Stem Cell Research & Therapy最新文献

Background: Periodontal tissue regeneration can be achieved by periodontal ligament stem cells (PDLSCs) through its regulating the immune system. However, the specific signal or molecular mechanism remains unreported. The interaction between MSCs and macrophages (Mφ) has been the focus of the research in recent years. The objective of this study is to examine the effect of direct co-culture of human periodontal ligament stem cells (hPDLSCs) and macrophages on the osteogenic differentiation of hPDLSCs and the polarization of macrophages, and to explore the potential involvement of the EphB4/ephrinB2 signaling pathway in the interaction of co-cultured hPDLSCs and macrophages.

Methods: hPDLSCs isolated from human periodontal ligament were co-cultured with non-activated M0 macrophages (M0-Mφ) induced from THP-1. Quantitative reverse transcription polymerase chain reaction (qRT-PCR), alkaline phosphatase (ALP) staining and assay, as well as Alizarin red staining (ARS) were carried out to evaluate hPDLSCs osteogenic differentiation. qRT-PCR and Enzyme-Linked Immunosorbent Assay (ELISA) were employed to detect the expression of macrophage polarization-related factors. Western Blot was utilized to detect the expression of EphB4, ephrinB2, ERK1/2 and STAT3.

Results: When M0-Mφ was directly co-cultured with hPDLSCs at a ratio of 5:1, the co-culture system significantly promoted the osteogenic differentiation of hPDLSCs, as demonstrated by enhanced ALP staining/activity, ARS mineralization and upregulated mRNA expression of osteogenic markers (RUNX2, ALP, OCN/BGLAP, and OPN/SPP1). Meanwhile, the co-culture system markedly increased anti-inflammatory factor expression (TGF-β1 and IL-10) and decreased the pro-inflammatory factors (TNF-α and IL-1β), indicating enhanced polarization of alternatively activated macrophages (M2-Mφ). The mRNA and protein expression of EphB4 and ephrinB2 showed a significant increase with the time extension of the two cells' co-culture. However, pharmacological interruption of EphB4/ephrinB2 signaling pathway was associated with a decrease of hPDLSC osteogenic differentiation, M2 macrophage polarization, and p-STAT3 expression in the co-culture system.

Conclusions: Our data suggest a potential mediatory role for the EphB4/ephrinB2 pathway in the osteogenic differentiation of hPDLSCs and the polarization of M2-Mφ within the co-culture system. Its regulatory effect on the osteogenic differentiation of hPDLSCs may involve the STAT3 signaling pathway.

Background: Mesenchymal stem cells (MSCs) possess immunomodulatory properties and are concurrently influenced by the local inflammatory microenvironment. Extracellular vesicles (EVs) derived from MSCs (MSCs-EVs) replicate the therapeutic effects of their parent cells while avoiding the limitations of cell therapy. Elucidating the impact of diverse inflammatory factors on the composition and functionality of MSCs-EVs is crucial for their optimal use, though this remains incompletely understood. The aim of this study was to explore the immunomodulatory effects and underlying mechanisms of EVs derived from adipose-derived stem cells (ADSCs) pretreated with TNF-α and TGF-β1 (α-EVs and β-EVs) on macrophages and tissue-engineered cartilage regeneration.

Methods: Isolation and identification of EVs from ADSCs cultured in 3D spheres. The effects on chondrocyte and macrophage proliferation, migration, and polarization were evaluated in vitro. In vivo, chondrocytes-laden porous GelMA hydrogel with EVs were implanted into mice to assess chondrocyte regeneration and macrophage infiltration. Small RNA sequencing revealed distinct EVs-miRNA expression profiles, and the target validation confirmed the molecular mechanism.

Results: In vitro, both α-EVs and β-EVs demonstrated the capacity to modulate macrophage phenotypes. α-EVs more effectively reduced M1 macrophage markers and enhanced M2 polarization. Besides, β-EVs exhibited a stronger inhibitory effect on macrophage proliferation and migration, while also promoting chondrocyte proliferation and extracellular matrix (ECM) formation. In vivo, β-EVs significantly improved ECM deposition and chondrocyte maintenance, while both EVs groups reduced M1 infiltration and increased M2 presence. Small RNA sequencing identified miR-378a-3p upregulation in both α-EVs and β-EVs, targeting Signal-regulatory protein alpha (SIRPα) to modulate the immune status of macrophages.

Conclusions: Both TNF-α and TGF-β1 enhanced the immunomodulatory effects of EVs, with TGF-β1 showing a stronger capacity to promote chondrocyte proliferation and ECM synthesis. The miR-378a-3p/SIRPα axis was identified as a key mechanism underlying the protective effects of both α-EVs and β-EVs. This study provides valuable insights into optimizing EVs-based regenerative strategies to regulate the local inflammatory microenvironment and promote the regeneration of engineered tissues.

Background: Desmin-related cardiomyopathies caused by mutations in the DES gene are characterized by cytoskeletal disorganization and impaired cardiomyocyte function. Viral infections, particularly with Coxsackievirus B3 (CVB3), have been implicated as environmental triggers for cardiac decompensation. However, the interaction between desmin mutations and viral infection has never been explored.

Methods: Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) from a healthy donor (control-CMs) and from patients carrying DES^S46Y, DES^D214-E245del, or DES^P419H mutations (DES^mut-CMs) were infected with the cardiovirulent enterovirus CVB3/28. Structural changes were assessed by immunofluorescence for sarcomeric proteins and desmin. Contractile function was evaluated through video-based motion tracking. Viral replication, protein expression and antiviral responses were measured via plaque assays, immunostaining, and qPCR. Coxsackievirus and Adenovirus Receptor (CAR) and cell-surface vimentin expression were quantified post-infection.

Results: DES^mut-CMs exhibited baseline sarcomeric disorganization and desmin aggregation, which were further aggravated by CVB3/28 infection in a mutation-specific manner. CVB3/28 significantly reduced spontaneous contractility in control-CMs, DES^S46Y, and DES^P419H-CMs, with minimal effect in DES^D214-E245del-CMs. Infected DES^mut-CMs showed enhanced viral replication, increased VP1 expression and elevated virion release. This was accompanied by a stunted IFN-β response, reduced APOBEC3A expression, and infection-induced upregulation of viral receptors CAR and cell-surface vimentin.

Conclusion: CVB3/28 infection compromises the structural integrity and contractile function of cardiomyocytes and exerts a more severe effect in cells harboring DES mutations. These findings underscore a pathogenic synergy between genetic cytoskeletal defects and viral infection, revealing a mechanistic basis for the heightened vulnerability of patients carrying mutation in DES gene to virus-induced cardiac decompensation. CVB3/28 infection disrupts cardiomyocyte structure and impairs contractility, with more severe effects in cells carrying DES mutations. By enhancing viral replication and weakening antiviral defenses, DES mutations act synergistically with CVB3/28 infection to increase the risk of cardiac dysfunction.

Background & aims: Macrophages related inflammation plays a pivotal role in the progression of acute-on-chronic liver failure (ACLF). Our previous study has found that mesenchymal stem cells (MSCs) alleviate inflammatory damage in ACLF mice by promoting polarization of M2 macrophages through upregulating the expression of Mer tyrosine kinase (MERTK). In this study, we investigate the specific mechanism by which MSCs regulate MERTK.

Methods: Bioinformatics was used to predicted the candidate transcription factors for Mertk gene and FOS like antigen 1(FOSL1) was chosen. After overexpressing or knocking down Fosl1, MERTK, iNOS and Arg-1 were analyzed in Raw264.7 and/or J774a.1 cells. Conditioned medium (CM) of MSCs was cocultured with macrophages and the expressions of TGF-β1 and FOSL1 were detected. Male Balb/c mice aged 5-6 weeks were used to establish ACLF mice model. And adeno-associated virus or MSCs-CM was injected through tail vein. Then mouse liver tissue was collected and analyzed.

Results: FOSL1 promotes M2 polarization of macrophages by upregulating the expression of MERTK in vivo and in vitro. The luciferase reporter assays indicate that FOSL1 acts as a transcription factor of Mertk gene. Furthermore, MSCs-CM promotes expression of FOSL1 and M2 polarization of macrophages through TGF-β1 receptor. After knocking down TGF-β1 in MSCs using shRNA, shRNA-CM could not upregulate the expression of FOSL1, and promote M2 polarization of macrophages as CM does.

Conclusions: Our findings show that TGF-β1 secreted by MSCs promotes M2 macrophages polarization via FOSL1/MERTK axis in ACLF mice, providing a novel therapeutic target for ACLF treatment.

Background: Over the past decade, forward programming of human pluripotent stem cells (hPSCs) using various transcription factor (TF) combinations has been widely applied in neuroscience research. Ectopic NGN2 expression in hPSCs has been widely used for rapidly generating in vitro models of induced neurons (iNs) that are predominantly composed of excitatory glutamatergic neurons. Achieving a more balanced synaptic communication between excitatory and inhibitory neurons is essential for physiologically relevant in vitro models. Additionally, incorporating hPSC-derived astrocytes into models, rather than commonly used primary astrocytes, would more closely mimic in vivo disease phenotypes, especially for those associated with astrocyte dysfunction.

Methods: Inducible hPSC lines were generated by targeting the AAVS1 safe harbor site with TF transgene cassettes using CRISPR/Cas9. Forward programming was achieved through forced expression of NGN2 for glutamatergic neurons (iGlutNs), ASCL1/DLX2 for GABAergic neurons (iGABANs) and SOX9/NFIB for astrocytes (iAstros). Cell identity was validated by immunocytochemistry and bulk RNA sequencing. Functional properties were characterized on multielectrode arrays (MEAs).

Results: Bulk RNA sequencing confirmed lineage-specific differentiation while revealing distinct transcriptomic profiles between iAstros and human primary astrocytes. Functional assays demonstrated robust inhibitory control of network dynamics in co-culture with iGABANs on MEA, with enhanced responses to GABA_A receptor-targeting drugs including picrotoxin, bicuculline and clonazepam. Neurons co-cultured with iAstros showed reduced spontaneous activity compared to those cultured with primary astrocytes.

Conclusion: We successfully generated hPSC-derived excitatory and inhibitory neurons to establish an appropriate E/I balance in vitro, supported by primary astrocytes. Although astrocyte identity was confirmed in our hPSC-derived astrocytes, further optimization is required to achieve full functional maturation. This approach to developing an isogenic co-culture system derived from a single hPSC line may more faithfully replicate native neural network dynamics, offering a physiologically relevant platform for studying neurological disorders and screening therapeutic compounds.

Redox balance is crucial for maintaining normal physiological functions. Its disruption by oxidative stress can trigger or exacerbate a series of pathological cascades, ultimately contributing to various chronic diseases, particularly inflammatory disorders. Inhibiting oxidative stress and its associated pathological cascades may alleviate these diseases, a process often linked to the activation of nuclear factor erythroid 2-related factor 2 (Nrf2). Initially characterized as a redox-sensitive transcription factor, Nrf2 is now recognized as a pivotal regulator of an extensive network of antioxidant genes, effectively counteracting oxidative stress and its detrimental effects. Consequently, advances in understanding Nrf2 activators and their regulatory mechanisms have accelerated the development of Nrf2-targeted therapies, demonstrating significant potential for preventing and treating chronic inflammation diseases. Many natural phytochemicals, particularly flavonoids, have been identified as Nrf2 activators that can ameliorate inflammatory responses. Furthermore, therapy with mesenchymal stromal/stem cells (MSCs) is a highly researched treatment approach with the potential to confer immunomodulatory, anti-inflammatory, anti-apoptotic and antimicrobial effects. Owing to their superior safety profile compared to conventional therapeutics, MSCs are gaining prominence as sustainable long-term treatment options, although their precise molecular mechanisms remain to be fully elucidated. This review focuses on the activation mechanisms of Nrf2 and its clinical and preclinical inducers, with particular emphasis on the mechanistic insights and therapeutic applications of natural flavonoids and MSCs in the prevention or treatment of inflammatory diseases. More importantly, it summarizes the profound role of flavonoid-MSCs combinatorial therapy in the intervention of inflammatory diseases, pointing out novel therapeutic strategies and future prospects for modulating the Nrf2 signaling pathway in the treatment of inflammatory disorders.

Background: Impaired osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) contributes to osteoporosis (OP) pathogenesis. While RNA modifications such as N6-methyladenosine (m⁶A) regulate stem cell differentiation, their role in OP remains unclear. Transcriptomic analysis revealed upregulated basic helix-loop-helix family member e22 (Bhlhe22) in OP-BMSCs. This study aimed to investigate whether dysregulated m⁶A modification contributes to OP by upregulating Bhlhe22, thereby impairing the osteogenic differentiation of BMSCs.

Methods: Female Sprague-Dawley rats were subjected to ovariectomy (OVX) to establish an OP model. BMSCs were isolated, characterized by flow cytometry and trilineage differentiation, and subjected to osteogenic induction. Bhlhe22 expression was modulated via lentiviral overexpression, siRNA-mediated knockdown, and adeno-associated virus (AAV)-mediated shRNA knockdown in vivo. Gene expression was assessed by immunofluorescence, quantitative PCR (qPCR), western blotting, and RNA sequencing. Osteogenic differentiation was evaluated via alkaline phosphatase (ALP), alizarin red S (ARS) staining, and osteogenic marker expression. Micro-computed tomography (micro-CT) and histology were used to assess bone structure. Mechanisms were investigated via PI3K-Akt pathway activation (recilisib), chromatin immunoprecipitation sequencing (ChIP-seq), ChIP-qPCR, dot blot, methylated RNA immunoprecipitation (MeRIP)-qPCR, luciferase reporter assays, and site-directed mutagenesis.

Results: Global m⁶A levels were dysregulated in OP-BMSCs. Bhlhe22 mRNA and protein levels were significantly elevated in OP-BMSC and OVX rat femurs. Bhlhe22 knockdown restored the osteogenic potential in OP-BMSCs and increased the bone volume/trabecular thickness in OVX rats, whereas Bhlhe22 overexpression inhibited osteogenesis. BHLHE22 repressed osteogenesis by directly transactivating phosphoinositide-3-kinase regulatory subunit 3 (Pik3r3), activating the PI3K-AKT-GSK3β pathway. Crucially, m⁶A modification at position 2023 stabilized Bhlhe22 mRNA, increasing its expression. Mutation of this m⁶A site reduced the inhibitory effect of Bhlhe22.

Conclusions: m⁶A modification stabilizes Bhlhe22 mRNA, leading to its overexpression in OP. Elevated BHLHE22 protein suppresses BMSC osteogenesis by transcriptionally activating Pik3r3 and the PI3K-AKT-GSK3β signaling axis. Targeting the m⁶A-Bhlhe22-PI3K axis represents a promising therapeutic strategy for osteoporosis.

Background: Normal tension glaucoma (NTG) causes progressive retinal ganglion cell (RGC) loss without elevated intraocular pressure, and currently lacks effective neuroprotective therapies. OPTN(E50K) mutation, a pathogenic gene of NTG, compromises paracrine trophic capacity in bone marrow (BM) stem cells. Allogeneic young BM stem cells can provide trophic support and exhibit therapeutic potential; however, their clinical application is limited by immunogenic rejection and tumorigenic risks, prompting a renewed interest in rejuvenate autologous BM stem cells strategy.

Methods: To address these challenges, we developed a composite chitosan-rapamycin (RAPA) carbon dots (CRCD) to restore the reparative potential of autologous BM stem cells. BM stem cells from aged OPTN(E50K) mice were isolated, clustered and treated with CRCD before validated their changes of neurotrophic factors expression as well as oxidative stress and autophagy indicators. Then we assessed their impact on co-cultured retinas in vitro. Transplantation were performed into lethally irradiated age-matched OPTN(E50K) hosts with CRCD-pretreated autologous BM stem cells via tail vein injection, and generated stable chimeric models. Neurotrophic factors and neuron apoptosis analyses were performed, followed by the visual behavioral tests.

Results: Our results demonstrated that CRCD pretreatment enhanced autophagy, reduced oxidative stress, and augmented paracrine neurotrophic release. In vitro, these cells reduced retinal ganglion cell apoptosis and promoted neurotrophic factor expression in co-cultured retinal explants. In vivo, comparative analysis revealed that CRCD intervention significantly elevated neurotrophic factor expression in retinal tissues compared to RAPA-treated and untreated chimeras. Functional assessments further confirmed superior visual performance in CRCD-Sca-1⁺ chimeras, correlating with improved RGC survival.

Conclusion: These findings suggest that CRCD improved paracrine neurotrophic support of autologous BM stem cells by enhancing their autophagy and paracrine functions, leading to structural and functional rescue of the glaucomatous retina. This approach offered a clinically relevant strategy for NTG, providing sustained neuroprotection while avoiding the risks of allogeneic transplantation and repeated intravitreal drug delivery.

Objective: Mesenchymal stem cells (MSCs) hold substantial promise in regenerative medicine owing to their immunomodulatory, neuroregenerative, and self-renewal properties. Adipose tissue (AT) serves as an optimal MSC source due to its high yield and rapid proliferation. This study evaluated the safety and exploratory clinical effects of non-cryopreserved, culture-expanded autologous AT-MSCs in patients with secondary progressive multiple sclerosis (SPMS).

Methods: High-dose fresh autologous AT-MSCs (4.4 × 10⁶ ± 1.7 × 10⁶ cells) were intravenously administered to 10 female patients with SPMS (Expanded Disability Status Scale [EDSS] score 4-6) in two doses, seven days apart. Adverse events were monitored for 9 months post-transplantation. Magnetic resonance imaging (MRI) assessments quantified lesion number, volume, and contrast-enhancing lesions. EDSS scores, depression, and quality-of-life measures were evaluated over 9 months. MSC immunomodulatory effects were assessed via gene expression of inflammatory and anti-inflammatory cytokines and peripheral blood regulatory T-cell (Treg) proportions.

Results: No serious adverse events occurred over 9 months. AT-MSC therapy reduced T2-FLAIR lesion number and volume, improved EDSS scores, and enhanced psychological outcomes. It also increased Treg cell proportions and anti-inflammatory cytokine expression while decreasing inflammatory cytokines.

Conclusion: High-dose fresh AT-MSCs appear safe and well-tolerated in SPMS patients, with promising exploratory clinical benefits. These findings support AT-MSCs as a potential multiple sclerosis therapy. Trial registration Registered with the Iranian Registry of Clinical Trials (Reference: IRCT20091127002778N1 at 2018-01-10).

Osteoporosis (OP) is among the most prevalent systemic skeletal disorders worldwide and is characterized by decreased bone mass and microarchitectural deterioration, leading to increased fracture risk and significant impairment of quality of life, particularly among elderly individuals. Recently, exosomes derived from bone marrow mesenchymal stem cells (BMSCs), termed BMSC-exosomes, have emerged as promising therapeutic agents for OP because of their regenerative and immunomodulatory potential. In this study, we used senescence-accelerated mouse prone 6 (SAMP6) mice, MC3T3-E1 osteoblastic cells, and CD4(+) T cells to investigate the effects of BMSC-exosomes on osteogenesis and to elucidate the underlying molecular mechanisms. Our results demonstrate that BMSC-derived exosomes enhance osteogenic differentiation in vitro and ameliorate age-related bone loss in vivo. We identified miR-21-5p as a highly enriched microRNA within BMSC-exosomes, which plays a central role in mediating their pro-osteogenic effects and protecting against OP progression. Flow cytometry analysis revealed that BMSC-exosome treatment effectively restored the imbalance between T helper 17 cells (Th17) and regulatory T cells (Treg cells)-a key immune dysregulation observed in OP-in both SAMP6 mice and cultured CD4(+) T cells. Through integrated bioinformatics analysis and experimental validation, we showed that BMSC-derived miR-21-5p directly targeted S-phase kinase-associated protein 2 (SKP2), leading to its downregulation. SKP2 then promotes the ubiquitination and subsequent degradation of Forkhead Box O1 (FoxO1), a transcription factor essential for maintaining Th17/Treg homeostasis. By suppressing SKP2, miR-21-5p stabilizes FoxO1, thereby promoting immune balance and enhancing osteogenic activity. Collectively, these findings indicate that miR-21-5p-enriched BMSC-exosomes alleviate OP by modulating the SKP2/ubiquitination/FoxO1 signalling axis and restoring the Th17/Treg balance. This dual action-promoting bone formation and correcting immune dysfunction-highlights the therapeutic potential of BMSC-exosomes. Thus, the use of miR-21-5p-loaded BMSC-exosomes represents a novel and promising strategy for the prevention and treatment of OP.