Protocol for intracellular and extracellular metabolite detection in human embryonic stem cells

Xu, Faxiang; Song, Chengcheng; Liu, Weiwei; Chen, Guokai

doi:10.1016/j.xpro.2021.100740

Cited by 7 publications

(2 citation statements)

References 9 publications

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“…Metabolite extraction for untargeted metabolomics was executed following the methodology published by Xu et al ( 31 ). RAW264.7 cells were infected and incubated overnight with LPS, S. anginosus or S. mitis .…”

Section: Methodsmentioning

confidence: 99%

Oral streptococci S. anginosus and S. mitis induce distinct morphological, inflammatory, and metabolic signatures in macrophages

Senthil Kumar,

Gunda,

Reinartz

et al. 2024

Infect Immun

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Oral streptococci, key players in oral biofilm formation, are implicated in oral dysbiosis and various clinical conditions, including dental caries, gingivitis, periodontal disease, and oral cancer. Specifically, Streptococcus anginosus is associated with esophageal, gastric, and pharyngeal cancers, while Streptococcus mitis is linked to oral cancer. However, no study has investigated the mechanistic links between these Streptococcus species and cancer-related inflammatory responses. As an initial step, we probed the innate immune response triggered by S. anginosus and S. mitis in RAW264.7 macrophages. These bacteria exerted time- and dose-dependent effects on macrophage morphology without affecting cell viability. Compared with untreated macrophages, macrophages infected with S. anginosus exhibited a robust proinflammatory response characterized by significantly increased levels of inflammatory cytokines and mediators, including TNF, IL-6, IL-1β, NOS2, and COX2, accompanied by enhanced NF-κB activation. In contrast, S. mitis -infected macrophages failed to elicit a robust inflammatory response. Seahorse Xfe96 analysis revealed an increased extracellular acidification rate in macrophages infected with S. anginosus compared with S. mitis . At the 24-h time point, the presence of S. anginosus led to reduced extracellular itaconate, while S. mitis triggered increased itaconate levels, highlighting distinct metabolic profiles in macrophages during infection in contrast to aconitate decarboxylase expression observed at the 6-h time point. This initial investigation highlights how S. anginosus and S. mitis , two Gram-positive bacteria from the same genus, can prompt distinct immune responses and metabolic shifts in macrophages during infection. IMPORTANCE The surge in head and neck cancer cases among individuals devoid of typical risk factors such as Human Papilloma Virus (HPV) infection and tobacco and alcohol use sparks an argumentative discussion around the emerging role of oral microbiota as a novel risk factor in oral squamous cell carcinoma (OSCC). While substantial research has dissected the gut microbiome’s influence on physiology, the oral microbiome, notably oral streptococci, has been underappreciated during mucosal immunopathogenesis. Streptococcus anginosus , a viridans streptococci group, has been linked to abscess formation and an elevated presence in esophageal cancer and OSCC. The current study aims to probe the innate immune response to S. anginosus compared with the early colonizer Streptococcus mitis as an important first step toward understanding the impact of distinct oral Streptococcus species on the host immune response, which is an understudied determinant of OSCC development and progression.

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Section: Methodsmentioning

confidence: 99%

Oral streptococci S. anginosus and S. mitis induce distinct morphological, inflammatory, and metabolic signatures in macrophages

Senthil Kumar,

Gunda,

Reinartz

et al. 2024

Infect Immun

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“…Quantification of metabolites and data processing were based on previous publications [ 19 , 22 ]. Briefly, hESCs were cultured in E8 medium with or without T3 for 3 days.…”

Section: Methodsmentioning

confidence: 99%

Thyroid hormone enhances stem cell maintenance and promotes lineage-specific differentiation in human embryonic stem cells

Deng

Zhang²,

Xu³

et al. 2022

Stem Cell Res Ther

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Background Thyroid hormone triiodothyronine (T3) is essential for embryogenesis and is commonly used during in vitro fertilization to ensure successful implantation. However, the regulatory mechanisms of T3 during early embryogenesis are largely unknown. Method To study the impact of T3 on hPSCs, cell survival and growth were evaluated by measurement of cell growth curve, cloning efficiency, survival after passaging, cell apoptosis, and cell cycle status. Pluripotency was evaluated by RT-qPCR, immunostaining and FACS analysis of pluripotency markers. Metabolic status was analyzed using LC–MS/MS and Seahorse XF Cell Mito Stress Test. Global gene expression was analyzed using RNA-seq. To study the impact of T3 on lineage-specific differentiation, cells were subjected to T3 treatment during differentiation, and the outcome was evaluated using RT-qPCR, immunostaining and FACS analysis of lineage-specific markers. Results In this report, we use human pluripotent stem cells (hPSCs) to show that T3 is beneficial for stem cell maintenance and promotes trophoblast differentiation. T3 enhances culture consistency by improving cell survival and passaging efficiency. It also modulates cellular metabolism and promotes energy production through oxidative phosphorylation. T3 helps maintain pluripotency by promoting ERK and SMAD2 signaling and reduces FGF2 dependence in chemically defined culture. Under BMP4 induction, T3 significantly enhances trophoblast differentiation. Conclusion In summary, our study reveals the impact of T3 on stem cell culture through signal transduction and metabolism and highlights its potential role in improving stem cell applications.

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Development of metabolome extraction strategy for metabolite profiling of skin tissue

Bu,

Kim

2024

Metabolomics

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Protocol for intracellular and extracellular metabolite detection in human embryonic stem cells

Cited by 7 publications

References 9 publications

Oral streptococci S. anginosus and S. mitis induce distinct morphological, inflammatory, and metabolic signatures in macrophages

Oral streptococci S. anginosus and S. mitis induce distinct morphological, inflammatory, and metabolic signatures in macrophages

Thyroid hormone enhances stem cell maintenance and promotes lineage-specific differentiation in human embryonic stem cells

Development of metabolome extraction strategy for metabolite profiling of skin tissue

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