Stem Cell Research & Therapy最新文献

Background: Small extracellular vesicles originating from adipose-derived mesenchymal stromal cells (ADSC-sEVs) have excellent therapeutic value in acute tendon injury. However, their mechanism and effects have not been fully elucidated. This study aimed to identify the key subsets and mechanisms of action of ADSC-sEVs involved in the repair of complete tendon tear caused by acute injury.

Methods: Based on our previous research demonstrating that ADSC-sEVs improve the quality of acute tendon injury repair, the present study utilized second-generation sequencing and bioinformatics to predict the key role of the TNFAIP6^- ADSC subgroup in acute tendon injury repair. We constructed different ADSC-sEVs through ADSC transfection and treated tendon stem cells for further exploration. EdU, cell scratch, and Transwell assays were used to evaluate cell proliferation and migration in vitro. Western blot and quantitative real-time polymerase chain reaction analyses were performed. Histopathological, immunohistochemical, and biomechanical testing were used for in vivo validation.

Results: TNFAIP6^- ADSC-sEVs significantly improved the therapeutic effect of ADSC-sEVs on acute tendon injury, which was related to the high expression of let-7c-5p. Application of different ADSC-sEVs in vitro and in vivo identified CRCT1/JAK2/STAT3 as a key downstream signaling pathway regulated by let-7c-5p.

Conclusions: Our findings enhance the current understanding of how TNFAIP6^- ADSC-sEVs exert healing properties in acute tendon injury through the let-7c-5p/CRCT1/JAK2/STAT3 signaling pathway. Furthermore, this study proposes a concept for constructing conditional ADSC-sEVs to enhance their inherent therapeutic effects.

Human mesenchymal stromal cells (hMSCs) are currently at the center of interest in many randomized and non-randomized clinical trials. According to the data, the number of trials on hMSCs has increased rapidly over time. However, the safety of this treatment, despite some available evidence, remains questionable. Routinely collected data (RCD) has become a helpful approach for gathering clinical data, especially in clinical trials. This method of data collection has helped investigators overcome the limitations of randomized controlled trials (RCTs), such as ensuring long-term follow-ups. Herein, the potential role of RCD in investigating the safety of hMSCs in RCTs, particularly concerns about their possible pro-tumorigenic potential, is discussed. The patterns of recent trials in this field suggest high feasibility and the potential for using RCD for this purpose.

Backgroud: This study systematically evaluated the immunomodulatory function of PD-L1-positive mesenchymal stem cells (PD-L1(+) MSCs) using single-cell RNA sequencing (scRNA-seq) and investigated their roles in suppressing inflammation and regulating pathological bone formation in curdlan-induced SKG ankylosing spondylitis (AS) mouse models.

Methods: scRNA-seq identified MSC subpopulations with high immunomodulatory capacity and key biomarker PD-L1 for subpopulation classification. In vitro co-culture experiments were conducted to evaluate the effects of MSC subpopulations on T-cell proliferation and TNF-α levels. In vivo experiments were performed in forty-eight SKG mouse models to analyze the effects of MSC subpopulations on joint inflammation scores, T-cell subset proportions, inflammatory cytokines, histopathology, and pathological bone formation.

Results: scRNA-seq revealed significant heterogeneity in MSCs under inflammatory stimulation, with the immunomodulatory subpopulation exhibiting high expression of PD-L1 and IDO. In vitro experiments demonstrated that PD-L1(+) MSCs significantly suppressed T-cell proliferation and reduced TNF-α levels. Joint redness and swelling scores showed that the PD-L1(+) MSC group exhibited the most significant improvement in arthritis, while the IL-17Ai, PD-L1(-) MSC, and MSC groups also effectively reduced inflammation, with significantly lower scores than the model control(MC) group. Histological analysis revealed severe inflammatory cell infiltration in the MC group, while the IL-17Ai, PD-L1(+) MSC, and MSC groups exhibited reduced infiltration. Immunohistochemical analysis further confirmed these findings, with PD-L1(+) MSCs exhibiting a significant reduction in TNF-α and IL-17A-positive cells (P < 0.0001 and P < 0.01, respectively).PD-L1(+) MSCs regulated immune responses by reducing Th17 cell proportions, increasing Th2 and Treg cell proportions, and significantly lowering pro-inflammatory cytokines IFN-γ, IL-17A, and TNF-α. MicroCT analysis indicated that the PD-L1(+) MSC, MSC, and IL-17Ai group effectively suppressed pathological bone formation through immunomodulation, whereas the PD-L1(-) MSC group showed weaker effects, underscoring the importance of PD-L1 in regulating bone formation.

Conclusion: hUC-MSCs demonstrated significant therapeutic effects in the AS mouse model, particularly the PD-L1(+) MSCs, which inhibited joint inflammation and pathological new bone formation through immunomodulatory mechanisms. These findings provide valuable insights into the therapeutic mechanisms of AS treatment.

Introduction: Direct reprogramming of fibroblasts into cardiomyocytes by overexpressing cardiac transcription factors Gata4, Mef2c, and Tbx5 (GMT) is a promising way for cardiac repair, however, the low reprogramming efficiency remains a significant challenge. Cellular senescence, an irreversible cell-cycle arrest occurring in mitotic cells, has been reported to influence the efficiency of induced pluripotent stem cell (iPSC) reprogramming.

Methods: We established an inducible GMT expression system in mouse embryonic fibroblasts (MEFs) and human fetal cardiac fibroblasts (hFCFs) using the PiggyBac transposon system. RNA sequencing was performed to identify genes associated with cellular senescence during reprogramming. Selected senescence-related genes were knocked down using shRNA, and their impact on reprogramming efficiency was assessed via flow cytometry, gene expression analysis, and staining for senescence and apoptosis markers.

Results: Direct cardiac reprogramming induced cellular senescence and apoptosis, evidenced by enhanced β-Gal staining, elevated expression of senescence markers P16 and GLB1, and increased apoptosis rates. RNA sequencing and gene set enrichment analysis (GSEA) revealed significant upregulation of senescence-related genes (RB1, RBBP4, RBBP7, CBX8, and CDKN1B). Knockdown of these genes, particularly RB1, significantly enhanced reprogramming efficiency, increasing the proportion of GFP + cells in MEFs and α-actinin + cells in hFCFs. RB1 inhibition also reduced senescence marker levels and upregulated endogenous cardiac transcription factors GATA4 and MEF2C.

Conclusions: Our findings demonstrate that cellular senescence might serves as a barrier to direct cardiac reprogramming and offer novel insights into the regulatory mechanisms involved in this process.

Background: Mesenchymal stem cells (MSCs) are highly sensitive to fluctuations in culture process parameters (CPPs), which remain a major barrier to consistent product quality in cell manufacturing. A mechanistic understanding of how cells respond to and encode these variations is essential to enable standardization under a quality-by-design paradigm.

Methods: To address this, we propose the concept of cell manufacturability, defined as the intrinsic ability of cells to maintain their functional phenotype in response to variable inputs. Drawing inspiration from the Japanese concept of yuragi (gentle, adaptive fluctuation), we profiled histone modifications (H3K4me3 and H3K27me3) at the promoters of critical quality attribute (CQA) genes using chromatin immunoprecipitation followed by quantitative PCR (ChIP-qPCR). We established a cell potency index based on the H3K4me3/H3K27me3 ratio. Weighted principal component analysis (PCA) was applied to derive two composite indices: the Cell Susceptibility Index (CSI), indicating environmental responsiveness, and the Cell Comparability Index (CCI), representing inter-donor and process consistency.

Results: The CSI and CCI captured distinct, condition-dependent patterns. Under low-stress conditions (e.g., early passages and low seeding density), a positive correlation between CSI and CCI reflected reproducible adaptive plasticity. Conversely, high-stress cultures exhibited a strong negative correlation, which was indicative of unstable epigenetic responses. These patterns were consistently observed across different MSC sources, underscoring the generalizability of the framework.

Conclusions: This study highlights CSI and CCI as quantitative, chromatin-based metrics that offer a mechanistic basis for characterizing MSC plasticity and manufacturing robustness. Integration of these indices into the evaluation of cell manufacturability offers a predictive and scalable approach to enhance standardization and batch comparability in MSC production processes.

Background: Heterotopic ossification (HO) pathogenesis involves ROS-driven stem cell differentiation. Carnosic acid (CA), a natural antioxidant, remains unexplored for HO.

Methods: In vitro, tendon-derived stem cells (TDSCs) were stimulated with IL-1β, and CA was used for intervention to assess its effects on differentiation and ROS production via real-time quantitative PCR (qPCR), western blotting (WB), and immunofluorescence. Additionally, a burn and Achilles tendon transection-induced mouse model of traumatic HO was established to evaluate the therapeutic potential of CA.

Results: In vitro, CA activated nuclear factor erythroid 2-related factor 2 (Nrf2) and inhibited nicotinamide adenine dinucleotide phosphate oxidase 1 (NOX1), leading to increased antioxidant enzyme activity and reduced intracellular ROS levels. CA also regulated the PTEN/AKT signaling pathway, suppressing osteogenic and chondrogenic differentiation of TDSCs. In vivo, micro-computed tomography (Micro-CT) and histological analyses demonstrated that CA activated Nrf2 and enhanced antioxidant enzyme expression, thereby inhibiting osteogenic and chondrogenic factor expression in Achilles tendon tissue and reducing HO formation.

Conclusions: CA is a novel HO therapeutic by dual targeting of oxidative stress and differentiation pathways.

Osteoarthritis (OA) is a complex degenerative joint disease with substantial global health implications, yet effective disease-modifying treatments remain elusive. This review explores next-generation models revolutionizing OA research, including microfluidic organ-on-a-chip (OOAC) platforms, organoid systems, computational modelling, finite element analysis (FEA), and artificial intelligence (AI). OOAC systems replicate joint microenvironments, integrating biomechanical stimulation and dynamic tissue interactions, thereby enabling precise investigations of inflammatory and degenerative processes. While organoid technologies capture cellular heterogeneity and self-organization, they primarily serve as static, multicellular models rather than dynamic biomechanical systems. FEA provides high-resolution, patient-specific simulations of joint mechanics and cartilage degeneration, offering insights into mechanical stress distribution and OA progression. Computational modelling and AI enhance predictive capabilities, facilitating precision medicine approaches and optimizing treatment strategies. Despite significant advancements, critical challenges remain, particularly regarding biological fidelity, cross-model integration, and clinical translation. Ensuring computer-based model validation against curated, high-quality datasets-including patient-derived biomechanical, imaging, and molecular data-is imperative for increasing accuracy and translatability. By improving early diagnosis, treatment personalization, and cost-effective therapeutic screening, these advanced technologies can inform healthcare policies, optimize resource allocation, and shape evidence-based guidelines for OA management. This review underscores the necessity of interdisciplinary collaboration to refine these advanced platforms, bridge the gap between preclinical and clinical research, and accelerate the development of patient-specific OA interventions while informing adequate healthcare policies.

With the intensification of global population aging, research on aging-related diseases has become increasingly critical. Bone marrow mesenchymal stromal cells (BMSCs), residing in bone marrow microvironment, serve as precursors for osteoblasts and adipocytes while playing unique roles in regulating hematopoietic and immune systems. However, BMSCs undergo progressive senescence during aging, characterized by diminished proliferative capacity, skewed differentiation potential, compromised immunomodulatory functions, and the development of a senescence-associated secretory phenotype (SASP). These age-related alterations exacerbate inflammatory responses within the bone marrow microenvironment, contributing to the pathogenesis of degenerative diseases such as osteoporosis and osteoarthritis, while potentially inducing hematopoietic dysfunction and oncogenic transformation. Notably, senescent BMSCs secrete pro-inflammatory cytokines that establish a chronic inflammatory milieu, which not only impairs hematopoietic stem cells (HSCs) functionality but also promotes bone marrow adipogenesis. Despite these insights, the intricate interplay between BMSC senescence and microenvironmental alterations remains incompletely understood. This narrative review Comprehensively synthesizes current knowledge on the molecular mechanisms underlying BMSCs senescence, with particular emphasis on telomere attrition, DNA damage accumulation, oxidative stress, epigenetic dysregulation, and bidirectional microenvironmental crosstalk. Furthermore, we critically evaluate emerging therapeutic strategies aimed at mitigating BMSCs senescence and optimizing their clinical applications in age-related disorders.

Microcarrier (µC) suspension systems enable closed, high-density manufacturing of human mesenchymal stem cells (MSCs), but current workflows remain labor-intensive because they require post-thaw cryoprotectant removal and static culture steps to allow cell attachment. To address these bottlenecks, we developed a cryogenic microcarrier-assisted stem cell storage (Cryo-MASCS) workflow that integrates cell attachment, cryopreservation, and thawing directly on surface-engineered µCs. MSCs were pre-seeded onto µCs coated with stacked heparan sulfate-collagen bilayers, cryopreserved on-carrier in either conventional dimethyl sulfoxide (DMSO)-supplemented medium or a DMSO-free, serum-free (SF) minimally supplemented medium, and then thawed and returned directly to suspension culture without intermediate processing. We optimized cell seeding density to maximize recovery. Following thaw, MSCs on engineered µCs and cryopreserved in either DMSO-containing or DMSO-free media retained viability comparable to traditional suspension cultures in DMSO-containing medium, while remaining attached to the carrier surface. Surface-engineered µCs show increased MSC yield within seven days in SF medium, significantly outperforming commercial collagen-coated µCs. Moreover, MSCs recovered metabolic activity and retained robust suppression of lipopolysaccharide-induced M1 macrophage polarization after IFN-γ priming. These findings demonstrate that direct cryopreservation of MSCs on heparan sulfate-collagen-coated µCs is compatible with both DMSO-free and DMSO-supplemented conditions and supports streamlined, scalable culture of undifferentiated MSCs for translational applications.

Background: Mesenchymal stromal cells (MSCs) have emerged as a promising disease-modifying therapy for the complications of diabetes mellitus (DM), including diabetic retinopathy (DR). However, the optimal treatment regimen remains unclear, and challenges persist regarding the timing, route of delivery and the mechanisms underlying the therapeutic effects. This study focused on human umbilical cord-derived mesenchymal stromal cells (hUC-MSCs), to elucidate their retinal protective effects, and investigate the underlying mechanisms by which a single intravenous injection might ameliorate the pathological alterations of DR.

Methods: Two time points after the development of DM were chosen for the in vivo experiments to study the effects of the intervention after different times of exposure to hyperglycemia. hUC-MSCs were injected via the tail vein at 8 and 16 weeks after STZ injection. Retinal samples were collected 2 weeks post-treatment to analyze the therapeutic effect of MSCs on DR. In vitro experiments were conducted using a Müller cell line and a retinal microvascular endothelial cell line cultured under high-glucose conditions, with treatment by hUC-MSCs conditioned media (MSC-CM), to explore the underlying mechanisms.

Results: After a single intravenous injection of hUC-MSCs at week 16 and not 8 weeks post-STZ injection, retinal tissue showed improved thickness of the inner nuclear layer. There was also an increase in the number of acellular capillaries observed in retinal flat mounts of diabetic animals which was improved in the DM and MSC treatment group. MSC treatment reduced high glucose induced activation markers (GFAP and Vimentin) of Müller cells and alleviated endoplasmic reticulum (ER) stress. VEGF expression was also reduced in the retina. MSC-conditioned media also reversed high glucose-induced expression of VEGF in Müller cells. Finally, in a retinal microvascular endothelial cell line, high glucose concentrations, demonstrated increased ER stress which was reduced by MSC conditioned media.

Conclusions: Single Intravenous injection of hUC-MSC to DM animals could alleviate DR via reducing Müller cell and endothelial cell activation and ER stress, and thus might represent a promising therapy for DR.