Priming mesenchymal stem cells with uric acid enhances neuroprotective properties in parkinsonian models
Mesenchymal stem cells (MSCs) are a potential source of cell-based disease-modifying therapy in Parkinsonian disorders. A promising approach to develop in vitro culture methods that mimic natural MSC niche is cell priming. Uric acid (UA), a powerful antioxidant, scavenges reactive oxygen species, which has a vital role in maintaining the self-renewal and differentiation potential of MSCs. Here, they demonstrated that UA treatment in naïve MSCs stimulated glycolysis and upregulated transcriptional factors responsible for the regulation of stemness, leading to an increase in the expression levels of osteogenesis-, adipogenesis-, and chondrogenesis-related genes. UA-primed MSCs had more enhanced neuroprotective properties in cellular and parkinsonian animal models compared to naïve MSCs by inhibiting apoptotic signaling pathways. Additionally, expression of miR-137 and miR-145 was decreased in UA-treated MSCs. Their data demonstrated that priming MSCs with UA augment neuroprotective properties through enhanced self-renewal and differentiation potential, suggesting a practical strategy for improving the application of MSCs in parkinsonian disorders.
Parkinson’s disease (PD) is characterized pathologically by the progressive loss of dopaminergic neurons in the substantia nigra (SN) and the presence of Lewy bodies, proteinaceous fibrillar cytoplasmic inclusions that are mainly composed of aggregated α-synuclein. The mainstay of PD management is symptomatic treatment with drugs that increase dopamine concentrations or directly stimulate dopamine receptors. Nevertheless, these therapies do not affect the progressive nature of PD and, moreover, they are ineffective against some axial parkinsonian symptoms and various non-motor symptoms. Thus, it is crucial to develop disease-modifying treatments that reduce the rate of neurodegeneration or stop the disease process in PD. Recently, the concept of stem cell therapy has been extended to adult stem cells, which secrete biologically active molecules, exerting beneficial effects on their surroundings. Previous studies have shown that mesenchymal stem cells (MSCs) can act as potent modulators of PD-related neurodegenerative microenvironments through the modulation of neuroinflammation, inhibition of apoptosis, increased neurogenesis, and neuronal differentiation, enhancement of autophagy, and modulation of α-synuclein propagation. However, the major challenge in MSC-based therapies is to develop in vitro culture systems that mimic the natural MSC niche, while allowing clinical scale cell expansion without compromising the quality and function of cells. To date, several studies have demonstrated that the modulation of biological, biochemical, and/or biophysical factors can influence the fate, lineage-specific differentiation, functions, and therapeutic potential of MSCs.One approach is cell priming. Many studies have demonstrated the effects of MSC priming with hypoxia, cytokines, growth factors, pharmacological or other chemical agents, biomaterials, and different culture conditions.
Uric acid (UA), a purine metabolite, is a powerful antioxidant, which can be found intracellularly and in all body fluids.17–19 It not only scavenges reactive oxygen species (ROS) but also blocks the reaction of the superoxide anion with nitric oxide, which can injure cells by nitrosylating the tyrosine residues of proteins. It also prevents extracellular superoxide dismutase degradation. Ample evidence has suggested that UA has neuroprotective properties in PD, showing that PD patients with higher UA levels have been linked to reduced risk of PD incidence as well as slower disease progression. The beneficial effects of UA have also been observed in other neurodegenerative diseases, such as amyotrophic lateral sclerosis, Alzheimer’s disease, and Huntington’s disease. Hence, UA has not only antioxidant properties but also neuroprotective properties against neurodegenerative conditions.
Stemness encompasses the maintenance of self-renewal and multi-lineage differentiation potential. The functions fulfilled by specialized stem cells, such as stem cell proliferation, lineage specification, and quiescence require a certain energy supply. Glycolysis is the enzymatic conversion of glucose to pyruvate, which generates two net ATP molecules per glucose molecule. However, cells in oxygen-rich environments may prefer oxidative phosphorylation (OXPHOS), which leads to a more efficient ATP production by oxidizing pyruvate to acetyl-CoA in the mitochondrial tricarboxylic acid cycle. Many types of stem cells rely on glycolysis when they are undifferentiated, but they activate the mitochondrial OXPHOS process during differentiation. Stimulation of glycolysis in pluripotent or adult stem cells by hypoxia or supplementation with insulin promotes stemness, while glycolysis inhibition halts proliferation and induces cell death. In terms of pluripotency genes, OCT4, NANOG, and SOX2 constitute the core regulatory network that suppresses differentiation-associated genes, thereby maintaining cell pluripotency OCT4, a key transcription factor essential for self-renewal and survival of MSC interacts with other embryonic regulators, such as SOX2 and NANOG, to regulate the network that maintains pluripotency and inhibits differentiation. Moreover, OCT4 has a number of targets associated with energy metabolism, which may impact the balance between glycolysis and oxidative metabolism.
ROS resulting from cellular metabolism is crucial for stemness and stem cell differentiation. Differentiation stimuli cause elevated ROS levels, thus inducing stem cell differentiation into specific lineages. However, glycolysis enhancement via hypoxia and OXPHOS suppression, which leads to concomitantly decreased ROS levels, promotes stem cell maintenance and proliferation, thereby repressing differentiation. On the other hand, ROS can also lead to stem cell dysfunction, leading to senescence with loss of stemness. Thus, antioxidants would be a core relationship between redox homeostasis and pluripotency of stem cells.
A schematic illustration of the biological action and mechanistic role of UA-priming of MSCs in parkinsonian models. UA treatment in naïve MSCs stimulated glycolysis and upregulated transcriptional factors responsible for regulation of stemness, which increased the expression levels of osteogenesis-, adipogenesis-, and chondrogenesis-related genes via modulation of MiR-137. Priming MSCs with UA increased neuroprotective properties relative to naïve MSCs. These data suggest that MSC priming with UA can provide a strategy to improve MSC application to the treatment of parkinsonian disorders.
In the present study, they hypothesized that UA can efficiently decrease ROS levels against oxidative stress and thus play a central role in stemness maintenance. To prove this, they evaluated whether UA treatment enhances stemness properties in MSCs. Moreover, they tested whether UA-primed MSCs exert a neuroprotective effect in PD animal models, thus providing a strategy to improve MSC application in tissue engineering.1
(a)–(f) Quantitative RT-PCR showed that UA-treated MSCs led to increased mRNA levels of glycolysis enzymes, such as HK2, PFKFB3, PKM, ALDOA, and LDH, compared to the control MSCs. (g) The L-lactate levels were increased in the UA-treated MSCs compared to those in control MSCs. All data are presented as the mean±SE. *p<0.05; **p<0.01.
(a) FACS analysis revealed that both control MSCs and 200 μM UA-treated MSCs expressed CD44 and CD105, but did not express CD34 and CD45, (b) MTS analysis showed that UA-treated MSCs significantly increased a cell density compared to control MSCs in a time-dependent manner, regardless of UA concentration, (c) Quantitative RT-PCR showed that the expression levels of the senescence markers p16 and p21 did not increase after 24 h incubation with 200 μM UA, (d) Quantitative RT-PCR and western blotting showed that the expression levels of the stemness markers OCT4, NANOG, and SOX2 increased after 24 h incubation with 200 μM UA, (e) Quantitative RT-PCR showed that UA-treated MSCs exhibited increased expression levels of osteogenesis (RUNX2, ALP), adipogenesis (PPARG, RHOA), and chondrogenesis (HAT1, BMP4)-related genes compared to naïve MSCs after induction with a differentiation medium, (f) Immunostaining showed that UA-treated MSCs markedly increased differentiation potential of MSCs toward osteogenesis, adipogenesis, and chondrogenesis after induction with a differentiation medium. Scale bar represents 200 μm, and (g) Quantitative RT-PCR and western blotting showed that expression levels of OCT4, NANOG, and SOX2 that were significantly increased after 24 h h of 200 μM UA treatment were dramatically decreased after UA removal.
(a) Cell viability showed that co-culture of MPP+-treated cells with primed MSCs resulted in significantly increased cell viability compared to the MPP+-treated, co-cultured with control MSCs, and co-cultured with primed MSCs with UA wash out groups. (b) ROS activity showed that co-culture of MPP+-treated cells with primed MSCs significantly inhibited ROS generation compared to the MPP+-treated, co-cultured with control MSCs, and co-cultured with primed MSCs with UA wash out groups. Scale bar represents 20 μm, (c) LDH cytotoxicity assay showed that co-culture of MPP+-treated cells with primed MSCs significantly inhibited ROS generation compared to the MPP+-treated, co-cultured with control MSCs, and co-cultured with primed MSCs with UA wash out-groups. (d)–(g) Western blotting for apoptosis markers showed that co-culture with primed MSCs significantly attenuated cleaved caspase-3, cytochrome c, and Bax, and increased Bcl-2 expression compared to the co-culture with control or primed MSCs with UA wash out.
1. Kim HN, Shin JY, Kim DY, Lee JE, Lee PH. Priming mesenchymal stem cells with uric acid enhances neuroprotective properties in parkinsonian models. Journal of Tissue Engineering. January 2021. doi:10.1177/20417314211004816