Transforming growth factor-β (TGF-β) and interleukin-6 (IL-6) are the pivotal cytokines to induce IL-17-producing CD4(+) T helper cells (TH17); yet their signalling network remains largely unknown. ...Here we show that the highly homologous TGF-β receptor-regulated Smads (R-Smads): Smad2 and Smad3 oppositely modify STAT3-induced transcription of IL-17A and retinoic acid receptor-related orphan nuclear receptor, RORγt encoded by Rorc, by acting as a co-activator and co-repressor of STAT3, respectively. Smad2 linker phosphorylated by extracellular signal-regulated kinase (ERK) at the serine 255 residue interacts with STAT3 and p300 to transactivate, whereas carboxy-terminal unphosphorylated Smad3 interacts with STAT3 and protein inhibitor of activated STAT3 (PIAS3) to repress the Rorc and Il17a genes. Our work uncovers carboxy-terminal phosphorylation-independent noncanonical R-Smad-STAT3 signalling network in TH17 differentiation.
This study was undertaken to determine whether metformin has anti-inflammatory effects in the collagen antibody-induced arthritis (CAIA) murine model. The effect of metformin on Th17 cell ...differentiation was also investigated.
CAIA mice were treated with 100 and 150mg/kgi.p. metformin (low- and high-dose groups, respectively). Arthritis activity and histological joint destruction were studied. Flow cytometry was used to (i) determine RORγt-expressing CD4+ percentages in draining axillary lymph nodes (ALNs) from metformin-treated and untreated mice with CAIA, (ii) determine Th17 percentages in splenic CD4+ T cells cultured ex vivo for 3days in Th17-differentiation-inducing conditions, and (iii) determine the percentages of RORγt+CD4+ T cells when normal splenic T cells from DBA/1 mice were cultured in Th17-differentiation-inducing conditions together with various metformin doses. Western blot analysis was used to assess the intracellular signaling of the metformin-treated splenocytes.
Metformin attenuated both arthritis scores and bone destruction in CAIA mice, decreased the serum levels of the pro-inflammatory cytokines, TNF-α and IL-1, and reduced the number of RORγt+CD4+ T cells in the ALNs. Splenocytes from metformin-treated CAIA mice differentiated less readily into Th17 cells upon ex vivo stimulation. Metformin treatment of normal cells cultured in Th17-differentiation-inducing conditions decreased the number of RORγt-expressing CD4+ cells in a dose-dependent manner and downregulated STAT3 phosphorylation via the AMPK pathway.
Metformin had an anti-inflammatory effect on murine autoimmune arthritis due to the inhibition of Th17 cell differentiation. Metformin may have a possible therapeutic value for treatment of rheumatoid arthritis.
•Metformin attenuated both arthritis scores and bone destruction in murine autoimmune arthritis.•Metformin inhibited Th17 cell differentiation in vivo.•Metformin treatment decreased the number of RORγt-expressing CD4+ cells in a dose-dependent manner.
Spinal cord injury (SCI) is an intractable neurological disease in which functions cannot be permanently restored due to nerve damage. Stem cell therapy is a promising strategy for neuroregeneration ...after SCI. However, experimental evidence of its therapeutic effect in SCI is lacking. This study aimed to investigate the efficacy of transplanted cells using stepwise combined cell therapy with human mesenchymal stem cells (hMSC) and induced pluripotent stem cell (iPSC)-derived motor neuron progenitor cells (iMNP) in a rat model of SCI.
A contusive SCI model was developed in Sprague-Dawley rats using multicenter animal spinal cord injury study (MASCIS) impactor. Three protocols were designed and conducted as follows: (Subtopic 1) chronic SCI + iMNP, (Subtopic 2) acute SCI + multiple hMSC injections, and (Main topic) chronic SCI + stepwise combined cell therapy using multiple preemptive hMSC and iMNP. Neurite outgrowth was induced by coculturing hMSC and iPSC-derived motor neuron (iMN) on both two-dimensional (2D) and three-dimensional (3D) spheroid platforms during mature iMN differentiation in vitro.
Stepwise combined cell therapy promoted mature motor neuron differentiation and axonal regeneration at the lesional site. In addition, stepwise combined cell therapy improved behavioral recovery and was more effective than single cell therapy alone. In vitro results showed that hMSC and iMN act synergistically and play a critical role in the induction of neurite outgrowth during iMN differentiation and maturation.
Our findings show that stepwise combined cell therapy can induce alterations in the microenvironment for effective cell therapy in SCI. The in vitro results suggest that co-culturing hMSC and iMN can synergistically promote induction of MN neurite outgrowth.
It is unclear how systemic administration of mesenchymal stem cells (MSCs) controls local inflammation. The aim of this study was to evaluate the therapeutic effects of human MSCs on inflammatory ...arthritis and to identify the underlying mechanisms. Mice with collagen antibody-induced arthritis (CAIA) received two intraperitoneal injections of human bone marrow-derived MSCs. The clinical and histological features of injected CAIA were then compared with those of non-injected mice. The effect of MSCs on induction of regulatory T cells was examined both in vitro and in vivo. We also examined multiple cytokines secreted by peritoneal mononuclear cells, along with migration of MSCs in the presence of stromal cell-derived factor-1 alpha (SDF-1α) and/or regulated on activation, normal T cell expressed and secreted (RANTES). Sections of CAIA mouse joints and spleen were stained for human anti-nuclear antibodies (ANAs) to confirm migration of injected human MSCs. The results showed that MSCs alleviated the clinical and histological signs of synovitis in CAIA mice. Peritoneal lavage cells from mice treated with MSCs expressed higher levels of SDF-1α and RANTES than those from mice not treated with MSCs. MSC migration was more prevalent in the presence of SDF-1α and/or RANTES. MSCs induced CD4+ T cells to differentiate into regulatory T cells in vitro, and expression of FOXP3 mRNA was upregulated in the forepaws of MSC-treated CAIA mice. Synovial and splenic tissues from CAIA mice receiving human MSCs were positive for human ANA, suggesting recruitment of MSCs. Taken together, these results suggest that MSCs migrate into inflamed tissues and directly induce the differentiation of CD4+ T cells into regulatory T cells, which then suppress inflammation. Thus, systemic administration of MSCs may be a therapeutic option for rheumatoid arthritis.
Alzheimer's disease (AD) is the most common condition in patients with dementia and affects a large population worldwide. The incidence of AD is expected to increase in future owing to the rapid ...expansion of the aged population globally. Researchers have shown that women are twice more likely to be affected by AD than men. This phenomenon has been attributed to the postmenopausal state, during which the level of estrogen declines significantly. Estrogen is known to alleviate neurotoxicity in the brain and protect neurons. While the effects of estrogen have been investigated in AD models, to our knowledge, they have not been investigated in a stem cell-based three-dimensional
system. Here, we designed a new model for AD using induced pluripotent stem cells (iPSCs) in a three-dimensional,
culture system. We used 5xFAD mice to confirm the potential of estrogen in alleviating the effects of AD pathogenesis. Next, we confirmed a similar trend in an AD model developed using iPSC-derived cerebral organoids, in which the key characteristics of AD were recapitulated. The findings emphasized the potential of estrogen as a treatment agent for AD and also showed the suitability of AD-recapitulating cerebral organoids as a reliable platform for disease modeling and drug screening.
Background Spinal cord injury (SCI) is a disease that causes permanent impairment of motor, sensory, and autonomic nervous system functions. Stem cell transplantation for neuron regeneration is a ...promising strategic treatment for SCI. However, selecting stem cell sources and cell transplantation based on experimental evidence is required. Therefore, this study aimed to investigate the efficacy of combination cell transplantation using the brain-derived neurotrophic factor (BDNF) over-expressing engineered mesenchymal stem cell (BDNF-eMSC) and induced pluripotent stem cell-derived motor neuron progenitor cell (iMNP) in a chronic SCI rat model. Method A contusive chronic SCI was induced in Sprague-Dawley rats. At 6 weeks post-injury, BDNF-eMSC and iMNP were transplanted into the lesion site via the intralesional route. At 12 weeks post-injury, differentiation and growth factors were evaluated through immunofluorescence staining and western blot analysis. Motor neuron differentiation and neurite outgrowth were evaluated by co-culturing BDNF-eMSC and iMNP in vitro in 2-dimensional and 3-dimensional. Results Combination cell transplantation in the chronic SCI model improved behavioral recovery more than single-cell transplantation. Additionally, combination cell transplantation enhanced mature motor neuron differentiation and axonal regeneration at the injured spinal cord. Both BDNF-eMSC and iMNP played a critical role in neurite outgrowth and motor neuron maturation via BDNF expression. Conclusions Our results suggest that the combined transplantation of BDNF- eMSC and iMNP in chronic SCI results in a significant clinical recovery. The transplanted iMNP cells predominantly differentiated into mature motor neurons. Additionally, BDNF-eMSC exerts a paracrine effect on neuron regeneration through BDNF expression in the injured spinal cord. Graphical Keywords: BDNF over-expressing engineered mesenchymal stem cell, Induced pluripotent stem cell-derived motor neuron progenitor cell, Chronic spinal cord injury, Combination cell transplantation
Alzheimer's disease (AD) is the most common neurodegenerative disorder in the elderly, resulting in gradual destruction of cognitive abilities. Research on the development of various AD treatments is ...underway; however, no definitive treatment has been developed yet. Herein, we present induced pluripotent stem cell (iPSC)-derived cortical neural stem cell secretome (CNSC-SE) as a new treatment candidate for AD and explore its efficacy.
We first assessed the effects of CNSC-SE treatment on neural maturation and electromagnetic signal during cortical nerve cell differentiation. Then to confirm the efficacy in vivo, CNSC-SE was administered to the 5×FAD mouse model through the nasal cavity (5 μg/g, once a week, 4 weeks). The cell-mediated effects on nerve recovery, amyloid beta (Aβ) plaque aggregation, microglial and astrocyte detection in the brain, and neuroinflammatory responses were investigated. Metabolomics analysis of iPSC-derived CNSC-SE revealed that it contained components that could exert neuro-protective effects or amplify cognitive restorative effects.
Human iPSC-derived CNSC-SE increased neuronal proliferation and dendritic structure formation in vitro. Furthermore, CNSC-SE-treated iPSC-derived cortical neurons acquired electrical network activity and action potential bursts. The 5×FAD mice treated with CNSC-SE showed memory restoration and reduced Aβ plaque accumulation.
Our findings suggest that the iPSC-derived CNSC-SE may serve as a potential, non-invasive therapeutic option for AD in reducing amyloid infiltration and restoring memory.
Methotrexate (MTX) is widely used for the treatment of rheumatoid arthritis (RA). The drug is cost-effective, but sometimes causes hepatotoxicity, requiring a physician's attention. In this study, we ...simulated hepatotoxicity by treating hepatocytes derived from RA patient-derived induced pluripotent stem cells (RA-iPSCs) with MTX.
RA-iPSCs and healthy control iPSCs (HC-iPSCs) were established successfully. RA-iPSCs were differentiated into hepatocytes in two-dimensional (2D) monolayers and three-dimensional (3D) hepatocyte spheroid cultures; this process required growth factors such as BMP4, bFGF, HGF, and OSM. Immunofluorescence staining and flow cytometry were performed to confirm that the mature hepatocytes expressed cytokeratin 18, anti-alpha-1 antitrypsin, and albumin. MTX toxicity was evaluated via monitoring of cell viability, alanine aminotransferase, and mitochondrial status after MTX treatment in 2D and 3D cultures.
RA-iPSCs generated from three RA patients suffering from MTX-induced hepatotoxicity differentiated into the endoderm lineage, hepatoblasts, and hepatocytes. In 2D culture, RA-iPSC-derived hepatocytes were more sensitive to MTX than healthy controls. A 3D culture system using hepatocyte spheroids also successfully recapitulated MTX-induced hepatotoxicity. The 3D culture system had several advantages, including longer culture periods under more complex conditions.
A patient-derived iPSC platform could recapitulate MTX toxicity. Simulation of drug toxicity in vitro may help clinicians choose safer drugs or less toxic doses.
Metabolomics is the systemic study of the unique fingerprints of metabolites involved in cellular processes and biochemical reactions. The metabolomic approach is useful in diagnosing and predicting ...the development of rheumatoid arthritis (RA) and osteoarthritis (OA) and is emerging as a useful tool for identifying disease biomarkers. The aim of this study was to compare the metabolic blueprint of fibroblast-like synoviocyte (FLS) cells and induced pluripotent stem cells (iPSCs) derived from RA and OA patients.
Somatic cells of RA patients (n = 3) and OA patients (n = 3) were isolated, transduced with a lentiviral plasmid, and reprogrammed into iPSCs displaying pluripotency. Metabolic profiling of RA and OA patient-derived FLS cells and iPSCs was performed using liquid chromatography/mass spectrometry and statistical analysis. After normalization by the sum of the peak intensities through LC/MS, 37 metabolites were detected across RA and OA patients.
The metabolites of RA and OA were distinguishable according to the PLS-DA analysis. LysoPC (20:4), 4-methoxychalcone, phosphorylcholine, and nicotinamide (NAM) were significantly higher in RA iPSCs than in OA iPSCs (p < 0.05). The NMNAT-3 enzyme, which catalyzes an important step in the biosynthesis of NAD
from adenosine triphosphate, was also upregulated in RA iPSCs. Interestingly, the proliferation of RA iPSCs was significantly greater than OA iPSC proliferation (p < 0.05). NAM played a critical role in the proliferation of RA iPSCs but not in OA iPSCs. When iPSCs were treated with 100 nM of the NAM inhibitor tannic acid (TA), the proliferation of RA iPSCs was significantly reduced (p < 0.001).
The metabolites of RA and OA FLS cells and RA and OA iPSCs were all clearly distinguishable from each other. NAM played a critical role in the proliferation of RA iPSCs but not in OA iPSCs. TA effectively inhibited the expression of NAM in RA iPSCs and is a possible effective treatment for RA patients.
The effects of several antihypertensive drugs on bone mineral density (BMD) and micro-architectural changes in ovariectomized (OVX) mice were investigated. Eight-week-old female C57/BL6 mice were ...used for this study. Three days after ovariectomy, mice were treated intraperitoneally with nifedipine (15 mg/kg), telmisartan (5 mg/kg), enalapril (20 mg/kg), propranolol (1 mg/kg) or hydrochlorothiazide (12.5 mg/kg) for 35 consecutive days. Uterine atrophy of all mice was confirmed to evaluate estrogen deficiency state. BMD and micro-architectural analyses were performed on tibial proximal ends by micro-computed tomography (micro-CT). When OVX mice with uterine atrophy were compared with mice without atrophy, BMD decreased (P < 0.001). There were significant differences in BMD loss between different antihypertensive drugs (P = 0.005). Enalapril and propranolol increased BMD loss in mice with atrophied uteri compared with control mice. By contrast, thiazide increased BMD in mice with uterine atrophy compared with vehicle-treated mice (P = 0.048). Thiazide (P = 0.032) and telmisartan (P = 0.051) reduced bone loss and bone fraction in mice with uterine atrophy compared with the control. Thiazide affects BMD in OVX mice positively. The reduction in bone loss by thiazide and telmisartan suggest that these drugs may benefit menopausal women with hypertension and osteoporosis.
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