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.

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

Abstract 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 βIII-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 βIII-tubulin; the fluorescent signal intensity was significantly higher in βIII-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.

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.

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.

Expanded CD133+ Cells from Human Umbilical Cord Blood Improved Heart Function in Rats after Severe Myocardial Infarction.

Pharmacological approaches are partially effective in limiting infarct size. Cell therapies using a cell population enriched with endothelial progenitor cells (EPCs) CD133+ have opened new perspectives for the treatment of ischemic areas after infarction. This preclinical study evaluated the effect of intramyocardial transplantation of purified or expanded human umbilical cord blood-derived CD133+ cells on the recovery of rats following acute myocardial infarction (AMI). Histology studies, electrocardiogram, and fluorescence in situ hybridization (FISH) were used to evaluate heart recovery. Purified CD133+ cells, enriched in endothelial progenitor cells, when expanded in vitro acquired an endothelial-like cell phenotype expressing CD31 and von Willebrand factor (vWF). The group of infarcted rats that received expanded CD133+ cells had a more significant recovery of contraction performance and less heart remodeling than the group that received purified CD133+ cells. Either purified or expanded CD133+ cells were able to induce neovascularization in the infarcted myocardium in an equivalent manner. Few human cells were detected in the infarcted myocardium of the rats 28 days after transplantation suggesting that the effects observed might be related primarily to paracrine activity. Although both cell populations ameliorated the infarcted heart and are suitable for regeneration of the vascular system, expanded CD133+ cells are more beneficial and promising candidates for vascular regeneration.

The Effect of Mesenchymal Stem Cells on Fertility in Experimental Retrocervical Endometriosis / O efeito das células-tronco mesenquimais na fertilidade em endometriose retrocervical experimental

Abstract Purpose To evaluate the effect of mesenchymal stem cells (MSCs) on fertility in experimental retrocervical endometriosis. Methods A total of 27 New Zealand rabbits were divided into three groups: endometriosis, in which endometrial implants were created; mesenchymal, in which MSCs were applied in addition to the creation of endometrial implants; and control, the group without endometriosis. Fisher's exact test was performed to compare the dichotomous qualitative variables among the groups. The quantitative variables were compared by the nonparametric Mann-Whitney and Kruskal-Wallis tests. The MannWhitney test was used for post-hoc multiple comparison with Boniferroni correction. Results Regarding the beginning of the fertile period, the three groups had medians of 14±12.7, 40±5, and 33±8.9 days respectively (p = 0.005). With regard to fertility (number of pregnancies), the endometriosis and control groups showed a rate of 77.78%, whereas the mesenchymal group showed a rate of 11.20% (p = 0.015). No differences in Keenan's histological classification were observed among the groups (p = 0.730). With regard to the macroscopic appearance of the lesions, the mesenchymal group showed the most pelvic adhesions. Conclusion The use of MSCs in endometriosis negatively contributed to fertility, suggesting the role of these cells in the development of this disease.

Resumo Objetivo Avaliar o efeito das células-tronco mesenquimais sobre a fertilidade na endometriose retrocervical experimental. Métodos Um total de 27 coelhas da raça Nova Zelândia foram divididas em três grupos: endometriose, em que os implantes endometriais foram criados; mesenquimal, em que as células-tronco mesenquimais foram aplicadas complementarmente à criação implantes endometriais; e controle, sem endometriose. O teste exato de Fisher foi realizado para comparar variáveis dicotômicas qualitativas entre os grupos. As variáveis quantitativas foram comparadas pelos testes não paramétricos de MannWhitney e Kruskal-Wallis. O teste de Mann-Whitney foi utilizado para a comparação múltipla pós-hoc com correção de Boniferroni. Resultados em relação ao início do período fértil, os grupos endometriose, mesenquimal e controle tiveram medianas de 14±12,7; 40±5; e 33±8,9 dias, respectivamente (p = 0,005). Sobre a taxa de fertilidade (número de gravidezes), os grupos endometriose e controle mostraram uma taxa de 77,78%, enquanto o grupo mesenquimal mostrou uma taxa de 11,20% (p = 0,015). Não foram observadas diferenças na classificação histológica de Keenan entre os grupos (p = 0,730). No que diz respeito à aparência macroscópica das lesões, o grupo mesenquimal mostrou maiores adesões pélvicas. Conclusão O uso de células-tronco mesenquimais na endometriose contribuiu negativamente para a fertilidade, sugerindo o papel dessas células no desenvolvimento da doença.

The Effect of Mesenchymal Stem Cells on Fertility in Experimental Retrocervical Endometriosis.

Purpose To evaluate the effect of mesenchymal stem cells (MSCs) on fertility in experimental retrocervical endometriosis. Methods A total of 27 New Zealand rabbits were divided into three groups: endometriosis, in which endometrial implants were created; mesenchymal, in which MSCs were applied in addition to the creation of endometrial implants; and control, the group without endometriosis. Fisher's exact test was performed to compare the dichotomous qualitative variables among the groups. The quantitative variables were compared by the nonparametric Mann-Whitney and Kruskal-Wallis tests. The Mann-Whitney test was used for post-hoc multiple comparison with Boniferroni correction. Results Regarding the beginning of the fertile period, the three groups had medians of 14 ± 12.7, 40 ± 5, and 33 ± 8.9 days respectively (p = 0.005). With regard to fertility (number of pregnancies), the endometriosis and control groups showed a rate of 77.78%, whereas the mesenchymal group showed a rate of 11.20% (p = 0.015). No differences in Keenan's histological classification were observed among the groups (p = 0.730). With regard to the macroscopic appearance of the lesions, the mesenchymal group showed the most pelvic adhesions. Conclusion The use of MSCs in endometriosis negatively contributed to fertility, suggesting the role of these cells in the development of this disease.

Objetivo Avaliar o efeito das células-tronco mesenquimais sobre a fertilidade na endometriose retrocervical experimental. Métodos Um total de 27 coelhas da raça Nova Zelândia foram divididas em três grupos: endometriose, em que os implantes endometriais foram criados; mesenquimal, em que as células-tronco mesenquimais foram aplicadas complementarmente à criação implantes endometriais; e controle, sem endometriose. O teste exato de Fisher foi realizado para comparar variáveis dicotômicas qualitativas entre os grupos. As variáveis quantitativas foram comparadas pelos testes não paramétricos de Mann-Whitney e Kruskal-Wallis. O teste de Mann-Whitney foi utilizado para a comparação múltipla pós-hoc com correção de Boniferroni. Resultados em relação ao início do período fértil, os grupos endometriose, mesenquimal e controle tiveram medianas de 14 ± 12,7; 40 ± 5; e 33 ± 8,9 dias, respectivamente (p = 0,005). Sobre a taxa de fertilidade (número de gravidezes), os grupos endometriose e controle mostraram uma taxa de 77,78%, enquanto o grupo mesenquimal mostrou uma taxa de 11,20% (p = 0,015). Não foram observadas diferenças na classificação histológica de Keenan entre os grupos (p = 0,730). No que diz respeito à aparência macroscópica das lesões, o grupo mesenquimal mostrou maiores adesões pélvicas. Conclusão O uso de células-tronco mesenquimais na endometriose contribuiu negativamente para a fertilidade, sugerindo o papel dessas células no desenvolvimento da doença.

Comparison of two surgical techniques for creating an acute myocardial infarct in rats / Comparação de duas técnicas cirúrgicas para criar um infarto agudo do miocárdio em ratos

Objective: To perform a comparative assessment of two surgical techniques that are used creating an acute myocardial infarc by occluding the left anterior descending coronary artery in order to generate rats with a left ventricular ejection fraction of less than 40%. Methods: The study was completely randomized and comprised 89 halothane-anaesthetised rats, which were divided into three groups. The control group (SHAM) comprised fourteen rats, whose left anterior descending coronary artery was not occluded. Group 1 (G1): comprised by 35 endotracheally intubated and mechanically ventilated rats, whose left anterior descending coronary artery was occluded. Group 2 (G2): comprised 40 rats being manually ventilated using a nasal respirator whose left anterior descending coronary artery was occluded. Other differences between the two techniques include the method of performing the thoracotomy and removing the pericardium in order to expose the heart, and the use of different methods and suture types for closing the thorax. Seven days after surgery, the cardiac function of all surviving rats was determined by echocardiography. Results: No rats SHAM group had progressed to death or had left ventricular ejection fraction less than 40%. Nine of the 16 surviving G1 rats (56.3%) and six of the 20 surviving G2 rats (30%) had a left ventricular ejection fraction of less than 40%. Conclusion: The results indicate a tendency of the technique used in G1 to be better than in G2. This improvement is probably due to the greater duration of the open thorax, which reduces the pressure over time from the surgeon, allowing occlusion of left anterior descending coronary artery with higher accuracy. .

Objetivo: Realizar uma avaliação comparativa de duas técnicas cirúrgicas que são usadas para criar um infarto agudo do miocárdio pela oclusão da artéria coronária descendente anterior esquerda, a fim de gerar ratos com uma fração de ejeção ventricular esquerda inferior a 40%. Métodos: O estudo foi completamente randomizado e composto por 89 ratos anestesiados com halotano, que foram divididos dentro de três grupos. O grupo controle (SHAM) composto por 14 ratos, cuja artéria coronária descendente anterior esquerda não foi ocluída. Grupo 1 (G1): composto por 35 ratos intubados endotraquealmente e ventilados mecanicamente, cuja artéria coronária descendente anterior esquerda foi ocluída. Grupo 2 (G2): constituído por 40 ratos sendo ventilados manualmente utilizando um respirador nasal, cuja artéria coronária descendente anterior esquerda foi ocluída. Outras diferenças entre as duas técnicas incluem o método de realizar a toracotomia e remover o pericárdio, a fim de expor o coração, e o uso de diferentes métodos e tipos de sutura para fechar o tórax. Sete dias após a cirurgia, a função cardíaca de todos os ratos sobreviventes foi determinada por ecocardiografia. Resultados: Nenhum rato do grupo SHAM foi a óbito ou teve fração de ejeção ventricular esquerda menor que 40%. Nove dos 16 ratos sobreviventes do G1 (56,3%) e seis dos 20 ratos sobreviventes do G2 (30%) tiveram uma fração de ejeção ventricular esquerda inferior a 40%. Conclusão: Os resultados indicam uma tendência da técnica utilizada no G1 ser melhor do que a do G2. Esta melhora deve-se provavelmente à maior duração do tórax aberto, o que reduz a pressão de tempo sobre o cirurgião, permitindo uma oclusão da artéria coronária descendente anterior esquerda com maior acurácia. .

Comparison of two surgical techniques for creating an acute myocardial infarct in rats.

OBJECTIVE: To perform a comparative assessment of two surgical techniques that are used creating an acute myocardial infarc by occluding the left anterior descending coronary artery in order to generate rats with a left ventricular ejection fraction of less than 40%. METHODS: The study was completely randomized and comprised 89 halothane-anaesthetised rats, which were divided into three groups. The control group (SHAM) comprised fourteen rats, whose left anterior descending coronary artery was not occluded. Group 1 (G1): comprised by 35 endotracheally intubated and mechanically ventilated rats, whose left anterior descending coronary artery was occluded. Group 2 (G2): comprised 40 rats being manually ventilated using a nasal respirator whose left anterior descending coronary artery was occluded. Other differences between the two techniques include the method of performing the thoracotomy and removing the pericardium in order to expose the heart, and the use of different methods and suture types for closing the thorax. Seven days after surgery, the cardiac function of all surviving rats was determined by echocardiography. RESULTS: No rats SHAM group had progressed to death or had left ventricular ejection fraction less than 40%. Nine of the 16 surviving G1 rats (56.3%) and six of the 20 surviving G2 rats (30%) had a left ventricular ejection fraction of less than 40%. CONCLUSION: The results indicate a tendency of the technique used in G1 to be better than in G2. This improvement is probably due to the greater duration of the open thorax, which reduces the pressure over time from the surgeon, allowing occlusion of left anterior descending coronary artery with higher accuracy.