Stem Cell Research & Therapy最新文献

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.

Background: The imbalance between osteogenic and adipogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) is a central pathological feature of osteoporosis (OP). The translocator protein (TSPO) is a multifunctional protein, yet its precise role in bone metabolism remains elusive. This study aimed to investigate the role and mechanism of TSPO in OP pathogenesis.

Methods: We integrated bioinformatic analyses of human and mouse OP-related datasets and validated TSPO expression in BMSCs from osteoporotic patients and mouse models. Gain- and loss-of-function experiments in human BMSCs (h-BMSCs) assessed the impact of TSPO on proliferation, senescence, migration, and lineage differentiation. RNA sequencing and mechanistic rescue experiments were employed to identify the involved signaling pathway. The therapeutic effect of Adeno-associated virus 9 (AAV-9)-mediated TSPO silencing was evaluated in ovariectomized (OVX) mice.

Results: TSPO was significantly upregulated in BMSCs from both OP patients and preclinical models. Functionally, TSPO overexpression suppressed h-BMSC proliferation, migration, and osteogenesis while promoting senescence and adipogenesis. Conversely, TSPO knockdown enhanced cellular fitness and osteogenic capacity. Mechanistically, TSPO functioned as a critical upstream regulator of the PI3K/AKT/GSK-3β signaling axis, suppressing the downstream phosphorylation cascade and ultimately inhibiting β-catenin-mediated osteogenic transcription. Crucially, local TSPO silencing in OVX mice effectively improved bone microarchitecture, enhanced bone formation, and reduced marrow adiposity, concomitant with the reactivation of the PI3K/AKT/GSK-3β/β-catenin pathway.

Conclusion: Our study identifies TSPO as a key pathogenic regulator that impairs osteogenesis by disrupting the PI3K/AKT/β-catenin pathway. Targeting TSPO presents a novel anabolic strategy for osteoporosis, potentially addressing the unmet clinical need for therapies that restore bone formation.

The rapidly growing diabetic population is at high risk of dental implant failure due to a disrupted peri-implant immune microenvironment. Mesenchymal stem cells-derived exosomes (MSC-Exos) have emerged as a potent nanotherapeutic platform to remodel this hostile niche. Their mechanisms involve reprogramming macrophage polarization to alleviate inflammation, delivering pro-angiogenic miRNAs to restore vascular-osteogenic coupling, and modulating neuro-immune crosstalk to reestablish homeostasis. Collectively, these actions break the vicious cycle of impaired healing. Furthermore, engineering strategies such as membrane modification, integration with biomaterials, and preconditioning of parent cells can enhance the targeting, stability, and controlled release of MSC-Exos, thereby improving osseointegration outcomes in diabetic models. These engineering innovations, which focus on precise delivery and controlled release, are as critical to therapeutic development as elucidating the underlying biological mechanisms. This review systematically delineates the mechanisms by which MSC-Exos recalibrate the diabetic bone immune niche to foster osseointegration and critically discusses the clinical translation prospects of engineered exosome-based therapies.

The issue of kidney disease represents a significant global health challenge. While current treatment options may provide symptomatic relief, they are limited by several factors. Consequently, there is a pressing need to create more effective therapeutic strategies. Mesenchymal stromal cell (MSCs) and their secretome have attracted considerable attention in the field of regenerative medicine owing to their multidirectional differentiation potential, immunomodulatory properties, and paracrine effects, which offer a promising solution to this challenge. However, direct transplantation of MSCs and their secretome faces problems such as low survival rate and unstable therapeutic effect in practical applications. These challenges have prompted researchers to explore strategies to enhance the therapeutic potential of MSCs and their secretory factors through pretreatment. This review summarizes the current research progress on pretreated MSCs and their secretome in the treatment of kidney diseases and discusses how various pretreatment approaches can enhance their therapeutic efficacy and clinical application in renal disorders, thereby providing insights for the future optimization and therapeutic use of MSCs.

Background: Early vascularization is one of the limitations of periodontal tissue engineering (PTE) based on mesenchymal stem cells (MSCs). Directed differentiation of endothelial progenitor cells (EPCs) into endothelial cells facilitates the osteogenic effect of MSCs. Therefore, this study constructed EPCs/peripheral blood derived-MSCs (EPCs/PBMSCs) sheets and evaluated their repair value and potential molecular mechanisms in bone regeneration.

Methods: Different ratios of EPCs and PBMSCs were co-cultured to prepare EPCs/PBMSCs sheets and the osteogenic differentiation was assessed. Exploring the bone regeneration properties of EPCs/PBMSC sheets in an animal model of alveolar bone defects. The effect of the SLIT3/ROBO1 axis on angiogenic-osteogenic coupling of EPCs/PBMSCs sheets was explored using exogenous modulation by shRNA lentivirus and neutralizing antibody.

Results: EPCs/PBMSCs sheets could form angiogenic-osteogenic coupling, and different ratios of EPCs/PBMSCs sheets had higher angiogenic and osteogenic differentiation properties than EPCs or PBMSCs alone, especially the ratio 4:6. Moreover, EPCs/PBMSCs sheets accelerated bone regeneration in the alveolar bone defect model and the treatment was superior to PBMSCs alone. The expression patterns of SLIT3 and ROBO1 were consistent with the angiogenic-osteogenic coupling of EPCs/PBMSCs sheets. Knockdown of SLIT3 in PBMSCs and/or neutralization of ROBO1 protein in EPCs effectively suppressed calcified nodule formation and markers expression of osteogenic differentiation and angiogenesis (ALP, RUNX2, OCN, Osx, EMCN, and CD31) in EPCs/PBMSCs sheets, and hindered its therapeutic effect in the alveolar bone defect model.

Conclusion: EPCs/PBMSCs sheets ameliorate the limitations of early vascularization in PTE and the SLIT3/ROBO1 axis mediates the angiogenic-osteogenic coupling of EPCs/PBMSCs sheets, thereby augmenting their osteogenic effects.

Background: Research on cartilage repair in the knee joint is crucial for treating knee arthritis or injuries. The application of mesenchymal stem cells (MSCs) for cartilage tissue regeneration represents a promising therapeutic approach. Among the critical aspects in cartilage formation, the enhancement of MSC chondrogenic differentiation stands as a pivotal challenge. WDR63 is a cytoplasmic dynein that plays a significant role in promoting stem cell differentiation and is closely associated with the cytoskeleton and energy metabolism processes. In the current study, our objective is to elucidate the phenotypic manifestations and mechanisms of WDR63 in relation to its chondrogenic differentiation function in MSCs.

Methods: Stem cells from apical papilla (SCAP) were used. The Alcian Blue staining technique, pellet culture system, and cell transplantation in rabbit knee cartilage defects were employed to assess the chondrogenic differentiation capabilities of MSCs. Western blot and real-time RT-PCR were utilized to investigate the molecular mechanisms involved.

Results: In vitro, WDR63 overexpression in SCAPs enhanced chondrogenic differentiation, as evidenced by upregulating collagen type II (COL2), collagen type V (COL5), and sex-determining region Y box protein 9 (SOX9), and robust pellet formation, whereas WDR63 knockdown produced opposite effects. In vivo, implantation of WDR63-overexpressing SCAP promoted cartilage repair in a rabbit osteochondral defect model, showing improved hyaline cartilage matrix deposition, higher COL2 expression, reduced collagen type X(COLX) expression, and increased collagen type Ι (COL1) expression in the subchondral bone. Mechanistically, WDR63 interacted and co-localized with vimentin (VIM), and its overexpression enhanced VIM expression and WDR63-VIM binding. WDR63 upregulates DRP1 expression, and rescues the Mdi-suppressed mitochondrial fission.

Conclusions: WDR63 may promote chondrogenic differentiation of SCAPs by interacting with VIM and enhancing its expression, potentially through facilitating mitochondrial fission.