Effects of high glucose conditions on the expansion and differentiation capabilities of mesenchymal stromal cells derived from rat endosteal niche

Abstract

BackgroundMesenchymal stromal cells in the endosteal niche lining compact bone (CB-MSCs) represent a heterogeneous population, all of which contribute to bone repair and remodelling. Hyperglycaemia associated with type 2 diabetes mellitus (T2DM) can delay and impair the bone healing process. Therefore, this study investigated the influences of high (25 mM) glucose conditions on CB-MSC populations isolated from male Wistar rats, versus normal (5.5 mM) glucose conditions; in terms of proliferation (population doublings, PDs), senescence characteristics, stem cell marker expression, colony forming efficiencies (CFEs); and osteogenic/adipogenic differentiation, following extended culture in vitro.ResultsCB-MSCs under both normoglycaemic and hyperglycaemic conditions demonstrated similar morphologies and rapid exponential growth to >300PDs, although high glucose conditions promoted more rapid and persistent proliferation beyond ~50PDs, with few indications of senescence. Limited senescence was confirmed by minimal SA-β-galactosidase staining, low senescence marker (p53, p21waf1, p16INK4a) expression and positive telomere maintenance marker (rTERT, TR) expression. However, telomere lengths varied throughout culture expansion, with hyperglycaemia significantly reducing telomere lengths at PD50 and PD200. Furthermore, CB-MSCs expanded in normal and high glucose conditions remained non-transformed, exhibiting similar MSC (CD73/CD90/CD105), multipotency (CD146) and embryonic (Slug, Snail) markers throughout extended culture, but negligible hematopoietic (CD34/CD45) or pluripotency (Nanog, Oct4) markers. Hyperglycaemia significantly increased CFEs at PD50 and PD100, which decreased at PD200. CB-MSC osteogenic differentiation was also inhibited by hyperglycaemia at PD15, PD100 and PD200, but not at PD50. Hyperglycaemia inhibited CB-MSC adipogenic differentiation to a lesser extent at PD15 and PD50, with reduced adipogenesis overall at PD100 and PD200.ConclusionThis study demonstrates the limited negative impact of hyperglycaemia on the proliferative and stem cell characteristics of heterogeneous CB-MSC populations, although minor sub-population(s) appear more susceptible to these conditions leading to impaired osteogenic/adipogenic differentiation capabilities. Such findings potentially highlight the impact of hyperglycaemia on CB-MSC bone repair capabilities in situ.

Role of Hyperglycemia in the Senescence of Mesenchymal Stem Cells

“…Moreover, the expression of the positive telomere maintenance marker (rTERT, TR) in MSCs in the endosteal niche lining compact bone was downregulated under high glucose conditions. Interestingly, telomere length is altered throughout in vitro expansion, with hyperglycemia markedly decreasing telomere lengths at PD50 and PD200 (Al-Qarakhli et al, 2019). Together, these studies recognize telomere attrition as one of the mechanisms of senescent MSCs in the hyperglycemic microenvironment.…”

“…Moreover, senescent human UCMSCs, also express comparable levels of CD73, CD90, CD105, and marker proteins (Kim et al, 2015). Additionally, when cultured in high and normal glucose conditions, MSCs in the endosteal niche lining compact bone were not altered, demonstrating the presence of analogous MSC (CD73/CD105/CD90), embryonic (Slug and Snail), and multipotency (CD146) markers throughout the extended culture (Al-Qarakhli et al, 2019).…”

“…However, MSCs under hyperglycemia showed negligible pluripotency (Nanog, Oct4) and hematopoietic (CD34/CD45) markers (Al-Qarakhli et al, 2019). Recently, one study showed that stemness markers were significantly lower in insulincontrolled GDM mothers-UCMSCs and diet-controlled GDM mothers-UCMSCs than in normal UCMSCs (Kong et al, 2019).…”

Recovering the osteoblastic differentiation potential of mesenchymal stem cells derived from diabetic rats by photobiomodulation therapy

“…However, these cells may not be completely therapeutically effective when they are exposed to pathological microenvironments that result in functional and molecular alterations. [ 2, 3 ]…”

Efeito da terapia de fotobiomodulação na diferenciação osteoblástica de células-tronco mesenquimais derivadas da medula óssea de ratos diabéticos

“…CTM derivadas da medula óssea (CTM-MO) apresentam elevado potencial de diferenciação osteoblástica e são, até o momento, consideradas o padrão-ouro nas estratégias de engenharia de tecidos ou terapia celular (Cavallo et al, 2011;Nancarrow-Lei et al, 2017), porém o uso dessas células destinadas à regeneração óssea e neovascularização pode ser impactado negativamente por fatores ou doenças pré-existentes (Wu et al, 2019). Logo, a utilização de CTM autólogas com vistas a regeneração óssea pode não ter a mesma eficácia terapêutica se estiverem expostas a microambientes patológicos, como o DM (Merlotti et al, 2010;van de Vyver, 2017;Al-Qarakhli et al, 2019). Estudos prévios evidenciaram que CTM obtidas de ratos diabéticos apresentam atividade celular e potencial osteogênico reduzidos, sugerindo que CTM obtidas de indivíduos com DM podem não representar uma alternativa como tratamento para regeneração óssea, exceto se combinadas com fatores de crescimento específicos (Lu et al, 2003;Qian et al, 2015;Flório et al, 2017 O estado hiperglicêmico pode afetar diversos órgãos e tecidos e problemas esqueléticos associados ao DM são frequentemente relatados (Merlotti et al, 2010;Weinberg et al, 2014;Jiao et al, 2015;Sundararaghavan et al, 2017).…”