Desenvolvimento do bioprocesso de co-cultivo de mioblastos esqueléticos e células-tronco mesenquimais para a regeneração do miocárdio : modelo em murinos

Orientador : Prof. Dr. Waldemiro GremskiCo-orientador : Prof. Dr. Carlos Ricardo SoccolTese (doutorado) - Universidade Federal do Paraná, Setor de Tecnologia, Programa de Pós-Graduação em Biotecnologia. Defesa: Curitiba, 20/12/2006Inclui referências : f. 87-95Área de concentração: Saúde animal e humanaResumo: No infarto do miocárdio e na doença de Chagas existem alguns mecanismos fisiopatológicos em comum: perda de cardiomiócitos devido à isquemia, à redução da contratilidade e à disfunção cardíaca. A terapia celular vem propondo o tratamento para a falência cardíaca usando vários tipos celulares. Objetivos: Desenvolver e avaliar o método de co-cultivo de mioblastos esqueléticos e célulastronco mesenquimais para a terapia celular no infarto do miocárdio (IM) e na doença de Chagas (DC). Materiais e métodos: IM- 39 ratos completaram o estudo após um mês, 17 ratos receberam a terapia com o produto celular do co-cultivo e 22 ratos, receberam apenas meio na cicatriz. Na DC- 15 ratos completaram o estudo após um mês, 07 ratos receberam o produto celular de co-cultivos autólogos e 08 receberam apenas o meio. Todos os animais realizaram ecocardiograma antes e após um mês de terapia. Os parâmetros analisados foram: fração de ejeção ventricular esquerda (FEVE); volume sistólico e diastólico finais. A análise estatística pela ANOVA. O cocultivo de mioblastos esqueléticos e células-tronco mesenquimais mantido por um período de 14 dias (DMEN, 15% SFB, 1% Antibiótico, IGF-I e dexametasona). Análises histológicas, de rotinas, foram realizadas. Resultados: Entre as provas funcionais verificou-se no grupo IM FEVE, nos animais, que receberam células do co-cultivo de 23,52 8,67 a 31,45 8, 87 (p=0,006) e no grupo 26,68 6,92 a 22,32 6, 94 (p=0,004) e no grupo DC FEVE, nos animais que receberam células do cocultivo de 31,10 5,78 a 53,37 5, 84 (p<0,001) e no grupo controle, 36,21 3,70 a 38,19 7,03 (p= 0,426). O exame histológico revelou, nos modelos de miocardiopatia estudados, miogênese e angiogênese naqueles animais, que receberam o produto celular de co-cultivo. Conclusão: Estes resultados validam o produto celular do co-cultivo de mioblastos esqueléticos e de células-tronco mesenquimais para o tratamento dessas duas doenças. DESCRITORES: Células musculares esqueléticas, células-tronco, regeneração, miocárdio.Abstract: In myocardial infarction and Chagas's disease some physiopathological aspects are common: cardiomyocyte loss due to ischemia leads to a reduction of contractility and heart function. Cell therapy has been proposed for the treatment of heart failure through transplant of various cells types. Objective: To develop and to evaluate the method of co-culture of skeletal muscle (SM) and mesenchymal stem cells (MSC) for cell therapy of heart failure in Myocardial Chagas's disease (MCD) and myocardial post-infarction (MI). Materials and methods: MI- 39 rats completed the study at one month. 17 rats received co-cultured cell therapy and 22 rats, only medium in the scar. MCD- 15 rats completed the study at one month. 7 rats received autogenous coculture cell therapy and 8 animals received only medium. All animals underwent ecocardiographic analysis at baseline and one month. The measures of Left Ventricular Ejection Fraction (LVEF), Left Ventricular End Systolic and Dyastolic Volume were registered and analyzed by ANOVA. The co-culture method of SM and MSC cells was performed at 14 days (medium culture: DMEN, with 15% FCS and 1% Antibiotic, IGF-I and dexamethasone). Standard stain analysis was done in the cells and tissue. Functional Results: MI- LVEF in the animals that had received the cocultured cells: 23,52 8,67 to 31,45 8, 87 p=0,006 versus control group: 26,68 6, 92 to 22,32 6, 94 p=0,004 and MCD- LVEF in animals that received the co-cultured cells: 31,10 5,78 to 53,37 5, 84 p<0,001 versus control group: 36,21 3,70 to 38,19 7,03 p= 0,426. Histopathogical Results: In both experimental model diseases, the analysis of the animals receiving co-cultured cells demonstrated a presence of myogenesis and angiogenesis. Conclusion: The results validate the product of the SM and MSC co-culture process for treatment in these diseases. KEY WORDS: Skeletal muscle cells, stem cell, regeneration, myocardium

Stem Cell Therapy in Heart Diseases: A Review of Selected New Perspectives, Practical Considerations and Clinical Applications

Degeneration of cardiac tissues is considered a major cause of mortality in the western world and is expected to be a greater problem in the forthcoming decades. Cardiac damage is associated with dysfunction and irreversible loss of cardiomyocytes. Stem cell therapy for ischemic heart failure is very promising approach in cardiovascular medicine. Initial trials have indicated the ability of cardiomyocytes to regenerate after myocardial injury. These preliminary trials aim to translate cardiac regeneration strategies into clinical practice. In spite of advances, current therapeutic strategies to ischemic heart failure remain very limited. Moreover, major obstacles still need to be solved before stem cell therapy can be fully applied. This review addresses the current state of research and experimental data regarding embryonic stem cells (ESCs), myoblast transplantation, histological and functional analysis of transplantation of co-cultured myoblasts and mesenchymal stem cells, as well as comparison between mononuclear and mesenchymal stem cells in a model of myocardium infarction. We also discuss how research with stem cell transplantation could translate to improvement of cardiac function

Macrolide-Clarithromycin Task-Force for the Treatment and Prophylaxis of Covid-19 as a Single Agent

SARS-CoV-2 is a novel RNA coronavirus responsible of a deadly pandemic: the clinical illness COVID-19. With only one authorized drug for emergency use in critically ill patients: Remdesivir, there is not any other approved drug or vaccine yet with proven potential to overcome this infection. We exposed here many scientific evidences to support our novel idea that a macrolide, basically Clarithromycin, could be effective as a single agent for treatment and prophylaxis of COVID-19. Clarithromycin could change the history of this pandemic. It could reduce the costs of treatment and the potential adverse effects when combining more than one drug such as with Hydroxychloroquine. Clarithromycin treatment and prophylaxis as a single agent could be much more simple, safe and cheaper as giving Chloroquine or Hydroxychloroquine alone or in combination with Azithromycin as well as other therapeutic options.Fil: Mansilla, Eduardo. Universidad Nacional de La Plata. Facultad de Ciencias Médicas; ArgentinaFil: Martínez, Ricardo Rangel. ExomePharma; MéxicoFil: Marin, Gustavo Horacio. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de La Plata. Facultad de Ciencias Médicas; ArgentinaFil: Filho, Idiberto Zotarelli. Faceres Medical School; BrasilFil: Rivas, Elsa. Hospital de la Amistad Perú - Corea San Rosa II; PerúFil: Rivas, Jaime. Hospital de la Amistad Perú - Corea San Rosa II; PerúFil: Carvalho, Katherine Athayde Teixeira de. Research Institute Pelé Little Prince; BrasilFil: Dayer, Mohammad Reza. Shahid Chamran University of Ahvaz; IránFil: Samadikuchaksaraei, Alí. Iran University of Medical Sciences; Irá

Why not &#x22;do simple things in a simple way&#x22;: Use of the Pap test as the first step in screening genetic stability for human cultured stem cell therapy?

The aim of this study was to analyze adipose tissue-derived mesenchymal stem cells (AT-MSCs) using the Pap test as a first screening step to evaluate genetic stability. Human adipose tissue from six healthy female donors was obtained from elective liposuction procedures. The cells were isolated, cultivated at P2/P3, characterized by flow cytometric analysis, and differentiation induced. The AT-MSCs were stained by Papanicolaou staining and analyzed according to the Bethesda classification, and viability-apoptosis relationships were evaluated. The results of the Pap test for Sample I indicated high-grade alterations consistent with genetic instability; for Samples II-V, atypical cells of undetermined significance; and for Sample VI, normal cells. These results demonstrate the potential of using the Pap test as an initial screening step to evaluate the genetic stability of cultured AT-MSCs and also suggest its use for other adherent cells such as embryonic stem cells or induced pluripotent stem cells

Echocardiographic assessment of myocardial infarction evolution in young and adult rats

BACKGROUND: The regeneration of cardiomyocytes after a myocardial infarction (MI) is more evident in young animals; however, it is not known whether it is associated with functional improvement. OBJECTIVE: To perform the functional analysis by echocardiography (echo) of young adult rats submitted to MI. METHODS: Seventy-two animals were included in the study: 35 young rats (group Y) that were 28 days old and 37 adult rats (group A) that were 153 days old. The rats were subdivided in two subgroups: infarcted (YI and AI) and control (YC and AC). The animals were assessed by echocardiogram on the 7thand 30th postoperative days for the analysis of the ejection fraction (EF) and the final systolic (FSV) and diastolic volume (FDV) of the left ventricle. Only animals with EF < 40% were included in the study. RESULTS: The comparison of the FDV and FSV between infarcted and control animals showed that there was a significant increase in infarcted adult animals at the two analyzed phases. Among young animals only the FSV was significantly higher on the 7th day. The intragroup evolution analysis showed an increase in FDV and FSV in the two young subgroups, which was proportional to growth and only increase in FDV in the infarcted adult group. There was an improvement in EF in young rats, whereas EF remained decreased in adult rats when compared to controls. CONCLUSION: The infarcted young rats presented improvement in the systolic function and ventricular volumes 30 days after the infarction, whereas the adult rats presented increased FDV with no improvement in systolic function.FUNDAMENTO: A regeneração dos cardiomiócitos após o infarto do miocárdio (IM) é mais evidente em animais jovens; entretanto, não se sabe se é acompanhada de melhora funcional. OBJETIVO: Realizar a análise funcional pela ecocardiografia (eco) de ratos jovens e adultos submetidos a IM. MÉTODOS: Setenta e dois animais foram incluídos no estudo: 35 ratos jovens (grupo J) com 28 dias, e 37 ratos adultos (grupo A) com 153 dias. Os ratos foram subdivididos em dois subgrupos: infartado (JI e AI) e controle (JC e AC). Os animais foram avaliados por meio de ecocardiograma no 7º e 30º dias de pós-operatório para análise da fração de ejeção (FE) e dos volumes sistólico (VSF) e diastólico (VDF) finais do ventrículo esquerdo. Foram incluídos no grupo de estudo somente animais com FE menor que 40%. RESULTADOS: Na comparação dos VDF e VSF entre infartados e controles, observou-se aumento significativo nos animais adultos infartados nas duas fases analisadas. Nos animais jovens, apenas o VSF, no 7º dia, foi significativamente maior. Na evolução intragrupo, observou-se aumento do VDF e do VSF nos dois subgrupos jovens, proporcional ao crescimento, e somente aumento do VDF no grupo adulto infartado. Houve melhora da FE nos ratos jovens, enquanto nos ratos adultos a FE permaneceu diminuída em relação aos controles. CONCLUSÃO: Os ratos jovens infartados apresentaram melhora da função sistólica e dos volumes ventriculares após 30 dias do infarto, enquanto nos ratos adultos houve aumento do VDF sem melhora da função sistólica.Pontifícia Universidade Católica do ParanáUniversidade Federal de São Paulo (UNIFESP)UNIFESPSciEL

Regeneration of Tracheal Tissue in Partial Defects Using Porcine Small Intestinal Submucosa

Background. Surgical correction of tracheal defects is a complex procedure when the gold standard treatment with primary end-to-end anastomosis is not possible. An alternative treatment may be the use of porcine small intestinal submucosa (SIS). It has been used as graft material for bioengineering applications and to promote tissue regeneration. The aim of this study was to evaluate whether SIS grafts improved tracheal tissue regeneration in a rabbit model of experimental tracheostomy. Methods. Sixteen rabbits were randomized into two groups. Animals in the control group underwent only surgical tracheostomy, while animals in the SIS group underwent surgical tracheostomy with an SIS graft covering the defect. We examined tissues at the site of tracheostomy 60 days after surgery using histological analysis with hematoxylin and eosin (H&E) staining and analyzed the perimeter and area of the defect with Image-Pro® PLUS 4.5 (Media Cybernetics). Results. The average perimeter and area of the defects were smaller by 15.3% (p=0.034) and 21.8% (p=0.151), respectively, in the SIS group than in the control group. Histological analysis revealed immature cartilage, pseudostratified ciliated epithelium, and connective tissue in 54.5% (p=0.018) of the SIS group, while no cartilaginous regeneration was observed in the control group. Conclusions. Although tracheal SIS engraftment could not prevent stenosis in a rabbit model of tracheal injury, it produced some remarkable changes, efficiently facilitating neovascularization, reepithelialization, and neoformation of immature cartilage

Benefit of stem cells and skeletal myoblast cells in dilated cardiomyopathies

Although some authors suggest that there is mitotic division in the heart, most cardiomyocytes do not have the capacity to regenerate after myocardial infarction and when this occurs there is a deterioration of contractile function, and if the area of ​​infarction is extensive ventricular remodeling may occur, leading to the development of heart failure. Cell transplantation into the myocardium with the goal of recovery of cardiac function has been extensively studied in recent years. The effects of cell therapy are based directly on the cell type used and the type of cardiac pathology. For myocardial ischemia in the hibernating myocardium, bone marrow cells have functional benefits, however these results in transmural fibrosis are not evident. In these cases there is a benefit of implantation with skeletal myoblasts, for treating the underlying cause of disease, the loss of cell contractility

Natural Membrane Differentiates Human Adipose-Derived Mesenchymal Stem Cells to Neurospheres by Mechanotransduction Related to YAP and AMOT Proteins

Adipose tissue-derived mesenchymal stem cells (ADMSCs) are promising candidates for regenerative medicine, as they have good cell yield and can differentiate into several cell lines. When induced to the neuronal differentiation, they form neurospheres composed of neural precursors (NPs) that can be an alternative in treating neurodegenerative diseases. This study aimed to characterize NPs from neurospheres obtained after seeding ADMSCs on a natural polyisoprene-based membrane. The ADMSCs were isolated from adipose tissue by enzymatic dissociation, were subjected to trilineage differentiation, and were characterized by flow cytometry for specific ADMSC surface markers. For neuronal differentiation, the cells were seeded on polystyrene flasks coated with the membrane and were characterized by immunocytochemistry and RT-PCR. The results demonstrated that the isolated cells showed characteristics of ADMSCs. At 15 to 25 days, ADMSCs seeded on the natural membrane developed neurospheres. Then, after dissociation, the cells demonstrated characteristic neuronal markers expressed on NPs: nestin, ß-III tubulin, GFAP, NeuN, and the YAP1/AMOT in the cytoplasm. In conclusion, it was demonstrated that this membrane differentiates the ADMSCs to NPs without any induction factors, and suggests that their differentiation mechanisms are related to mechanotransduction regulated by the YAP and AMOT proteins