Caracterização da célula-tronco mesenquimal expandida in vitro em condições de hipóxia e normóxia

Introdução: As células-tronco mesenquimais (CTMs) são consideradas células estromais multipotentes não hematopoiéticas com propriedades de autorrenovação e de diferenciação. Condições ótimas para cultura das CTMs tem sido alvo de investigação. A tensão de oxigênio utilizada no cultivo vem sendo estudada e parece exercer um papel importante no comportamento biológico de células mesenquimais. Objetivos: Caracterizar as CTMs em condições de hipóxia e normóxia comparando suas características morfológicas e funcionais. Métodos: Foram cultivadas células-tronco mesenquimais obtidas de 15 doadores saudáveis de medula óssea. As CTMs obtidas de cada doador foram separadas em duas condições de cultivo, normóxia (21% de O2) e hipóxia (três doadores em 1%, três doadores em 2%, cinco doadores em 3% e quatro doadores em 4% de O2) até segunda passagem. As CTM foram avaliadas quanto à proliferação, diferenciação, imunofenotipagem, tamanho e complexidade celular, estresse oxidativo, atividade mitocondrial e autofagia. Resultados: As condições de cultivo empregadas não pareceram afetar as características imunofenotípicas e plasticidade celular, no entanto as células submetidas à hipóxia apresentaram menor tamanho e maior complexidade celular, além de menor proliferação (p<0.002). Além disso, as células cultivadas em baixas tensões de O2 apresentaram menor atividade mitocondrial (p<0.03) e uma menor tendência à autofagia embora o stress oxidativo não tenha diferido entre os grupos (p< 0.39). Conclusão: A tensão de oxigênio parece ser um regulador chave na adaptação celular in vitro e efeitos metabólicos subjacentes a esta variável continuam a ser descritos. A heterogeneidade ou até mesmo escassez de resultados acerca do impacto da concentração de oxigênio utilizada na expansão de CTM sublinha a necessidade de novos estudos, a fim de aperfeiçoar as condições de cultivo e expansão e obter maior segurança e eficácia terapêutica.Introduction: Mesenchymal stem cells (MSC) are considered multipotent stromal, nonhematopoietic cells with properties of self-renovation and differentiation. Optimal conditions for culture of MSC have been under investigation. The oxygen tension used for cultivation has been studied and appears to play an important role in biological behavior of mesenchymal stem cells. Objectives: Characterize MSC in hypoxia and normoxia conditions comparing their morphological and functional characteristics. Methods: bone marrow-derived mesenchymal stem cells obtained from 15 healthy donors and cultured. MSC obtained from each donor were separated into two cultivation conditions normoxia (21 % O2) and hypoxia (three donors at 1 %, three donors at 2 %, five donors at 3 %, and four donors at 4 % O2) up to passage two. MSC were evaluated for proliferation, differentiation, immunophenotyping, size and cell complexity, oxidative stress, mitochondrial activity, and autophagy. Results: Culture conditions applied didn't seem to affect immunophenotypic features and cellular plasticity. However, cells subjected to hypoxia showed smaller size and greater cellular complexity, besides lower proliferation (p<0.002). Furthermore, cells cultured in low O2 tension had lower mitochondrial activity (p<0.03) and a reduced tendency to autophagy, although oxidative stress didn't vary among groups (p< 0.39). Conclusion: Oxygen tension seems to be a key regulator of cellular adaptation in vitro, and metabolic effects underlying this variable remain undescribed. Heterogeneity or even lack of results on the impact of oxygen concentration used for expanding MSC highlights the need for further research, in order to optimize conditions of cultivation and expansion and achieve greater safety and therapeutic efficacy

Bone marrow mesenchymal stem cells in acute-on-chronic liver failure grades 2 and 3 : a phase I-II randomized clinical trial

Introduction: Acute-on-chronic liver failure (ACLF) is an acute liver decompensation in cirrhotic patients, which leads to organ failures and high short-term mortality. The treatment is based on the management of complications and, in severe cases, liver transplantation. Since specific treatment is unavailable, we aimed to evaluate the safety and initial efficacy of bone marrow mesenchymal stem cells (BM-MSC) in patients with ACLF Grades 2 and 3, a population excluded from previous clinical trials. Methods: This is a randomized placebo-controlled phase I-II single center study, which enrolled 9 cirrhotic patients from 2018 to 2020, regardless of the etiology. The control group (n=5) was treated with standard medical therapy (SMT) and placebo infusion of saline. The intervention group (n=4) received SMT plus 5 infusions of 1 × 10^6 cells/kg of BM-MSC for 3 weeks. Both groups were monitored for 90 days. A Chi-square test was used for qualitative variables, and the t-test and Mann–Whitney U test for quantitative variables. The Kaplan–Meier estimator was used to build survival curves. In this study, we followed the intention-totreat analysis, with a significance of 5%. Results: Nine patients with a mean Child–Pugh (CP) of 12.3, MELD of 38.4, and CLIF-C score of 50.7 were recruited. Hepatitis C and alcohol were the main etiologies. )e average infusion per patient was 2.9 and only 3 patients (2 in control and 1 in the BM-MSC group) received all the protocol infusions. There were no infusion-related side effects, although one patient in the intervention group presented hypernatremia and a gastric ulcer, after the third and fifth infusions, respectively. )e survival rate after 90 days was 20% (1/5) for placebo versus 25% (1/4) for the BM-MSC. The patient who completed the entire MSC protocol showed a significant improvement in CP (C-14 to B-9), MELD (32 to 22), and ACLF (grade 3 to 0). Conclusion: BM-MSC infusion is safe and feasible in patients with ACLF Grades 2 and 3

ß-globin gene cluster haplotypes and clinical severity in sickle cell anemia patients in southern Brazil

Hematopoietic stem cell transplantation (HSCT) has emerged as a curative strategy for sickle cell anemia (SCA); it is necessary to find markers of SCA clinical severity to spare those SCA patients whose clinical course is mild from the morbidity and mortality associated with HSCT. Haplotypes have been correlated with the severity of clinical manifestations in SCA patients, and fetal hemoglobin (HbF) and socioeconomic status (SeS) have also been described as negative factors. We studied these factors and their impact on clinical manifestations in a population of Southern Brazilian patients attending the Center for Sickle Cell Anemia at Hospital de Clínicas de Porto Alegre/RS, Brazil. Clinical severity was defined as two or more veno-occlusive episodes per year. The βS haplotypes were determined by PCR in 75 SCA patients. Among the 150 βS chromosomes analyzed, 99 (66%) were identified as Bantu (Ban), 41 (27%) as Benin (Ben), and 10 (7%) as other haplotypes. Most patients in our sample (62.7%) belonged to lower SeS groups, precluding meaningful statistical analysis of SeS impact on clinical severity. There was no correlation between haplotypes or HbF level and SCA clinical severity. Gene polymorphisms and environmental issues have to be taken into consideration

Células-tronco mesenquimais – caracterização, cultivo, propriedades imunológicas e aplicações clínicas

As células-tronco mesenquimais (CTM) são consideradas células multipotentes não hematopoéticas com propriedades de autorrenovação e capacidade de diferenciação em tecidos mesenquimais e, possivelmente em não mesenquimais. Vários estudos recentes tem reforçado o carater multipotente destas células pela capacidade de diferenciarem-se em células derivadas da mesoderma embrionário: osteócitos, condroblastos e adipócitos.Devido ao fácil isolamento e cultivo, potencial de diferenciação e produção de fatores de crescimento e citocinas, as CTM têm se tornado as candidatas ideais para os protocolos da medicina regenerativa.Este artigo revisa as principais características desta célula, forma de obtenção e cultivo, propriedades imunológicas e aplicações clínicas