal Stromal Cells

Abstract: Asthma is a chronic respiratory disease affecting 300 million people worldwide. It results in several structural changes in the airways, which are minimally accessible in clinical practice. Cell therapy using mesenchymal stromal cells (MSCs) is a promising strategy for treating asthma due to the paracrine activity of MSCs, which influences tissue regeneration and modulates the immune response. Studies using extracellular vesicles (EV) released by MSCs have demonstrated their regenerative properties in animal models. The aim of this study was to evaluate the potential of EVs isolated from human bone marrow MSCs (hBM-MSCs) to control lung tissue remodeling in ovalbumin-induced allergic asthma in Balb/c mice. We isolated hBM-MSCs from a single donor, expanded and characterized them, and then isolated EVs. Asthma was induced in 43 male Balb/c mice, divided into four groups: control, asthmatic (AS), asthmatic plus systemic EVs (EV-S), and asthmatic plus intratracheal EVs (EV-IT). Upon completion of asthma induction, animals were treated with EVs either locally (EV-IT) or intravenously (EV-S). Seven days after, we performed bronchoalveolar lavage (BAL) and the total nuclear cells were counted. The animals were euthanized, and the lungs were collected for histopathological analysis of the airways. The EV-S group showed improvement in only the total BAL cell count compared with the AS group, while the EV-IT group showed significant improvement in almost all evaluated criteria. Therefore, we demonstrate that the local application of EVs derived from hBM-MSCs may be a potential treatment in controlling asthma.