Stem Cell Research & Therapy最新文献

Acute gastroenteritis viruses, such as rotavirus, human norovirus, human astrovirus, human adenovirus, human sapovirus, represent significant threats to global public health. Research on these pathogens has long been hampered by the limitations of conventional models. Animal and cell-based systems, widely used in virological studies, show limited efficiency in supporting rotavirus replication, while noroviruses remain largely non-cultivable in these settings. Organoids-complex, three-dimensional multicellular structures derived from stem cells-exhibit organ-specific characteristics and spatial organization, making them promising tools for viral research. Intestinal organoids, in particular, recapitulate key features of the gut epithelium and have emerged as versatile platforms for investigating viral pathogenesis and developing intervention strategies. This review systematically outlines the cultivation and functional properties of human intestinal organoids, as well as the evolution and progress of their application in studying acute gastroenteritis viruses. However, current intestinal organoid models are primarily composed of epithelial cells and lack immune and other non-epithelial components, thereby limiting their ability to fully simulate host-pathogen interactions and immune responses following infection. Future efforts should focus on incorporating emerging technologies, such as CRISPR/Cas9 gene editing, to develop more physiologically relevant intestinal models that better mimic in vivo conditions.

Background: Variants in OTUD5 are associated with neurodevelopmental disorders (NDDs), yet the underlying molecular mechanisms remain unclear. This study aimed to investigate the pathogenicity of a novel OTUD5 variant (c.697G > A, p.Val233Met) and elucidate its regulatory role in neural progenitor cell (NPC) proliferation and differentiation, thereby uncovering the function of OTUD5 in neurodevelopment.

Methods: The OTUD5 variant was identified in two NDD patients via exome sequencing. Patient-derived induced pluripotent stem cells (iPSCs) and CRISPR/Cas9-corrected isogenic controls were generated. NPC proliferative activity was assessed by Ki67 immunofluorescence staining, cell-cycle distribution was analyzed by flow cytometry, and neuronal differentiation was evaluated by Tuj1/MAP2 immunofluorescence staining. Substrate screening was conducted in HEK293T cells using co-immunoprecipitation (Co-IP) and mass spectrometry. Deubiquitination capacity and protein stability were validated through ubiquitination assays and cycloheximide (CHX) chase experiments.

Results: The p.Val233Met variant, located within the catalytic OTU domain, induced a marked conformational alteration in the OTUD5 protein. Functionally, the variant caused aberrant NPC proliferation (1.8-fold increase in Ki67⁺ cells, accompanied by release of G1 arrest) and impaired neuronal differentiation (60% reduction in Tuj⁺ cells). Mechanistically, wild-type OTUD5 stabilized GSK3β by removing K48-linked ubiquitin chains, whereas the mutant isoform exhibited diminished deubiquitinase activity, accelerating GSK3β degradation and shortening its half-life by 40%.

Conclusion: This study establishes a novel disease mechanism whereby OTUD5 mutations disrupt NPC homeostasis through GSK3β destabilization, highlighting the critical role of ubiquitination regulation in neurodevelopment. Our iPSC model provides a platform for testing GSK3β-targeted therapies in OTUD5-related NDDs.

Neuroinflammation is a key pathogenic factor for neurodegenerative diseases. Mesenchymal stem cell (MSC) transplantation, as a potential strategy for regulating neuroinflammation, has received extensive attention. Our previous research revealed that compared with ordinary MSC, MSC pretreated with tanshinone IIA (TIIA), referred to as TIIA-MSC, exhibited superior anti-neuroinflammatory activity, but the mechanism of action remains unclear. To clarify the underlying mechanism, this study integrated in vitro and in vivo experiments and evaluated the therapeutic effect of TIIA-MSC in a triple-transgenic Alzheimer's disease mouse model (3×Tg-AD mice) and explored its mechanism of action in a lipopolysaccharide (LPS)-induced BV2 microglial cell inflammation model. The results showed that TIIA-MSC could significantly improve the cognitive function of 3×Tg-AD mice, increase brain glucose metabolism levels, promote the recovery of synaptic and mitochondrial structures, and effectively alleviate neuroinflammatory responses. In vitro experiments further verified the superior inhibitory effect of TIIA-MSC on microglial cell activation and proinflammatory factor release. Mechanistic studies have indicated that the triggering receptor expressed on myeloid cells 2 (TREM2) is the key molecule that mediates this process. The knockdown of TREM2 expression significantly weakened the anti-inflammatory effect of TIIA-MSC, suggesting that TREM2 plays a central role in this process. Further analysis revealed that by activating the phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt) signaling pathway downstream of TREM2, TIIA-MSC may promote the transformation of the functional state of microglia from mainly proinflammatory to having neuroprotective and repair properties. This study systematically revealed the molecular mechanism by which TIIA-MSC regulate microglial cell phenotypic transformation through the TREM2/PI3K/Akt pathway and exert anti-neuroinflammatory effects, providing new ideas and an experimental basis for expanding the application of MSC in the treatment of neurodegenerative diseases.

Background: Alcoholic hepatic fibrosis (AHF) ultimately leads to liver cirrhosis and even hepatocellular carcinoma. The aim of this study was to investigate the specific mechanism by which high mobility group protein B1 (HMGB1) regulated the activation of hepatic stellate cells (HSCs) in AHF.

Methods: CCK-8, EdU staining and flow cytometry assays were utilized to evaluate the viability, proliferation and apoptosis of LX-2 cells stimulated by ethanol. The effect of HMGB1 on cell glycolysis was assessed by cellular energy metabolism assays. The levels of Fe²⁺/Fe³⁺, ROS, GSH and MDA were detected to evaluate the effect of HMGB1 on ferroptosis. In addition, clinical liver tissue samples and an AHF mouse model were employed to further investigate the effect of HMGB1 on AHF.

Results: Ethanol stimulation significantly upregulated HMGB1 and HSC activation markers, enhanced glycolysis, and inhibited ferroptosis in LX-2 cells. Knockdown of HMGB1 suppressed ethanol-induced effects, including HSC activation and glycolysis promotion. However, these effects of HMGB1 knockdown were negated by an oxidative phosphorylation inhibitor. Furthermore, a ferroptosis inducer impeded ethanol-induced HSC activation. Overexpression of HMGB1 decreased the ferroptosis level in ethanol-stimulated LX-2 cells, which was reversed by a glycolysis inhibitor. These in vitro findings demonstrated that upregulated HMGB1 inhibited ferroptosis by enhancing glycolysis, thereby promoting HSC activation. In vivo validation data further confirmed that HMGB1 knockdown inhibited glycolysis, increased ferroptosis level, reduced HSC activation, and alleviated liver fibrosis in AHF mice. Ferroptosis inhibitor counteracted the impacts of HMGB1 knockdown on ferroptosis, HSC activation and liver fibrosis in AHF mice.

Conclusion: In summary, HMGB1 promoted the metabolism of HSCs towards glycolysis to inhibit ferroptosis, which eventually led to the activation of HSCs and the progression of AHF.

Intervertebral disc (IVD) degenerative disease is a prevalent and debilitating spinal disease. Current treatments only focus on symptomatic relief but fail to halt disease progression or restore the native biomechanical function of the spine. Regenerative medicine strategies, particularly those harnessing endogenous progenitor cells, offer a promising avenue for achieving biological repair and functional homeostasis. The identification of intervertebral disc progenitor cells (IVD-PCs) has unveiled a potential cellular reservoir for self-repair, given their demonstrated stemness attributes, including clonogenicity and multipotent differentiation. However, the clinical translation of IVD-PCs is significantly hampered by an incomplete understanding of their inherent heterogeneity, hierarchical organization, and, most critically, the dynamic interplay with their unique microenvironment, which dictates their fate decisions. This review synthesizes recent advances in deciphering the molecular signatures and functional plasticity of IVD-PCs. We place a particular emphasis on how key physicochemical, mechanical, and cellular cues within the IVD niche orchestrate progenitor cell behavior-ranging from maintenance and activation to aberrant differentiation-during both homeostasis and degeneration. Furthermore, we propose forward-looking insights to bridge critical knowledge gaps, aiming to propel the development of novel progenitor cell-based therapeutics for IVD degeneration.

Background: Osteoarthritis (OA) is a degenerative joint disease characterized by progressive cartilage breakdown and limited intrinsic repair capacity. Recent single-cell RNA sequencing (scRNA-seq) studies have revealed remarkable chondrocyte heterogeneity, identifying multiple functionally distinct subpopulations. Increasing evidence suggests that articular cartilage harbors progenitor-like chondrocytes with regenerative potential.

Methods: Articular chondrocytes were isolated from knee cartilage of six end-stage OA patients and profiled using droplet-based scRNA-seq (~ 14,000 cells). Unsupervised clustering, differential gene expression, and gene ontology (GO) enrichment analyses were performed to define subpopulations and their functional characteristics. Pseudotime trajectory analysis (Monocle) was used to infer lineage relationships and differentiation hierarchies.

Results: Twelve transcriptionally distinct chondrocyte clusters were identified, including seven previously described subsets-proliferative, prehypertrophic, hypertrophic, fibrochondrocytic, effector, regulatory, and homeostatic chondrocytes-and three novel ones: NRF2⁺ regulatory chondrocytes enriched in antioxidant pathways, secretory chondrocytes, and progenitor-like chondrocytes(PLCs). Cluster 11 (PLCs) accounted for approximately 2-5% of total chondrocytes and exhibited high expression of stemness-associated genes such as RGS5, PDGFRB, THY1 (CD90), MCAM (CD146), TAGLN, SPARCL1, COL4A1, and ID3. Gene ontology (GO) enrichment revealed activation of developmental and extracellular matrix organization programs, suggesting that these cells are transcriptionally primed for tissue remodelling. Pseudotime mapping positioned PLCs at an early bifurcation upstream of differentiated chondrocyte states, consistent with their progenitor-like role.

Conclusion: This study delineates the single-cell transcriptomic landscape of OA cartilage and identifies a distinct progenitor-like chondrocyte (PLC) subpopulation with progenitor-associated gene signatures. While functional and spatial validation are still required, the unique molecular features of PLCs raise the hypothesis that they may participate in both intrinsic attempts at cartilage repair and osteoarthritis pathophysiology. These findings provide a conceptual and molecular framework for future studies aimed at isolating PLCs, defining their in vivo behaviour, and exploring their potential as targets for cartilage regeneration or OA modulation.

Background: The dentin-pulp complex (DPC) is composed of the odontoblastic layer and associated stromal components. It serves key functions in immunological homeostasis and tissue regeneration of dental tissues. Human dental pulp stem cells (hDPSCs) have emerged as pivotal cells for DPC regeneration. Current research frontiers primarily focus on developing novel strategies to increase the odontogenic differentiation potential and regenerative efficacy of hDPSCs. This study aims to boost the capacity of hDPSCs to regenerate DPC through mitochondrial transplantation.

Methods: Mitochondria were isolated from donor hDPSCs and transplanted into recipient hDPSCs (Mito-hDPSCs) in the same passage. Subsequently, cell viability and mitochondrial transplantation efficiency were evaluated via CCK-8, β-galactosidase staining, mitochondrial imaging, and flow cytometry. Furthermore, Mito-hDPSCs' metabolic capacity was assessed by mitochondrial membrane potential assays and cellular oxidative phosphorylation assays. Moreover, Alkaline Phosphatase (ALP) activity, Alizarin Red S (ARS) staining, RT-qPCR, and Western blotting (WB) were performed to assess Mito-hDPSC's odontogenic differentiation potential. Moreover, a nude mouse model was used to assess how Mito-hDPSCs induce DPC regeneration in vivo. RNA-Seq analysis was conducted to examine the expression of signaling pathways in Mito-hDPSCs. In addition, ALP, ARS, WB, and Ca²⁺ fluorescence staining were carried out to analyze the underlying mechanisms between mitochondria and the Ca²⁺/Transcription factor activating protein 2α (TFAP2A) signaling axis.

Results: The results revealed that mitochondrial transplantation enhanced the viability of Mito-hDPSCs. Furthermore, an increased mitochondrial transplant rate was observed at a recipient-to-donor cell ratio of 1:3. Moreover, Mito-hDPSCs demonstrated increased odontogenic differentiation and formed more dentin-pulp-like tissue in vivo. Ca²⁺ signaling and odontogenesis were significantly enriched in Mito-hDPSCs. TFAP2A was identified as a key transcription factor in the odontogenic differentiation of Mito-hDPSCs. Knockdown array revealed that mitochondrial transplantation effectively upregulated TFAP2A expression in Mito-hDPSCs. Furthermore, mitochondrial transplantation elevated intracellular Ca²⁺ concentration, which in turn increased TFAP2A expression.

Conclusions: Mitochondrial transplantation may promote DPC regeneration by regulating the Ca²⁺/TFAP2A signaling axis in Mito-hDPSCs.

Background: Hirschsprung disease (HSCR) is a congenital condition featuring aganglionosis in the distal colon, causing functional obstruction. While EGF and bFGF are well-characterized neurogenic factors, the precise mechanistic role of GDNF in modulating enteric glial cell plasticity remains incompletely understood.

Methods: EGCs were identified via proteomic profiling and immunofluorescence in Ednrb⁻/⁻ mice modeling HSCR. EGC/PK060399egfr and primary EGCs were induced with neural stem cell-inducing medium (NSC-Med). Morphological changes, EdU assay, immunofluorescence, RT‒qPCR, and Western blotting were employed to assess the expression of stemness- and neuron-associated markers. Metabolomic and transcriptomic analyses were performed to evaluate metabolic remodeling and signaling pathways.

Results: Following treatment with NSC-Med, immunofluorescence analysis revealed that neurospheres expressed high proportions of Nestin-positive (97.09%), Sox2-positive (50.11%), and p75^NTR-positive (77.87%) cells. Metabolomic profiling revealed a significant enhancement of the Warburg effect in the NSC-Med group. Western blot analysis further revealed elevated expression of PKM2, along with significant increases in both extracellular and intracellular lactate levels following NSC-Med treatment. NSC-Med treatment significantly enhanced proliferation, as demonstrated by a 2.3-fold increase in EdU incorporation (P < 0.05). Transcriptomic analysis revealed the activation of the calcium signaling pathway in the GDNF group. Western blotting revealed a significant increase in CaMKII phosphorylation, and treatment with the calcium chelator BAPTA-AM attenuated GDNF-induced NeuroD1 upregulation.

Conclusion: NSC-Med promotes stem cell-associated features and gene expression in enteric glial cells. GDNF-a key component of NSC-Med-activates a neurogenic cascade via the calcium signaling pathway (CaMKII-NeuroD1 axis), which offers a potential targeted molecular strategy for HSCR therapy.

Background: Mesenchymal stem cells (MSCs) are often considered hypoimmunogenic. However, a transient fever observed after intracerebroventricular (ICV) administration in a clinical trial suggests an acute host response. This study examines the mechanisms underlying this reaction, with a focus on MSC migration and the role of matrix metalloproteinase-9 (MMP9).

Methods: We analyzed cerebrospinal fluid (CSF) from Alzheimer's disease (AD) patients treated with saline (n = 3) or human MSCs (hMSCs) (n = 6) using an exploratory protease array, followed by enzyme-linked immunosorbent assay (ELISA). The function of MMP9 was examined further through in-vitro migration and lipopolysaccharide (LPS) stimulation assays in MMP9-silenced hMSCs (siMMP9-hMSCs). In-vivo, siMMP9-hMSCs were delivered ICV into 5xFAD mice to evaluate cell distribution and immune responses.

Results: CSF protease profiling of AD patients revealed that MSC administration increased MMP9 levels. MMP9 knockdown reduced hMSC migration and attenuated LPS induced cytokine increase in the conditioned media (TNF-α and IL-1β) or in the hMSC lysates (IL-1β, IL-6, and CRP) in-vitro. In 5xFAD mice, siMMP9-hMSCs exhibited altered migration and inflammation signatures, characterized by restricted periventricular distribution accompanied by increased CD45 leukocyte accumulation and caspase-3 activity. Naïve hMSCs, on the other hand, dispersed more broadly.

Conclusions: MMP9 promotes the migration of hMSCs and influences the initial interactions between the host and the graft after ICV delivery. Loss of MMP9 activity limits dispersion and is associated with increased local immune activation. This highlights the importance of MMP9-dependent processes in the early post-transplantation phase. These findings may inform strategies to optimize the safety of central nervous system-directed stem cell therapies.

Trial registration number: ClinicalTrials.gov Identifier: NCT02054208.