Stem Cell Research & Therapy最新文献

Background: Cardiac fibrosis represents a significant health burden, with endothelial dysfunction and damaged perivascular microenvironment increasingly recognized as key contributors to fibrotic remodeling. The urokinase plasminogen activator receptor (uPAR), a critical component of the urokinase system, plays a pivotal role in vascular remodeling and fibrosis. While prior evidence indicates that uPAR deficiency leads to microvascular dysfunction and perivascular fibrosis, the underlying mechanisms remain poorly defined. This study investigates how uPAR deficiency contributes to fibrotic remodeling of the cardiac perivascular-like microenvironment.

Methods: Single-cell RNA sequencing data analysis and immunofluorescence staining on mouse heart cryosections were performed to characterize uPAR expression within the cardiac perivascular microenvironment. To model this microenvironment in vitro, cardiospheres (CSs) were generated from non-myocyte cardiac cells of wild-type and uPAR-knockout mice. CRISPR/Cas9-generated Plaur knockout (KO) 3T3 fibroblasts (FBs) were employed as model stromal cells. Pro-fibrotic activation of FBs was induced by TGFβ1 treatment. Comparative analyses of extracellular matrix (ECM) deposition, fibrotic cell transformation, and comprehensive secretome profiling was conducted using western blotting.

Results: Our findings demonstrated that uPAR was expressed by endothelial cells (ECs) and FBs within the cardiac perivascular microenvironment. uPAR deficiency exacerbated profibrotic stimuli in CSs, including elevated active TGFβ1, impaired integrin functions, and altered cell secretome. These alterations collectively disrupt critical cell-cell and cell-matrix interactions, leading to increased ECM deposition, EC loss and decreased cell viability. Using Plaur KO FBs, we demonstrated that uPAR deficiency amplified TGFβ1-mediated Akt signaling pathway and ECM deposition.

Conclusions: Our study reveals that uPAR loss drives fibrotic remodeling of the cardiac perivascular-like microenvironment and exacerbates TGFβ1-mediated effects, highlighting its potential as a therapeutic target for cardiac fibrosis.

Background: Inflammatory pain is a hallmark symptom of osteoarthritis (OA), characterized by spontaneous hypersensitivity resulting from tissue damage and chronic inflammation. This study investigates the pain-relieving and cartilage-protective potential of extracellular vesicles (EVs) derived from human adipose-derived stem cells (hASCs) as a cell-free therapeutic approach for OA.

Methods: hASC-EVs were isolated via multi-filtrations based on tangential flow filtration (TFF) and characterized using transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA), dynamic light scattering (DLS), zeta potential measurement, flow cytometry and Liquid chromatography-mass spectrometry (LC-MS/MS)-based proteomic analysis. An in vitro inflammatory OA model was established by treating human osteoarthritic chondrocytes (HC-OA) with interleukin-1β (IL-1β). The expression of inflammation- and pain-related genes was assessed by quantitative PCR (qPCR), and modulation of the Phosphoinositide 3-kinase / Protein kinase B (PI3K/Akt) signaling pathway was analyzed using an antibody array. In vivo therapeutic effects were evaluated in seven-week-old male Wistar rats using a monosodium iodoacetate (MIA)-induced OA model following intra-articular injection of hASC-EVs. Pain behavior was assessed via paw withdrawal latency (PWL), paw withdrawal threshold (PWT), and weight-bearing tests. Cartilage protection was evaluated by histological and immunohistochemical stainings (IHC).

Results: hASC-EVs were efficiently internalized into chondrocytes and significantly suppressed IL-1β-induced expression of pain and inflammatory markers (TRPA1, COX-2, MMP-2, MMP-3, and MMP-9). Additionally, hASC-EVs down-regulated key PI3K/Akt signaling genes, such as PIK3CA and AKT1. In vivo, hASC-EV treatment markedly improved PWL, PWT, and weight-bearing performance compared with untreated OA rats. Histological and immunohistochemical analyses revealed reduction of inflammatory cytokine expression and preservation of collagen type II, indicating both anti-inflammatory and cartilage-protective effects.

Conclusions: hASC-EVs exhibited robust pain-relieving and cartilage-preserving effects in an OA rat model, highlighting their potential as a promising cell-free therapeutic strategy for the management of OA-related pain and joint degeneration.

Acute kidney injury (AKI) is a serious condition marked by a rapid decline in renal function, often leading to long-term complications. Mesenchymal stromal cells (MSCs) and their derivatives, including conditioned medium (CM) and extracellular vesicles (EVs), show promise as regenerative therapies. However, the comparative efficacy of CM and EVs and the development of clinically translatable interventions remains underexplored. This study systematically compared the renoprotective effects of CM and EVs derived from human umbilical cord MSCs in a murine cisplatin-induced AKI model, using a therapeutically feasible dose. Both treatments improved renal function, reduced histological damage, preserved mitochondrial integrity, energy metabolism, and antioxidant response. Notably, EVs induced the greatest proliferative response in renal tubular cells. To further enhance the regenerative potential of EVs, we engineered MSCs to overexpress nicotinamide phosphoribosyltransferase (NAMPT), a metabolic enzyme that plays a key role in NAD⁺ biosynthesis. NAMPT-transfected MSCs released NAMPT-enriched EVs, which more effectively enhanced cell viability, reduced apoptosis, and protected mitochondria in cisplatin-damaged tubular cells in vitro compared to EV-GFP. In mice with AKI, NAMPT-enriched EVs improved renal function and repaired damage by enhancing renal NAMPT and NAD⁺ levels, promoting tubular cell regeneration. Mechanistically, the amelioration of mitochondrial function was related to increased PGC1α and SIRT3 and consequently SOD2 and ATP5i expression. These findings highlight the therapeutic potential of EVs, particularly NAMPT-enriched EVs, in renal repair, supporting their promise as a clinically translatable approach for promoting recovery from AKI and other kidney diseases.

Background: Renal ischemia-reperfusion injury (RIRI) refers to kidney damage following blood flow restoration, with limited effective treatments available. Bone mesenchymal stem cell (BMSC) derived exosomes exhibit therapeutic potential via targeted molecular delivery, though limited by isolation challenges and transient retention, while curcumin demonstrates multi-organ protective capacities in RIRI.

Methods: In vivo, RIRI mice received tail vein injections of BMSC exosomes (exo) or curcumin preconditioned BMSC exosomes (cur-exo). Biodistribution was tracked via bioluminescence/immunofluorescence, while therapeutic efficacy was evaluated through renal function parameters, histopathology, and ferroptosis biomarkers. In vitro, ferroptosis-induced renal tubular epithelial cells were treated with exo and cur-exo, with subsequent quantification of Fe²⁺, lipid peroxidation, glutathione, mitochondrial ultrastructure, ROS levels, and ferroptosis-related protein/mRNA expression. Mechanistic studies integrated transcriptomics, siRNA/overexpression systems, ChIP, dual-luciferase assays, SPR, Co-IP and bioinformatics to delineate anti-ferroptosis pathways of cur-exo and the effect of curcumin on miR-16-5p.

Results: Curcumin preconditioning can enhance the targeted delivery capability of BMSC exosomes to injured kidneys and improves the restoration of renal function and tissue damage in mice with ischemia-reperfusion injury by inhibiting ferroptosis. In vitro, TCMK-1 cells can take up both exo and cur-exo, with cur-exo significantly enhancing the survival rate of TCMK-1 cells induced by ferroptosis compared to exo. This is achieved by downregulating lipid peroxidation levels, improving iron overload and ROS accumulation, and restoring mitochondrial structure to exert anti-ferroptosis effects. Mechanistically, curcumin increases the expression of miR-16-5p in cur-exo by regulating the activity of CYP1B1, and cur-exo inhibits the translation of Smad3 by delivering miR-16-5p that targets the 3'UTR of Smad3, leading to the downregulation of myoglobin (Mb) transcriptional activity and thereby antagonizing ferroptosis in TCMK-1 cells.

Conclusion: Our research indicates that curcumin preconditioned BMSC exosomes can exert a therapeutic effect on RIRI by inhibiting cellular ferroptosis. The primary mechanism behind this effect involves curcumin increasing the expression of miR-16-5p by modulating CYP1B1 activity, and cur-exo promoting the alleviation of ferroptosis in TCMK-1 cells through the miR-16-5p/Smad3/Mb axis. This study provides a new strategy for enhancing the biological functions of exosomes and presents new targets and ideas for the treatment of RIRI.

Background: Lung ischemia-reperfusion injury (IRI) is a major contributor to primary graft dysfunction (PGD) after lung transplantation. Mesenchymal stem cell-derived extracellular vesicles (MSC-EVs) have emerged as promising therapeutic agents in inflammatory diseases by ameliorating tissue damage and promoting repair. However, the anti-inflammatory efficacy of these approaches and the underlying mechanisms in lung ischemia-reperfusion injury remain incompletely understood.

Methods: The protective effects of mesenchymal stromal cell-derived extracellular vesicles (MSC-EVs) against lung ischemia-reperfusion injury were evaluated using two delivery approaches, inhalation and intravenous injection. Both in vivo and in vitro models were employed to assess the biological activity of MSC-EVs and to elucidate the underlying molecular mechanisms. In addition, a rat orthotopic lung transplantation (OLT) model was established to further examine the translational relevance of MSC-EVs.

Results: MSC-EVs treatment significantly ameliorated lung IRI, with inhalation showing superior efficacy over intravenous delivery. Mechanistically, miR-22-3p within MSC-EVs targeted macrophage NLRP3, suppressing activation of the NLRP3/Caspase-1/IL-1β pathway and promoting M2 polarization. The protective efficacy was confirmed in a clinically relevant rat OLT model, underscoring their translational potential CONCLUSIONS: Our findings indicate that inhaled MSC-derived extracellular vesicles attenuate lung ischemia-reperfusion injury by promoting macrophage polarization via the miR-22-3p/NLRP3/IL-1β pathway, supporting their potential as a cell-free therapeutic approach to mitigate primary graft dysfunction after lung transplantation.

Background: Retinal organoids (ROs) derived from human embryonic stem cells (hESCs) hold immense potential for modeling retinal development and diseases. However, current differentiation protocols often overlook the physiological hypoxic microenvironment of early embryogenesis, potentially compromising developmental fidelity. In this study, we investigated how staged oxygen modulation-hypoxic priming followed by normoxic transition-optimizes RO development by mimicking in vivo oxygen dynamics.

Methods: The H9 hESC line was differentiated into ROs using a modified serum-free floating culture of embryoid body-like aggregates with quick reaggregation (SFEBq) protocol under four oxygen regimens: constant normoxia (20% O₂), chronic hypoxia (5% O₂), hypoxia-to-normoxia transition (5% → 20% O₂), and normoxia-to-hypoxia transition (20% → 5% O₂). RO morphology, retinal progenitor cell (RPC) and retinal ganglion cell (RGC) marker expression using immunofluorescence, and transcriptomic profiles of ROs were assessed at key developmental stages.

Results: Early hypoxia (5% O₂, Days 0-6) significantly increased embryoid body volume (+ 55%, P < 0.001) and antigen Kiel 67 (Ki67)-positive proliferating RPCs (2.78-fold, P < 0.001) compared with normoxia. Early hypoxia also delayed class Ⅲ β-tubulin (TUJ1) expression but enhanced atonal homolog 7 (ATOH7)-positive RGC precursors (2.46-fold, P < 0.001). Upon transition to normoxia (Days 6-60), RPC expansion, indicated by a higher ratio of Ki67-positive proliferating cells, was maintained, and robust RGC differentiation was induced, yielding 38% larger ROs than those formed under chronic hypoxia (P < 0.001). Normoxic conditions also reduced the decline in the ratio of outer-layer CHX10-positive cells and increased the mature TUJ1-positive neurite density of RGC. In contrast, chronic hypoxia markedly impeded paired box 6 (PAX6)-positive RGC differentiation. Transcriptomic analyses showed significant enrichment of sensory and visual system development pathways (P < 0.01) in hypoxia-to-normoxia ROs, supporting distinct developmental patterns influenced by staged oxygen exposure.

Conclusion: Staged oxygen modulation-hypoxic priming followed by normoxic transition-synergistically enhanced RO development by expanding progenitor reservoirs and promoting RGC maturation. This protocol offers a physiologically relevant framework for generating high-fidelity ROs for disease modeling and regenerative applications.

The convergence of CRISPR genome editing, patient-derived organoids, and induced pluripotent stem cells (iPSCs) has reshaped in vitro disease modeling by enabling mechanistic investigations of human pathophysiology within genetically matched, tissue-relevant systems. Together, these technologies provide a synergistic platform for precise manipulation of disease-associated variants and support the generation of isogenic organoid models that reproduce key phenotypic and functional hallmarks across cancer, neurodegenerative, inflammatory, and monogenic disorders. In this review, we highlight how diverse CRISPR modalities-including knock-out, knock-in, CRISPRa/i, and genome-scale screening-have been applied to dissect gene function, model disease progression, and guide therapeutic development using iPSC- and organoid-based systems. We further discuss the application of these platforms in genotype- and phenotype-driven precision medicine, enabling patient stratification, drug-response prediction, and individualized treatment design. We illustrate these convergent applications with representative case studies spanning mechanistic research and early clinical translation. By combining the scalability of genome engineering with the physiological fidelity of organoids, CRISPR-integrated platforms are redefining the frontiers of experimental medicine. These approaches accelerate the discovery of disease mechanisms and actionable therapeutic targets while establishing individualized clinical strategies for complex human diseases. Collectively, they position CRISPR-enabled organoid systems as a foundational infrastructure that bridges genome editing to individualized therapy and supports next-generation precision medicine.

Alopecia Areata (AA) is a chronic inflammatory disorder characterized by non-scarring, patchy hair loss that may progress to the entire scalp (alopecia totalis) or body (alopecia universalis), significantly impairing patients' quality of life and psychological health. Although the exact pathogenesis of AA remains unclear, current evidence suggests that the breakdown of hair follicle immune privilege (IP) and subsequent autoimmune-mediated follicular attack play a pivotal role. Conventional therapeutic modalities, including corticosteroid and Janus kinase (JAK) inhibitors, are often limited by suboptimal efficacy in severe cases and high relapse rates following treatment cessation. In recent years, stem cell-based therapy has emerged as a novel treatment for AA, showing therapeutic potential through multiple mechanisms. Preliminary clinical trials have indicated significant efficacy in promoting hair regrowth among AA patients. However, comprehensive evaluation of long-term safety and therapeutic efficacy remains imperative. This review article aims to give a comprehensive overview of the recent advances in stem cell-based therapies for AA and explore their underlying mechanisms and clinical application prospects, hoping to provide a framework and reference for future research and clinical practice.

Mesenchymal stromal cells (MSCs) are widely recognized for their immunomodulatory properties, which underpin their therapeutic potential in inflammatory and immune-mediated diseases. Although MSC therapies have consistently proven safe, clinical efficacy remains inconclusive, maybe due to incomplete understanding of MSC interactions with the immune environment. This review evaluates current trends in MSC immunomodulation research, based on 318 studies published since 2019 until medio 2024. The most frequently used assays included characterization, proliferation, and polarization, employing methods such as flow cytometry, enzyme-linked immunosorbent assays and colorimetric assays, and polymerase chain reaction. Many studies incorporated strategies for priming of MSCs or included immune cells, most commonly peripheral blood mononuclear cells, T cells, and macrophages. We identify key sources of variability and propose a minimum reporting checklist including MSC source, priming conditions, assay design, and immune cell characteristics. We further recommend implementation of multi-assay workflows combining phenotypic characterization with at least one functional assay. These measures may improve transparency, comparability across studies, and guide robust assay design.

Background: Protein tyrosine phosphatases (PTPs) play key roles in β-cell function and diabetes development. PTPN2 is a candidate gene for type 1 diabetes (T1D) that negatively regulates JAK/STAT signalling. However, the impact of PTPN2 deficiency on the differentiation and functionality of human stem cell-derived somatic metabolic cells remains unclear.

Methods: PTPN2 expression in β cells from T1D organ donors and during the differentiation of human stem cell-derived islets (SC-islets) was evaluated using single-cell RNA-Sequencing (scRNA-Seq) datasets. We differentiated CRISPR-Cas12a genome-edited PTPN2-deficient H1 human embryonic stem cells (H1-hESCs) into SC-islets, and scRNA-Seq was performed. The maturation and functionality of PTPN2-deficient SC-islets were assessed by implantation under the kidney capsule of NOD-SCID mice.

Results: scRNA-Seq analysis showed that PTPN2 expression was increased in β cells from recently diagnosed T1D and decreased in long-standing T1D organ donors compared with controls. Conversely, we found that PTPN2 expression was decreased at the early stages of SC-islet differentiation and reconstituted at the later stages, suggesting a developmental dynamic. PTPN2 deficiency exacerbated interferon-induced inflammatory signalling in stem cells and differentiated somatic metabolic cells. Interestingly, PTPN2 deficiency increased hedgehog signalling and reduced SC-islet differentiation efficiency in vitro. In addition, PTPN2-knockout SC-islets exhibited reduced glycaemic control after implantation in vivo, mediated by reduced endocrine cell identity and enhanced interferon signalling.

Conclusions: Our study postulates a key role of PTPN2 in preserving β-cell function during inflammatory and metabolic stress in SC-islets.