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

Ahmed Makki A Al-Qarakhli,Rachel J Waddington,Norhayati Yusop,Ryan Moseley

doi:10.1186/s12860-019-0235-y

Ahmed Makki A Al-Qarakhli, Rachel J Waddington + Show 2 more

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https://doi.org/10.1186/s12860-019-0235-y

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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.

Highlights

Mesenchymal stromal cells in the endosteal niche lining compact bone (CB-mesenchymal stromal cells (MSCs)) represent a heterogeneous population, all of which contribute to bone repair and remodelling
High glucose conditions have no negative impact on CBMSC population doublings and expansion As high glucose conditions have primarily been shown to reduce proliferative capabilities in bone marrowderived MSCs (BM-MSCs) due to the induction of replicative senescence [30,31,32, 37], initial studies assessed the effects of normal (5.5 mM) and high (25 mM) glucose condition on Compact bone-derived (CB-MSCs) expansion over ~ 350 days in culture, expressed as cumulative Peroxisomal proliferator-activated receptor-γ (PDs) (Fig. 1a)
Extended CB-MSC culture in high and low glucose conditions alters cell size and morphology To further examine whether extended CB-MSC expansion over ~ 350 days in culture under normoglycaemic and hyperglycaemic condition induced changes in cell size or morphology, CB-MSC morphological characteristics were assessed by cytoskeletal staining with fluorescein isothiocyanate (FITC)-phalloidin and fluorescence microscopy

Summary

Introduction

Mesenchymal stromal cells in the endosteal niche lining compact bone (CB-MSCs) represent a heterogeneous population, all of which contribute to bone repair and remodelling. The endosteal niche exists at the interface between trabecular bone and bone marrow, organised around hematopoietic cells and uncommitted MSCs interacting with pre-osteoblasts and osteoblasts lining the compact bone [9, 10] These more committed, lineage restricted bone lining progenitor cells have been proposed to represent the first responder cells during mineralised tissue repair and remodelling, with the immature MSCs subsequently proliferating to replace the former cell population [11, 12]. More prolonged culture expansion resulted in the more highly proliferative immature MSCs and transit amplifying (TA) cells dominating the cell population, which is commensurate with increased bi-potentiality for osteogenic and adipogenic induction These cells were able to continue to proliferate to beyond PD100 without exhibiting any classical characteristics of cellular senescence [12]

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