High glucose concentration in cell culture medium does not acutely affect human mesenchymal stem cell growth factor production or proliferation

Abstract

Optimizing the function and proliferative capacity of stem cells is essential to maximize their therapeutic benefits. High glucose concentrations are known to have detrimental effects on many cell types. We hypothesized that human mesenchymal stem cells (hMSCs) cultured in high glucose-containing media would exhibit diminished proliferation and attenuated production of VEGF, hepatocyte growth factor (HGF), and FGF2 in response to treatment with TNF-alpha, LPS, or hypoxia. hMSCs were plated in medium containing low (5.5 mM) and high (20 mM or 30 mM) glucose concentrations and treated with TNF-alpha, LPS, or hypoxia. Supernatants were collected at 24 and 48 h and assayed via ELISA for VEGF, HGF, and FGF2. In addition, hMSCs were cultured on 96-well plates at the above glucose concentrations, and proliferation at 48 h was determined via bromo-2'-deoxy-uridine (BrdU) incorporation. At 24 and 48 h, TNF-alpha, LPS, and hypoxia-treated hMSCs produced significantly higher VEGF, HGF, and FGF2 compared with control. Hypoxia-induced VEGF production by hMSCs was the most pronounced change over baseline. At both 24 and 48 h, glucose concentration did not affect production of VEGF, HGF, or FGF2 by untreated hMSCs and those treated with TNF-alpha, LPS, or hypoxia. Proliferation of hMSCs as determined via BrdU incorporation was unaffected by glucose concentration of the media. Contrary to what has been observed with other cells, hMSCs may be resistant to the short-term effects of high glucose. Ongoing efforts to characterize and optimize ex vivo and in vivo conditions are critical if the therapeutic benefits of MSCs are to be maximized.

Fig. 1.

Human mesenchymal stem cells (hMSCs) treated with TNF-α, LPS, or hypoxia demonstrated statistically significant increased levels of VEGF, hepatocyte growth factor (HGF), and FGF2 production in all cases except HGF production by hypoxia-treated hMSCs at both 24 and 48 h. Notably, the most striking difference occurred in VEGF expression by hypoxia-treated hMSCs with levels ∼8 times that of control. A: 24 h; B: 48 h. *P < 0.05 vs. control.

Fig. 2.

In untreated hMSCs, glucose concentration did not affect levels of growth factor production at 24 or 48 h.

Fig. 3.

In TNF-α-treated hMSCs, glucose concentration did not affect levels of growth factor production at 24 or 48 h.

Fig. 4.

In LPS-treated hMSCs, glucose concentration did not affect levels of growth factor production at 24 or 48 h.

Fig. 5.

In hypoxia-treated hMSCs, glucose concentration did not affect levels of growth factor production at 24 or 48 h.

Fig. 6.

Glucose concentration had no effect on proliferation at 48 h as measured by 5-bromo-2′-deoxy-uridine (BrdU) incorporation (A). Conversely, renal tubular epithelial cell (RTEC) proliferation at 48 h was significantly diminished at the 20 mM and 30 mM glucose concentrations (B). Medium containing equimolar concentrations of mannitol had no effect on RTEC proliferation. Absorbance is measured at a wavelength of 405 nm with a reference wavelength of 490 nm. *P < 0.05 vs. 5.5 mM group.

Fig. 7.

STAT3 expression in cellular lysates of hMSCs treated with no stimulus (control), TNF-α, LPS, or hypoxia for 24 h (A) or 48 h (B). The concentration of glucose in the cell culture medium was 5.5, 20, and 30 mM. Western blots are representative of typical results. Corresponding control bands and experimental bands are from the same blots.

Fig. 8.

p38 MAPK expression in cellular lysates of hMSCs treated with no stimulus (control), TNF-α, LPS, or hypoxia for 24 h (A) or 48 h (B). The concentration of glucose in the cell culture medium was 5.5, 20, and 30 mM. Western blots are representative of typical results. Corresponding control bands and experimental bands are from the same blots.