Caracterização das células-tronco mesenquimais de medula óssea humana de pacientes com osteoartrite purificadas por adesão diferencial

• Autor(a) principal: Claudino, Gustavo Paris
• Data de Publicação: 2019
• Tipo de documento: Dissertação
• Idioma: por
• Título da fonte: Biblioteca Digital de Teses e Dissertações da UERJ
• Texto Completo: http://www.bdtd.uerj.br/handle/1/7807
• topic: Mesenchymal stem cells; Purification; Perivascular; Adhesion; CD146; Célula-tronco mesenquimal; Purificação; Adesão; Perivascular; CD146; Células-tronco; Células-Tronco Mesenquimais; Osteoartrite; CNPQ::CIENCIAS BIOLOGICAS::MORFOLOGIA

Bone marrow mesenchymal stem cells (MSC) have been thoroughly explored due to its therapeutic effects as multipotent, immunosuppressing and anti-apoptotic cells. However, since MSC are isolated and identified from marrow cells using plastic adhesion and precise cell markers are absent, marked heterogeneity persists in the so-called MSC population. Therefore, progenitors and differentiated cells are mixed in MSC population, hampering true multipotent MSC isolation and characterization. The present study proposes an adhesion-based protocol to isolate bone marrow MSC by culturing the commonly discarded supernatant after 3 days culture, named here as late-adhesion MSC (MSC-LA). MSC-LA formed larger primary colonies, in addition to larger secondary colonies and frequency than the standard isolation protocol (MSC-RA). Besides, MSC-LA displayed clearly more adipogenic capacity while maintaining equal osteogenic potential as MSC-RA. In cytometry characterization, most cells presented the CD45-/CD31-/CD73+/CD90+/CD29+/CD44+ phenotype for both populations. While MSC-RA showed greater CD45+, HLA+ and CD184+ cells, MSC-LA displayed enhanced purity in CD24+, CD49d+ and CD146+ cells. Also, CD146+ MSC-LA cells also had higher median fluorescent intensity. Moreover, smooth muscle -actin was present in cells from both populations. Since CD146+ and smooth muscle -actin+ are highly correlated to perivascular cells, which are enriched for undifferentiated MSC, we sought to define their role in vessel formation and development. Human microvascular endothelial cells-GFP+ were co-cultivated in matrigel with MSC-AR or MSC-AL PKH26red+ (5:1), for 72hours. Throughout the whole experiment, both MSCs were observed surrounding vessels at the abluminal side in tight proximity to endothelial cells. Both MSC populations induced anti-angiogenic effects during 72 hours. Therefore, a higher proliferation potential, less CD45+ and HLA+ contamination while enriched for CD146+ cells was seen in the MSC-LA group. As noted, MSC-LA is capable of regulating and stabilizing the endothelial vessels. Thus, MSC-LA correlates to a perivascular MSC population, which presents more undifferentiated characteristics, confirming enhanced purity obtained by the adhesion-based protocol. Future work will determine the therapeutic potential and precise phenotype for primitive MSC.