Mesenchymal Stromal Cell Growth and Glycocalyx Morphology are Regulated by Media Glucose Levels and Hypoxia
Authors: Khan Saad , Florian Maria , Davila Liliana , Courtman David 
Published: November 2023

Physiological conditions such as hypoxia and/or chemical environments such as glucose levels in the media function as key regulators of cell behavior. Mesenchymal stromal cells (MSCs) are unique cells which have played an important role in many regenerative medicine technologies. They are characterized by a multi‐lineage potential, ability to self‐renew and immunomodulatory properties. The microenvironment of stem cells may play a crucial role in maintaining their stemness and regenerative potential, and the cellular glycocalyx may be an important cell based regulator within this microenvironment. Hyaluronan (HA), a non‐sulfonated linear glycosaminoglycan found in the glycocalyx of MSCs and other cell types has been implicated in regulation of various physiological processes including inflammation, tissue regeneration, cell proliferation and migration.Here we investigated the effect of hypoxic environments (1% O2 or 5% O2) as well as different glucose concentrations in minimal media on MSC identity, viability and potency as well as production of HA levels in the glycocalyx.MSCs were derived from bone marrow and maintained in different oxygen conditions (1% O2, 5% O2 or room air (~21% O2)). Additionally, bone marrow derived MSCs were also grown in alpha minimal essential media (αMEM) containing either low (1 mg/mL) or high (4.5 mg/mL) levels of glucose. Cell viability was measured microscopically using Trypan blue dye exclusion as well by flow cytometry with annexin V and propidium iodide staining. MSCs were characterized for identity through flow cytometry and potency through the induction of IDO by IFN‐gamma. HA levels were detected with biotin‐conjugated HA binding protein (HABP) followed by FITC‐conjugated streptavidin.Significantly higher numbers of MSCs were derived from bone marrow in 5% O2 (21067 ±1579 cells/cm2) when compared to 1% (14000 ± 5327 cells/cm2) or 21% O2 (10721 ± 3782 cells/cm2). Additionally, cells grown in 5% O2 had significantly lower doubling times of 42.5 ± 3.8 h in passage 1 in comparison to 52.8 ± 4.9 h for cells grown in 21% O2. However, no significant differences in cell morphology, cell identity and cell viability were observed between cells grown in 1%, 5% or 21% O2.Cells grown in high glucose media resulted in significantly enhanced cell yields (39333 ± 6839 cells/cm2) in comparison to those cultured in low glucose media (13111 ± 1736 cells/cm2). MSCs also displayed large intricate networks of high molecular weight (HMW) HA in the glycocalyx as shown by HABP binding and fluorescence microscopy. In comparison, MSCs grown in low glucose media displayed disperse networks of HA. Additionally, there was a 3‐fold increase observed in levels of HA when MSCs were cultured in high glucose media versus cells grown in low glucose media. MSCs in high glucose also displayed enhanced levels of CD44, one of the receptors of HA. Finally, Hyaluronidase treated cells grown in high glucose media displayed a lack of network formation of high molecular HA in the glycocalyx and a 2‐fold decrease in cell yield.These results suggest that the physiological and chemical environment of MSCs can play an important role in their growth characteristics. The balance between HMW and LMW HA as observed through the presence or absence of intricate networks of HA as a part of the cellular glycocalyx could be a significant factor regulating MSC growth and potency.