Thermostable Basic Fibroblast Growth Factor Enhances the Production and Activity of Human Wharton's Jelly Mesenchymal Stem Cell-Derived Extracellular Vesicles.

Wharton's jelly-derived mesenchymal stem cell (WJ-MSC)-derived exosomes contain a diverse cargo and exhibit remarkable biological activity, rendering them suitable for regenerative and immune-modulating functions. However, the quantity of secretion is insufficient. A large body of prior work has investigated the use of various growth factors to enhance MSC-derived exosome production. In this study, we evaluated the utilization of thermostable basic fibroblast growth factor (TS-bFGF) with MSC culture and exosome production. MSCs cultured with TS-bFGF displayed superior proliferation, as evidenced by cell cycle analysis, compared with wild-type bFGF (WT-bFGF). Stemness was assessed through mRNA expression level and colony-forming unit (CFU) assays. Furthermore, nanoparticle tracking analysis (NTA) measurements revealed that MSCs cultured with TS-bFGF produced a greater quantity of exosomes, particularly under three-dimensional culture conditions. These produced exosomes demonstrated substantial anti-inflammatory and wound-healing effects, as confirmed by nitric oxide (NO) assays and scratch assays. Taken together, we demonstrate that utilization of TS-bFGF for WJ-MSC-derived exosome production not only increases exosome yield but also enhances the potential for various applications in inflammation regulation and wound healing.

Role of Telomeres and Telomerase in Parkinson's Disease-A New Theranostics?

Parkinson's disease (PD) is a complex condition that is significantly influenced by oxidative stress and inflammation. It is also suggested that telomere shortening (TS) is regulated by oxidative stress which leads to various diseases including age-related neurodegenerative diseases like PD. Thus, it is anticipated that PD would result in TS of peripheral blood mononuclear cells (PBMCs). Telomeres protect the ends of eukaryotic chromosomes preserving them against fusion and destruction. The TS is a normal process because DNA polymerase is unable to replicate the linear ends of the DNA due to end replication complications and telomerase activity in various cell types counteracts this process. PD is usually observed in the aged population and progresses over time therefore, disparities among telomere length in PBMCs of PD patients are recorded and it is still a question whether it has any useful role. Here, the likelihood of telomere attrition in PD and its implications concerning microglia activation, ageing, oxidative stress, and the significance of telomerase activators are addressed. Also, the possibility of telomeres and telomerase as a diagnostic and therapeutic biomarker in PD is discussed.

Thermostable Human Basic Fibroblast Growth Factor (TS-bFGF) Engineered with a Disulfide Bond Demonstrates Superior Culture Outcomes in Human Pluripotent Stem Cell.

Human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs) can differentiate into various tissues and are an essential source of various disease models and therapeutics. Various growth factors are required in order to culture pluripotent stem cells, among which basic fibroblast growth factor (bFGF) is essential for maintaining stem cell ability. However, bFGF has a short half-life (8 h) under normal mammalian cell culture conditions, and its activity decreases after 72 h, posing a serious problem in the production of high-quality stem cells. Here, we evaluated the various functions of pluripotent stem cells (PSCs) by utilizing an engineered thermostable bFGF (TS-bFGF) that is thermally stable and maintains activity longer under mammalian culture conditions. PSCs cultured with TS-bFGF showed better proliferation, stemness, morphology, and differentiation than cells cultured with wild-type bFGF. In light of the importance of stem cells in a wide range of applications in the medical and biotechnology fields, we anticipate that TS-bFGF, as a thermostable and long-acting bFGF, can play a key role in securing high-quality stem cells through various sets of stem cell culture processes.

Ductal delivery of extracellular vesicles promote the recovery from salivary gland inflammation.

Salivary gland dysfunction worsens the quality of life, but treatment for restoration of salivary gland function is limited. Although previous reports have demonstrated the therapeutic potentials of extracellular vesicles (EVs) in different preclinical models, the role of EVs in salivary glands remains elusive. Furthermore, little is known about the roles of salivary gland-derived EVs in tissue repair or regeneration compared to other EVs. In this study, EVs secreted from salivary gland-derived mesenchymal stem cells (sgMSCs) were comparatively analyzed with those from Wharton's jelly-derived MSC (wjMSCs). sgMSCs secreted more significant amounts of EVs than wjMSCs, and salivary gland epithelial cells showed a more efficient uptake of sgMSC-EVs than wjMSC-EVs. The possibility of immune regulation was tested via macrophage polarization and LPS-induced epithelial inflammation, resulting in an M1-to-M2 shift and reversal of acinar-to-ductal metaplasia by sgMSC-EV. Furthermore, the roles of sgMSC-EV-mediated immune regulation and tissue repair were clarified in vivo via retroductal delivery of sgMSC-EVs in a mouse model of obstructive sialadenitis. Collectively, our data demonstrate the superior role of sgMSC-EVs in the recovery from salivary gland inflammation and injury and suggest EVs as therapeutic tools for salivary gland dysfunction.

Improved Wound Healing and Skin Regeneration Ability of 3,2'-Dihydroxyflavone-Treated Mesenchymal Stem Cell-Derived Extracellular Vesicles.

Flavonoids enhance the self-renewal and differentiation potential of mesenchymal stem cells (MSCs) and have therapeutic activities, including regenerative, anti-oxidative, and anti-inflammatory effects. Recent studies have revealed that MSC-derived extracellular vesicles (MSC-EVs) have therapeutic effects on tissue regeneration and inflammation. To facilitate further research on the therapeutic potential of MSC-EVs derived from flavonoid-treated MSCs, we surveyed the production of EVs and their therapeutic applications in wound regeneration. MSCs treated with flavonoids enhanced EV production twofold compared with naïve MSCs. EVs produced by MSCs treated with flavonoids (Fla-EVs) displayed significant anti-inflammatory and wound-healing effects in vitro. The wound-healing capacity of EVs was mediated by the upregulation of mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) signaling. Interestingly, the protein level of p-ERK under inhibition of MEK signals was maintained in Fla-EV-treated fibroblasts, suggesting that Fla-EVs have a higher therapeutic potential than naïve MSC-EVs (Cont-EVs) in wound healing. Moreover, the in vivo wound closure effect of the Fla-EVs showed significant improvement compared with that of the flavonoid-only treatment group and the Cont-EVs. This study provides a strategy for the efficient production of EVs with superior therapeutic potential using flavonoids.

The Orphan GPR50 Receptor Regulates the Aggressiveness of Breast Cancer Stem-like Cells via Targeting the NF-kB Signaling Pathway.

The expression of GPR50 in CSLC and several breast cancer cell lines was assessed by RT-PCR and online platform (UALCAN, GEPIA, and R2 gene analysis). The role of GPR50 in driving CSLC, sphere formation, cell proliferation, and migration was performed using shGPR50 gene knockdown, and the role of GPR50-regulated signaling pathways was examined by Western blotting and Luciferase Assay. Herein, we confirmed that the expression of G protein-coupled receptor 50 (GPR50) in cancer stem-like cells (CSLC) is higher than that in other cancer cells. We examined that the knockdown of GPR50 in CSLC led to decreased cancer properties, such as sphere formation, cell proliferation, migration, and stemness. GPR50 silencing downregulates NF-kB signaling, which is involved in sphere formation and aggressiveness of CSLC. In addition, we demonstrated that GPR50 also regulates ADAM-17 activity by activating NOTCH signaling pathways through the AKT/SP1 axis in CSLC. Overall, we demonstrated a novel GPR50-mediated regulation of the NF-κB-Notch signaling pathway, which can provide insights into CSLC progression and prognosis, and NF-κB-NOTCH-based CSLC treatment strategies.

Rapid production method with increased yield of high-purity extracellular vesicles obtained using extended mitochondrial targeting domain peptide.

Extracellular vesicles (EVs) are nano-sized membranous structures involved in intercellular communication and various physiological and pathological processes. Here, we present a novel method for rapid (within 15 min), large-scale production of high-purity EVs using eMTDΔ4, a peptide derived from Noxa. The treatment of mesenchymal stem cells derived from human Wharton's jelly after trypsinization and subsequent eMTDΔ4 stimulation in a chemically defined sucrose buffer with orbital shaking led to a substantial increase (approximately 30-fold) in EV production with markedly high purity (approximately 45-fold). These EVs (TS-eEVs) showed higher regenerative and immunomodulatory potential than natural EVs obtained from the culture media after 48 h. The calcium chelator BAPTA-AM and calpain inhibitor ALLM, but not the natural EV biogenesis inhibitor GW4869, blocked the TS-eEV production induced by eMTDΔ4, indicating that the eMTDΔ4-mediated regulation of intracellular calcium levels and calpain activity are closely associated with the rapid, mass production of TS-eEVs. The present study may lead to considerable advances in EV-based drug development and production of stem cell-derived EVs for cell therapy.

TTYH3 Modulates Bladder Cancer Proliferation and Metastasis via FGFR1/H-Ras/A-Raf/MEK/ERK Pathway.

Tweety family member 3 (TTYH3) is a calcium-activated chloride channel with a non-pore-forming structure that controls cell volume and signal transduction. We investigated the role of TTYH3 as a cancer-promoting factor in bladder cancer. The mRNA expression of TTYH3 in bladder cancer patients was investigated using various bioinformatics databases. The results demonstrated that the increasingly greater expression of TTYH3 increasingly worsened the prognosis of patients with bladder cancer. TTYH3 knockdown bladder cancer cell lines were constructed by their various cancer properties measured. TTYH3 knockdown significantly reduced cell proliferation and sphere formation. Cell migration and invasion were also significantly reduced in knockdown bladder cancer cells, compared to normal bladder cancer cells. The knockdown of TTYH3 led to the downregulation of H-Ras/A-Raf/MEK/ERK signaling by inhibiting fibroblast growth factor receptor 1 (FGFR1) phosphorylation. This signaling pathway also attenuated the expression of c-Jun and c-Fos. The findings implicate TTYH3 as a potential factor regulating the properties of bladder cancer and as a therapeutic target.

Modulation of Osteogenic Differentiation of Adipose-Derived Stromal Cells by Co-Treatment with 3, 4'-Dihydroxyflavone, U0126, and N-Acetyl Cysteine.

Background and Objectives: Flavonoids form the largest group of plant phenols and have various biological and pharmacological activities. In this study, we investigated the effect of a flavonoid, 3, 4'-dihydroxyflavone (3, 4'-DHF) on osteogenic differentiation of equine adipose-derived stromal cells (eADSCs). Methods and Results: Treatment of 3, 4'-DHF led to increased osteogenic differentiation of eADSCs by increasing phosphorylation of ERK and modulating Reactive Oxygen Species (ROS) generation. Although PD98059, an ERK inhibitor, suppressed osteogenic differentiation, another ERK inhibitor, U0126, apparently increased osteogenic differentiation of the 3, 4'-DHF-treated eADSCs, which may indicate that the effect of U0126 on bone morphogenetic protein signaling is involved in the regulation of 3, 4'-DHF in osteogenic differentiation of eADSCs. We revealed that 3, 4'-DHF could induce osteogenic differentiation of eADSCs by suppressing ROS generation and co-treatment of 3, 4'-DHF, U0126, and/or N-acetyl cysteine (NAC) resulted in the additive enhancement of osteogenic differentiation of eADSCs. Conclusions: Our results showed that co-treatment of 3, 4'-DHF, U0126, and/or NAC cumulatively regulated osteogenesis in eADSCs, suggesting that 3, 4'-DHF, a flavonoid, can provide a novel approach to the treatment of osteoporosis and can provide potential therapeutic applications in therapeutics and regenerative medicine for human and companion animals.

Superior therapeutic activity of TGF-ß-induced extracellular vesicles against interstitial cystitis.

Interstitial cystitis/bladder pain syndrome (IC/BPS) is a chronic disease characterized by incapacitating pelvic pain. Mesenchymal stem cell-derived extracellular vesicles (MSC-EVs) are considered key mediators of the paracrine action of MSCs and show better biological activities than the parent MSCs, especially in the bladder tissue, which may be unfavorable for MSC survival. Here, we produced MSC-EVs using advanced three-dimensional (a3D) culture with exogenous transforming growth factor-ß3 (TGF-ß3) (T-a3D-EVs). Treatment with T-a3D-EVs led to significantly enhanced wound healing and anti-inflammatory capacities. Moreover, submucosal layer injection of T-a3D-EVs in chronic IC/BPS animal model resulted in restoration of bladder function, superior anti-inflammatory activity, and recovery of damaged urothelium compared to MSCs. Interestingly, we detected increased TGF-ß1 level in T-a3D-EVs, which might be involved in the anti-inflammatory activity of these EVs. Taken together, we demonstrate the excellent immune-modulatory and regenerative abilities of T-a3D-EVs as observed by recovery from urothelial denudation and dysfunction, which could be a promising therapeutic strategy for IC/BPS.

New therapeutic approach with extracellular vesicles from stem cells for interstitial cystitis/bladder pain syndrome.

Interstitial cystitis/bladder pain syndrome (IC/BPS) is a debilitating chronic disorder characterized by suprapubic pain and urinary symptoms such as urgency, nocturia, and frequency. The prevalence of IC/BPS is increasing as diagnostic criteria become more comprehensive. Conventional pharmacotherapy against IC/BPS has shown suboptimal effects, and consequently, patients with end-stage IC/BPS are subjected to surgery. The novel treatment strategies should have two main functions, anti-inflammatory action and the regeneration of glycosaminoglycan and urothelium layers. Stem cell therapy has been shown to have dual functions. Mesenchymal stem cells (MSCs) are a promising therapeutic option for IC/BPS, but they come with several shortcomings, such as immune activation and tumorigenicity. MSC-derived extracellular vesicles (MSC-EVs) hold numerous therapeutic cargos and are thus a viable cell-free therapeutic option. In this review, we provide a brief overview of IC/BPS pathophysiology and limitations of the MSC-based therapies. Then we provide a detailed explanation and discussion of therapeutic applications of EVs in IC/BPS as well as the possible mechanisms. We believe our review will give an insight into the strengths and drawbacks of EV-mediated IC/BPS therapy and will provide a basis for further development. [BMB Reports 2022; 55(5): 205-212].