Serum-free medium and hypoxic preconditioning synergistically enhance the therapeutic effects of mesenchymal stem cells on experimental renal fibrosis

Abstract

Background Mesenchymal stem cells (MSCs) repair injured tissue in a paracrine manner. To enhance their therapeutic properties, preconditioning with various factors has been researched. We have previously showed that MSCs cultured in serum-free medium (SF-MSCs) promote their immunosuppressive ability, thereby enhancing their anti-fibrotic effect. Here, we examined whether serum-free medium and hypoxic preconditioning synergistically enhance the therapeutic effects of MSCs on renal fibrosis in rats with ischemia–reperfusion injury (IRI). Methods SF-MSCs were incubated under 1% O2 conditions (hypo-SF-MSCs) or 21% O2 conditions (normo-SF-MSCs) for 24 h before collection. After IRI procedure, hypo-SF-MSCs or normo-SF-MSCs were injected through the abdominal aorta. At 7 or 21 days post-injection, the rats were killed and their kidneys were collected to evaluate inflammation and fibrosis. In in vitro experiments, we investigated whether hypo-SF-MSCs enhanced secretion of anti-fibrotic humoral factors using transforming growth factor (TGF)-β1-stimulated HK-2 cells incubated with conditioned medium from hypo-SF-MSCs or normo-SF-MSCs. Results Normo-SF-MSCs showed attenuation of senescence, which increased their proliferative capacity. Although no significant difference in cellular senescence was found between normo-SF-MSCs and hypo-SF-MSCs, hypo-SF-MSCs further increased their proliferative capacity compared with normo-SF-MSCs. Additionally, administration of hypo-SF-MSCs more strongly ameliorated renal fibrosis than that of normo-SF-MSCs. Moreover, although hypo-SF-MSCs strongly attenuated infiltration of inflammatory cells compared with the control rats, which were treated with PBS, this attenuation was almost equal between normo-SF-MSCs and hypo-SF-MSCs. In vitro experiments revealed that hypo-SF-MSCs more significantly inhibited transforming growth factor (TGF)-β/Smad signaling compared with normo-SF-MSCs. Moreover, hypoxic preconditioning increased hepatocyte growth factor (HGF) secretion even under serum-free conditions, whereas knockdown of HGF in hypo-SF-MSCs attenuated inhibition of TGF-β/Smad signaling. Conclusions These results indicate that administration of ex vivo-expanded, hypoxia-preconditioned SF-MSCs may be a useful cell therapy to prevent renal fibrosis.

Simultaneous Visualization of Dual Intercellular Signal Transductions via SERS Imaging of Membrane Proteins Dimerization on Single Cells

“…Furthermore, stem cells (e.g., BMSCs) are capable of secreting a variety of cytokines, such as HGF and TGFβ1, to regulate various biological processes in organisms; however, highly sensitive visualization of the complex intercellular signal transductions and intracellular signaling pathways in this process is a big challenge. The dimerizations of cell membrane proteins induced by the stem cell secretions were imaged by SERS to reveal the key role of stem cells in bioregulation.…”

Enhancing Mesenchymal Stromal Cell Potency: Inflammatory Licensing via Mechanotransduction

“…The concept of inflammatory licensing pervades the field of MSC biology. Previous studies by our group and others have evaluated the effects of inflammatory cytokines ( 4 , 12 , 14 – 16 , 21 , 24 , 39 , 51 ), hypoxia ( 23 , 24 ), serum starvation ( 25 , 26 ), and numerous other culture manipulations. Mechanotransduction of MSC is particularly interesting for several reasons.…”

“…Not wholly anti-inflammatory, the MSC response elicited by these cytokines can include classical pro-inflammatory signals, such as chemokines, metalloproteins, and TNF-α ( 4 , 10 , 12 , 13 , 22 ). Other priming studies have employed culture manipulations to pre-activate MSC including the use of hypoxia ( 23 , 24 ) and low serum ( 25 , 26 ). While promising, these approaches carry additional associated costs as well as down-stream complications to large-scale cell manufacturing regulatory approval ( 5 , 8 , 12 ).…”

Strategies for the induction of anti-inflammatory mesenchymal stem cells and their application in the treatment of immune-related nephropathy

“…In addition, Ishiuchi's study found that serum-free culture and hypoxic pretreatment synergistically enhanced the therapeutic effects of MSCs on renal fibrosis and that hypoxia transformed cellular metabolism into glycolysis. Therefore, it is speculated that the production of antiinflammatory MSCs can be induced by regulating the metabolic reprogramming of MSCs (26).…”