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.