Adipose Tissue-Derived Stromal/Stem Cells Transplantation with Cholecalciferol Supplementation in Recent-Onset Type 1 Diabetes Patients: Twelve Months Follow-Up.

To evaluate safety and therapeutic effect along 12 months of allogenic adipose tissue-derived stromal/stem cells (ASCs) transplantation with cholecalciferol (VITD) in patients with recent-onset type 1 diabetes (T1D). Prospective, phase II, open trial, pilot study in which patients with recent onset T1D received ASCs (1xKgx106 cells) and VITD 2000UI/day for 12 months (group 1) and were compared to controls with standard insulin therapy (group 2). Adverse events, C-peptide area under the curve (CPAUC), insulin dose, HbA1c and frequency of FoxP3+ in CD4+ or CD8+ T-cells(flow cytometry) were evaluated at baseline(T0), after 3(T3), 6(T6) and 12 months(T12). Eleven patients completed follow up (7:group 1;4:group 2). Group 1 had lower insulin requirement at T3(0.24±0.18vs0.53±0.23UI/kg,p=0.04), T6(0.24±0.15vs0.66±0.33 UI/kg,p=0.04) and T12(0.39±0.15vs0.74±0.29 UI/Kg,p=0.04).HbA1c was lower at T6 (50.57±8.56vs72.25±10.34 mmol/mol,p=0.01), without differences at T12 (57.14±11.98 in group 1 vs. 73.5±14.57 mmol/min in group 2, p=0.16). CPAUC was not significantly different between groups at T0(p=0.07), higher in group 1 at T3(p=0.04) and T6(p=0.006), but similar at T12(p=0.23). IDAA1c was significantly lower in group 1 than group 2 at T3,T6 and T12 (p=0.006, 0.006 and 0.042, respectively). IDDA1c was inversely correlated to FoxP3 expression in CD4 and CD8+ T cells at T6 (p<0.001 and p=0.01, respectively). In group 1, one patient had recurrence of a benign teratoma that was surgically removed, not associated to the intervention. ASCs with VITD without immunosuppression were safe and associated lower insulin requirements, better glycemic control, and transient better pancreatic function in recent onset T1D, but the potential benefits were not sustained.

Quality Control Optimization for Minimizing Security Risks Associated with Mesenchymal Stromal Cell-Based Product Development.

Mesenchymal stromal cells (MSCs) have been considered a therapeutic strategy in regenerative medicine because of their regenerative and immunomodulatory properties. The translation of MSC-based products has some challenges, such as regulatory and scientific issues. Quality control should be standardized and optimized to guarantee the reproducibility, safety, and efficacy of MSC-based products to be administered to patients. The aim of this study was to develop MSC-based products for use in clinical practice. Quality control assays include cell characterization, cell viability, immunogenicity, and cell differentiation; safety tests such as procoagulant tissue factor (TF), microbiological, mycoplasma, endotoxin, genomic stability, and tumorigenicity tests; and potency tests. The results confirm that the cells express MSC markers; an average cell viability of 96.9%; a low expression of HLA-DR and costimulatory molecules; differentiation potential; a high expression of TF/CD142; an absence of pathogenic microorganisms; negative endotoxins; an absence of chromosomal abnormalities; an absence of genotoxicity and tumorigenicity; and T-lymphocyte proliferation inhibition potential. This study shows the relevance of standardizing the manufacturing process and quality controls to reduce variability due to the heterogeneity between donors. The results might also be useful for the implementation and optimization of new analytical techniques and automated methods to improve safety, which are the major concerns related to MSC-based therapy.

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.

Chromosomal aberrations after induced pluripotent stem cells reprogramming.

Induced pluripotent stem cells (iPSCs) are generated from adult cells that have been reprogrammed to pluripotency. However, in vitro cultivation and genetic reprogramming increase genetic instability, which could result in chromosomal abnormalities. Maintenance of genetic stability after reprogramming is required for possible experimental and clinical applications. The aim of this study was to analyze chromosomal alterations by using the G-banding karyotyping method applied to 97 samples from 38 iPSC cell lines generated from peripheral blood or Wharton's jelly. Samples from patients with long QT syndrome, Jervell and Lange-Nielsen syndrome and amyotrophic lateral sclerosis and from normal individuals revealed the following chromosomal alterations: acentric fragments, chromosomal fusions, premature centromere divisions, double minutes, radial figures, ring chromosomes, polyploidies, inversions and trisomies. An analysis of two samples generated from Wharton's jelly before and after reprogramming showed that abnormal clones can emerge or be selected and generate an altered lineage. IPSC lines may show clonal and nonclonal chromosomal aberrations in several passages (from P6 to P34), but these aberrations are more common in later passages. Many important chromosomal aberrations were detected, showing that G-banding is very useful for evaluating genetic instability with important repercussions for the application of iPSC lines.

Tissue-Derived Signals for Mesenchymal Stem Cell Stimulation: Role of Cardiac and Umbilical Cord Microenvironments.

The tissue microenvironment regulates such stem cell behaviors as self-renewal and differentiation. Attempts to mimic components of these microenvironments could provide new strategies for culturing and directing the behavior of stem cells. The aim of the present study was to mimic cardiac and umbilical cord tissue microenvironments in vitro and compare the resulting bone marrow-derived mesenchymal stem cell (BM-MSC) behaviors. We generated tissue microenvironments using conditioned medium (CM) and extracellular matrix (ECM) samples obtained from human heart and umbilical cord tissue explant cultures and by tissue decellularization. Mass spectrometry and immunostaining were used to characterize and determine the specific protein profiles of the ECMs and CMs. We demonstrated that the ECMs and CMs were not cytotoxic to BM-MSCs and could thus be tested via cell culture. The BM-MSCs showed a higher proliferation rate when cultured with umbilical cord-derived CM compared with the other analyzed conditions. Furthermore, the ECMs increased cell adhesion and migration. However, although the conditions tested in this work were able to maintain the viability and affect the proliferation, adhesion and migration of BM-MSCs in vitro, mimicking tissue microenvironments using ECM and CM was not sufficient to induce the cardiomyogenic differentiation of BM-MSCs. The present study provides a thorough characterization of the biological activity of these ECMs and CMs in human BM-MSC cultures.

Nasal septum-derived chondroprogenitor cells control mandibular condylar resorption consequent to orthognathic surgery: a clinical trial.

Condylar resorption is an aggressive and disability form of temporomandibular joint (TMJ) degenerative disease, usually non-respondent to conservative or minimally invasive therapies and often leading to surgical intervention and prostheses implantation. This condition is also one of the most dreaded postoperative complications of orthognathic surgery, with severe cartilage erosion and loss of subchondral bone volume and mineral density, associated with a painful or not inflammatory processes. Because regenerative medicine has emerged as an alternative for orthopedic cases with advanced degenerative joint disease, we conducted a phase I/IIa clinical trial (U1111-1194-6997) to evaluate the safety and efficacy of autologous nasal septal chondroprogenitor cells. Ten participants underwent biopsy of the nasal septum cartilage during their orthognathic surgery. The harvested cells were cultured in vitro and analyzed for viability, presence of phenotype markers for mesenchymal stem and/or chondroprogenitor cells, and the potential to differentiate into chondrocytes, adipocytes, and osteoblasts. After the intra-articular injection of the cell therapy, clinical follow-up was performed using the Diagnostic Criteria for Temporomandibular Disorders (DC/TMD) and computed tomography (CT) images. No serious adverse events related to the cell therapy injection were observed during the 12-month follow-up period. It was found that autologous chondroprogenitors reduced arthralgia, promoted stabilization of mandibular function and condylar volume, and regeneration of condylar tissues. This study demonstrates that chondroprogenitor cells from the nasal septum may be a promise strategy for the treatment of temporomandibular degenerative joint disease that do not respond to other conservative therapies.

Evaluation of type 1 diabetes' partial clinical remission after three years of heterologous adipose tissue derived stromal/stem cells transplantation associated with vitamin D supplementation.

BACKGROUND: Mesenchymal stem cell infusion and vitamin D supplementation may have immunomodulatory actions that could prolong the preservation of residual insulin secretion in patients with type 1 diabetes (T1D). Intervention with these agents after onset of T1D could favor the development of a remission phase, with potential clinical impact. We aimed to compare the presence of clinical remission (CR), glycemic control and daily insulin requirement at 6, 12, 18, 24 and 36 months after the diagnosis of T1D using IDAA1c in patients who received therapy with adipose tissue-derived mesenchymal stem cell (ASC) infusion and vitamin D supplementation and a control group. METHODS: This retrospective cohort study analyzed data from the medical records of patients with T1D diagnosed between 15 and 40 years. Partial CR was defined as an IDAA1c index < 9. Patients in the intervention group received an infusion of adipose tissued-derived mesenchymal stem cells (ASCs) within 3 months after diagnosis and supplementation with 2000 IU of cholecalciferol for 1 year, started on the day following the infusion. Partial CR was also determined using the ISPAD criteria, to assess its agreement with IDAA1c. RESULTS: A total of 28 patients were evaluated: 7 in the intervention group (group 1) and 21 in the control group (group 2). All patients in group 1 evolved with partial CR while only 46.7% of patients in group 2 had this outcome. Group 1 had a higher frequency of CR when evaluated with IDAA1c and ISPAD criteria. The mean duration of CR varied between the two criteria. Although HbA1c was similar between groups during follow-up, group 1 had a lower total daily insulin requirement (p < 0.005) at all time points. At 36 months, group 1 used 49% of the total daily insulin dose used by group 2 with similar glycemic control. CONCLUSION: The intervention with infusion of ASC + vitamin D supplementation was associated with partial CR at 6 months. Although there were no differences in CR established by the IDAA1c and ISPAD criteria after three years of follow-up, patients who underwent intervention had nearly the half insulin requirement of controls with conventional treatment, with similar glycemic control. TRIAL REGISTRATION: 37001514.0.0000.5257.

Human Adipose-Derived Stem Cells Reduce Cellular Damage after Experimental Spinal Cord Injury in Rats.

Traumatic spinal cord injury (SCI) is a devastating condition without an effective therapy. Cellular therapies are among the promising treatment strategies. Adult stem cells, such as mesenchymal stem cells, are often used clinical research for their immunomodulatory and regenerative potential. This study aimed to evaluate the effect of human adipose tissue-derived stem cells (ADSC) infusion through the cauda equina in rats with SCI. The human ADSC from bariatric surgery was isolated, expanded, and characterized. Wistar rats were subjected to blunt SCI and were divided into four groups. Two experimental groups (EG): EG1 received one ADSC infusion after SCI, and EG2 received two infusions, the first one after SCI and the second infusion seven days after the injury. Control groups (CG1 and CG2) received infusion with a culture medium. In vivo, cell tracking was performed 48 h and seven days after ADSC infusion. The animals were followed up for 40 days after SCI, and immunohistochemical quantification of myelin, neurons, and astrocytes was performed. Cellular tracking showed cell migration towards the injury site. ADSC infusion significantly reduced neuronal loss, although it did not prevent the myelin loss or enhance the area occupied by astrocytes compared to the control group. The results were similar when comparing one or two cell infusions. The injection of ADSC distal to the injured area was shown to be a safe and effective method for cellular administration in spinal cord injury.

Mesenchymal stromal cells derived from exfoliated deciduous teeth express neuronal markers before differentiation induction.

OBJECTIVE: This study aimed to evaluate neuronal markers in stromal cells from human exfoliated deciduous teeth (SHED) and standardize the isolation and characterization of those cells. METHODOLOGY: Healthy primary teeth were collected from children. The cells were isolated by enzymatic digestion with collagenase. By following the International Society for Cell and Gene Therapy (ISCT) guidelines, SHED were characterized by flow cytometry and differentiated into osteogenic, adipogenic, and chondrogenic lineages. Colony-forming unit-fibroblasts (CFU-F) were performed to assess these cells' potential and efficiency. To clarify the neuronal potential of SHED, the expression of nestin and ßIII-tubulin were examined by immunofluorescence and SOX1, SOX2, GFAP, and doublecortin (DCX), nestin, CD56, and CD146 by flow cytometry. RESULTS: SHED showed mesenchymal stromal cells characteristics, such as adhesion to plastic, positive immunophenotypic profile for CD29, CD44, CD73, CD90, CD105, and CD166 markers, reduced expression for CD14, CD19, CD34, CD45, HLA-DR, and differentiation in three lineages confirmed by staining and gene expression for adipogenic differentiation. The average efficiency of colony formation was 16.69%. SHED expressed the neuronal markers nestin and ßIII-tubulin; the fluorescent signal intensity was significantly higher in ßIII-tubulin (p<0.0001) compared to nestin. Moreover, SHED expressed DCX, GFAP, nestin, SOX1, SOX2, CD56, CD146, and CD271. Therefore, SHED had a potential for neuronal lineage even without induction with culture medium and specific factors. CONCLUSION: SHEDs may be a new therapeutic strategy for regenerating and repairing neuronal cells and tissues.

Biocompatibility of ABS and PLA Polymers with Dental Pulp Stem Cells Enhance Their Potential Biomedical Applications.

Polylactic Acid (PLA) and Acrylonitrile-Butadiene-Styrene (ABS) are commonly used polymers in 3D printing for biomedical applications. Dental Pulp Stem Cells (DPSCs) are an accessible and proliferative source of stem cells with significant differentiation potential. Limited knowledge exists regarding the biocompatibility and genetic safety of ABS and PLA when in contact with DPSCs. This study aimed to investigate the impact of PLA and ABS on the adhesion, proliferation, osteogenic differentiation, genetic stability, proteomics, and immunophenotypic profile of DPSCs. A total of three groups, 1- DPSC-control, 2- DPSC+ABS, and 3- DPSC+PLA, were used in in vitro experiments to evaluate cell morphology, proliferation, differentiation capabilities, genetic stability, proteomics (secretome), and immunophenotypic profiles regarding the interaction between DPSCs and polymers. Both ABS and PLA supported the adhesion and proliferation of DPSCs without exhibiting significant cytotoxic effects and maintaining the capacity for osteogenic differentiation. Genetic stability, proteomics, and immunophenotypic profiles were unaltered in DPSCs post-contact with these polymers, highlighting their biosafety. Our findings suggest that ABS and PLA are biocompatible with DPSCs and demonstrate potential in dental or orthopedic applications; the choice of the polymer will depend on the properties required in treatment. These promising results stimulate further studies to explore the potential therapeutic applications in vivo using prototyped polymers in personalized medicine.

HLA-G and CD152 Expression Levels Encourage the Use of Umbilical Cord Tissue-Derived Mesenchymal Stromal Cells as an Alternative for Immunosuppressive Therapy.

Mesenchymal stromal cells (MSCs) have been used in immunosuppressive therapy due to their therapeutic effects, with the HLA-G molecule seeming to play a fundamental role. This work evaluated alternative MSC sources to bone marrow (BM), namely, umbilical cord tissue (UC), adipose tissue (AD) and dental pulp tissue (DP), and the influence of interferon-γ (IFN-γ) and hypoxia on the cultivation of these cells for use in immunosuppression therapies. Expression of costimulatory markers CD40, CD80 and CD86 and immunosuppressive molecules CD152 and HLA-G was analyzed. Lymphocyte inhibition assays were also performed. Sequencing of the HLA-G gene from exons 1 to 5 was performed using next-generation sequencing to determine the presence of alleles. UC-derived MSCs (UCMSCs) expressed higher CD152 and HLA-G1 under standard cultivation. UCMSCs and DP-derived MSCs (DPSCs) secreted similar levels of HLA-G5. All MSC sources inhibited the proliferation of peripheral blood mononuclear cells (PBMCs); growth under regular versus hypoxic conditions resulted in similar levels of inhibition. When IFN-γ was added, PBMC growth was inhibited to a lesser extent by UCMSCs. The HLA-G*01:04:01:01 allele appears to generate a more efficient MSC response in inhibiting lymphocyte proliferation. However, the strength of this conclusion was limited by the small sample size. UCMSCs are an excellent alternative to BM in immunosuppressive therapy: they express high concentrations of inhibitory molecules and can be cultivated without stimuli, which minimizes cost.

Safety and long-term improvement of mesenchymal stromal cell infusion in critically COVID-19 patients: a randomized clinical trial.

BACKGROUND: COVID-19 is a multisystem disease that presents acute and persistent symptoms, the postacute sequelae (PASC). Long-term symptoms may be due to consequences from organ or tissue injury caused by SARS-CoV-2, associated clotting or inflammatory processes during acute COVID-19. Various strategies are being chosen by clinicians to prevent severe cases of COVID-19; however, a single treatment would not be efficient in treating such a complex disease. Mesenchymal stromal cells (MSCs) are known for their immunomodulatory properties and regeneration ability; therefore, they are a promising tool for treating disorders involving immune dysregulation and extensive tissue damage, as is the case with COVID-19. This study aimed to assess the safety and explore the long-term efficacy of three intravenous doses of UC-MSCs (umbilical cord MSCs) as an adjunctive therapy in the recovery and postacute sequelae reduction caused by COVID-19. To our knowledge, this is one of the few reports that presents the longest follow-up after MSC treatment in COVID-19 patients. METHODS: This was a phase I/II, prospective, single-center, randomized, double-blind, placebo-controlled clinical trial. Seventeen patients diagnosed with COVID-19 who require intensive care surveillance and invasive mechanical ventilation-critically ill patients-were included. The patient infusion was three doses of 5 × 105 cells/kg UC-MSCs, with a dosing interval of 48 h (n = 11) or placebo (n = 6). The evaluations consisted of a clinical assessment, viral load, laboratory testing, including blood count, serologic, biochemical, cell subpopulation, cytokines and CT scan. RESULTS: The results revealed that in the UC-MSC group, there was a reduction in the levels of ferritin, IL-6 and MCP1-CCL2 on the fourteen day. In the second month, a decrease in the levels of reactive C-protein, D-dimer and neutrophils and an increase in the numbers of TCD3, TCD4 and NK lymphocytes were observed. A decrease in extension of lung damage was observed at the fourth month. The improvement in all these parameters was maintained until the end of patient follow-up. CONCLUSIONS: UC-MSCs infusion is safe and can play an important role as an adjunctive therapy, both in the early stages, preventing severe complications and in the chronic phase with postacute sequelae reduction in critically ill COVID-19 patients. Trial registration Brazilian Registry of Clinical Trials (ReBEC), UTN code-U1111-1254-9819. Registered 31 October 2020-Retrospectively registered, https://ensaiosclinicos.gov.br/rg/RBR-3fz9yr.

Chromosomal Instability in Acute Myeloid Leukemia.

Chromosomal instability (CIN), the increasing rate in which cells acquire new chromosomal alterations, is one of the hallmarks of cancer. Many studies highlighted CIN as an important mechanism in the origin, progression, and relapse of acute myeloid leukemia (AML). The ambivalent feature of CIN as a cancer-promoting or cancer-suppressing mechanism might explain the prognostic variability. The latter, however, is described in very few studies. This review highlights the important CIN mechanisms in AML, showing that CIN signatures can occur largely in all the three major AML types (de novo AML, secondary-AML, and therapy-related-AML). CIN features in AML could also be age-related and reflect the heterogeneity of the disease. Although most of these abnormalities show an adverse prognostic value, they also offer a strong new perspective on personalized therapy approaches, which goes beyond assessing CIN in vitro in patient tumor samples to predict prognosis. Current and emerging AML therapies are exploring CIN to improve AML treatment, which includes blocking CIN or increasing CIN beyond the limit threshold to induce cell death. We argue that the characterization of CIN features, not included yet in the routine diagnostic of AML patients, might provide a better stratification of patients and be extended to a more personalized therapeutic approach.

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.

Adipose tissue-derived stromal/stem cells + cholecalciferol: a pilot study in recent-onset type 1 diabetes patients.

OBJECTIVE: Adipose tissue-derived stromal/stem cells (ASCs) and vitamin D have immunomodulatory actions that could be useful for type 1 diabetes (T1D). We aimed in this study to investigate the safety and efficacy of ASCs + daily cholecalciferol (VIT D) for 6 months in patients with recent-onset T1D. METHODS: In this prospective, dual-center, open trial, patients with recent onset T1D received one dose of allogenic ASC (1 × 106 cells/kg) and cholecalciferol 2,000 UI/day for 6 months (group 1). They were compared to patients who received chol-ecalciferol (group 2) and standard treatment (group 3). Adverse events were recorded; C-peptide (CP), insulin dose and HbA1c were measured at baseline (T0), after 3 (T3) and 6 months (T6). RESULTS: In group 1 (n = 7), adverse events included transient headache (all), mild local reactions (all), tachycardia (n = 4), abdominal cramps (n = 1), thrombophlebitis (n = 4), scotomas (n = 2), and central retinal vein occlusion at T3 (n = 1, resolution at T6). Group 1 had an increase in basal CP (p = 0.018; mean: 40.41+/-40.79 %), without changes in stimulated CP after mixed meal (p = 0.62), from T0 to T6. Basal CP remained stable in groups 2 and 3 (p = 0.58 and p = 0.116, respectively). Group 1 had small insulin requirements (0.31+/- 0.26 UI/kg) without changes at T6 (p = 0.44) and HbA1c decline (p = 0.01). At T6, all patients (100%; n = 7) in group 1 were in honeymoon vs 75% (n = 3/4) and 50% (n = 3/6) in groups 2 and 3, p = 0.01. CONCLUSION: Allogenic ASC + VIT D without immunosuppression was safe and might have a role in the preservation of ß-cells in patients with recent-onset T1D. ClinicalTrials.gov: NCT03920397.

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.

Recovery of motricity and micturition after transplantation of mesenchymal stem cells in rats subjected to spinal cord injury.

The objective was to evaluate the effect of human adipose-derived stem cell (hADSC) infusion on impaired hindlimb function and urinary continence after spinal cord contusion in rats. hADSCs were transplanted into the injured spinal cords of rats 7 and 14 days after injury in two groups (B and C). Group C also received methylprednisolone sodium succinate (MPSS) after 3 h of injury. The control group (group A) did not receive corticoids or stem cells. Voiding and motor performance evaluations were performed daily for 90 days post-transplantation. Cells were labeled with PKH26 or PKH67 for in vitro monitoring. For in vivo screening, the cells were evaluated for bioluminescence. The levels of some cytokines were quantified in different times. Euthanasia was performed 90 days post-transplant. ß-tubulin III expression was evaluated in the spinal cord of the animals from all groups. As a result, we observed a recovery of 66.6 % and 61.9 % in urinary continence of animals from groups B and C, respectively. Partial recovery of motor was observed in 23.8 % and 19 % of the animals from groups B and C, respectively. Cells remained viable at the site up to 90 days after transplantation. No significant difference was observed in levels of cytokines and thickness of urinary bladders between groups. A smaller percentage of tissue injury and higher concentrations of neuropils were observed in the spinal cords of the animals from groups B and C than control group. Thus, hADSCs transplantation with or without MPSS, contributed to the improvement in voiding and motor performance of Wistar rats submitted to compressive spinal cord injury.

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.

Use of rosiglitazone before and after vascular injury in hypercholesterolemic rabbits: Assessment of neointimal formation.

OBJECTIVES: To analyse the effects of rosiglitazone administered at different times on neointimal formation in hypercholesterolemic rabbits following vascular injury. METHODS: Thirty-nine rabbits on a hypercholesterolemic diet were included. The animals underwent balloon catheter injury to the right iliac artery on day 14. They were divided into three groups as follows: control group, 13 rabbits without rosiglitazone; group I, 13 rabbits treated with rosiglitazone (3 mg/Kg body weight/day) for 28 days after the vascular injury; and group II, 13 rabbits treated with rosiglitazone (3 mg/Kg body weight/day) during all the experiment (42 days). Histological analysis was done by an experienced pathologist who was unaware of the rosiglitazone treatment. Histomorphometric parameters were performed by calculation of the luminal and intimal layer area, and intima/media layer area ratio (the area of the intimal layer divided by the area of the medial layer). RESULTS: Intimal area was significantly lower in group II vs. CG (p = 0.024) and group I (p = 0.006). Luminal layer area was higher in group II vs. CG (p < 0.0001) and group I (p < 0.0001). Intima/media layer area ratio was equal between CG and group I. Intima/media layer ratio area was significantly lower in group II vs. control group (p < 0.021) and group I (p < 0.003). There was a significant reduction of 65% and 71% in intima/media layer area ratio in group II vs. control group and group I, respectively. CONCLUSION: Pretreatment with rosiglitazone in hypercholesterolemic rabbits submitted to vascular injury significantly reduces neointimal formation.