Stem Cell Research & Therapy最新文献

Traditional toxicological assessment relied heavily on 2D cell cultures and animal models of study, which were inadequate for the precise prediction of human response to chemicals. Researchers have now shifted focus on organoids for toxicological assessment. Organoids are 3D structures produced from stem cells that mimic the shape and functionality of human organs and have a number of advantages compared to traditional models of study. They have the capacity to replicate the intricate cellular microenvironment and in vivo interactions. They offer a physiologically pertinent platform that is useful for the researchers to monitor cellular responses in a more realistic manner and evaluate drug toxicity. Additionally, organoids can be created from cells unique to a patient, allowing for individualized toxicological research and providing understanding of the inter-individual heterogeneity in drug responses. Recent developments in the use of gut and liver organoids for assessment of the xenobiotics (environmental toxins and drugs) is reviewed in this article. Gut organoids can reveal potential damage to the digestive system and how xenobiotics affect nutrient absorption and barrier function. Liver is the primary site of detoxification and metabolism of xenobiotics, usually routed from the gut. Hence, these are linked and crucial for evaluating chemical or pollutant induced organ toxicity, forecasting their metabolism and pharmacokinetics. When incorporated into the drug development process, organoid models have the potential to improve the accuracy and efficiency of drug safety assessments, leading to safer and more effective treatments. We also discuss the limitations of using organoid-based toxicological assays, and future prospects, including the need for standardized protocols for overcoming reproducibility issues.

Background: Alternative splicing not only expands the genetic encoding of genes but also determines cellular activities. This study aimed to elucidate the regulation mechanism and biological functions of lincRNA-ASAO in the process of odontogenesis-related genes alternative splicing mediated odontogenic differentiation of hDPSCs.

Methods: RACE, RNA-seq, FISH and bioinformatics techniques were used to identify novel lincRNA-ASAO. ALP staining, alizarin red staining, qRT-PCR and western blot were used to identify the role of lincRNA-ASAO in regulating the odontoblast differentiation of hDPSCs. The binding protein PTBP1 of lincRNA-ASAO was screened by RNA-Pulldown, protein profiling and bioinformatics. The target gene ALPL of lincRNA-ASAO/PTBP1 was identified by RNA-seq, bioinformatics technology and DNA agarose gel electrophoresis. FISH, IF, PAR-CLIP and bioinformatics techniques were used to determine the roles of lincRNA-ASAO, PTBP1 and ALPL pre-mRNA in the odontoblast differentiation of hDPSCs.

Results: We identified a novel lincRNA-ASAO that could promote the odontogenic differentiation of human Dental Pulp Stem Cells (hDPSCs). And, the interaction between lincRNA-ASAO and alternative splicing factor PTBP1 promoted the odontoblast differentiation of hDPSCs. In addition, lincRNA-ASAO forms duplexes with ALPL pre-mRNA, targeting PTBP1 to exonic splicing silencer (ESS) of ALPL and regulating exon 2 skipping. Notably, lincRNA-ASAO/PTBP1 regulated ALPL production to increase the type 2 splice variant, which promoted the odontoblast differentiation of hDPSCs.

Conclusions: We have identified the novel lincRNA-ASAO, which can promote the odontoblast differentiation of hDPSCs. The mechanism study found that lincRNA-ASAO/PTBP1 mediated the exon 2 skipping of ALPL pre-mRNA, resulting in the type 2 splice variant of ALPL. Our results enrich the understanding of lncRNAs and alternative splicing in regulating the odontoblast differentiation of hDPSCs, and provide clues to improve the clinical therapeutic potential of hDPSCs for dental pulp restoration.

Background: Pulmonary fibrosis (PF) is a disease with high morbidity and mortality rates, but effective treatment options are extremely limited. Mesenchymal stem cells (MSCs) and their derivatives show promise as potential therapeutics for PF. However, the underlying mechanisms responsible for these beneficial effects remain poorly understood. The objective of this study was to elucidate the specific mechanism through which microvesicles derived from human umbilical cord MSCs (MSC-MVs) alleviate PF.

Methods: The effects of MSC-MVs on PF in bleomycin (BLM)-induced mice were assessed via histological staining, flow cytometry, and enzyme-linked immunosorbent assays (ELISAs). The potential therapeutic target was identified via RNA sequencing (RNA-seq) analysis, followed by validation via real-time quantitative polymerase chain reaction (RT‒qPCR), ELISAs, scratch testing, and western blotting (WB).

Results: MSC-MVs significantly attenuated collagen fiber deposition and downregulated the expression of extracellular matrix components in the lungs of the BLM-induced mice. Moreover, this treatment substantially ameliorated lung inflammation by reducing the monocyte‒macrophage ratio and the TNF-α and IL-6 levels. Further analyses revealed that MSC-MVs inhibited the classic chemotactic CCL2/CCR2 axis of monocyte‒macrophages, leading to reduced recruitment of monocytes‒macrophages to the lungs, which decreased lung inflammation and prevented fibrotic progression. Both in vitro and in vivo findings demonstrated that MSC-MVs suppressed ERK1/2 phosphorylation followed by decreased CCL2 production to modulate monocyte-macrophage migration.

Conclusions: Our findings demonstrate that the protective effect of MSC-MVs against BLM-induced lung toxicity was achieved through the inhibition of the ERK1/2 signaling pathway, leading to the suppression of CCL2 expression and subsequent modulation of monocyte-macrophage migration, thereby establishing a theoretical basis for the effect of MSC-MVs in PF.

Background: Tracheal replacement is a promising approach for treating tracheal defects that are caused by conditions such as stenosis, trauma, or tumors. However, slow postoperative epithelial regeneration often leads to complications, such as infection and granulation tissue formation. Ferroptosis, which is an iron-dependent form of regulated cell death, limits the proliferation of tracheal basal cells (TBCs), which are essential for the epithelialization of tissue-engineered tracheas (TETs). This study explored the potential of ferrostatin-1 (FER-1), which is a ferroptosis inhibitor, to increase TBC proliferation and accelerate the epithelialization of 3D-printed TETs.

Methods: TBCs were isolated from rabbit bronchial mucosal tissues and cultured in vitro. Ferroptosis was induced in TBCs at passage 2, as shown by increased reactive oxygen species (ROS) levels, Fe²⁺ accumulation, decreased ATP contents, and mitochondrial damage. TBCs were treated with FER-1 (1 μM) for 48 h to inhibit ferroptosis. The effects on ROS levels, Fe²⁺ levels, ATP contents, and mitochondrial morphology were measured. For in vivo experiments, FER-1-treated TBCs were seeded onto 3D-printed polycaprolactone (PCL) scaffolds, which were implanted into rabbits with tracheal injury. Epithelial regeneration and granulation tissue formation were evaluated 6 months after surgery.

Results: FER-1 treatment significantly reduced ferroptosis marker levels in vitro; that is, FER-1 treatment decreased ROS and Fe²⁺ accumulation, ameliorated mitochondrial structures, and increased ATP levels. TBC proliferation and viability were increased after ferroptosis inhibition. In vivo, the group that received 3D-printed scaffolds seeded with TBCs exhibited accelerated TET epithelialization and reduced granulation tissue formation compared with the control groups. These results suggest that inhibiting ferroptosis with FER-1 improves TBC function, leading to more efficient tracheal repair.

Conclusions: Ferrostatin-1 effectively inhibits ferroptosis in tracheal basal cells, promoting their viability and proliferation. This results in faster epithelialization of tissue-engineered tracheas, offering a promising strategy for improving tracheal reconstruction outcomes and reducing complications such as infection and granulation tissue formation. Future studies are needed to further investigate the molecular mechanisms underlying ferroptosis in TBCs and its potential clinical applications.

Glioblastoma remains one of the most lethal malignancies, largely due to its resistance to standard chemotherapy such as temozolomide. This study investigates a novel resistance mechanism involving glioblastoma stem cells (GSCs) and the polarization of M2-type macrophages, mediated by the extracellular vesicle (EV)-based transfer of Clusterin. Using 6-week-old male CD34⁺ humanized huHSC-(M-NSG) mice (NM-NSG-017) and glioblastoma cell lines (T98G and U251), we demonstrated that GSC-derived EVs enriched with Clusterin induce M2 macrophage polarization, thereby enhancing temozolomide resistance in glioblastoma cells. Single-cell and transcriptome sequencing revealed close interactions between GSCs and M2 macrophages, highlighting Clusterin as a key mediator. Our findings indicate that Clusterin-rich EVs from GSCs drive glioblastoma cell proliferation and resistance to temozolomide by modulating macrophage phenotypes. Targeting this pathway could potentially reverse resistance mechanisms, offering a promising therapeutic approach for glioblastoma. This study not only sheds light on a critical pathway underpinning glioblastoma resistance but also lays the groundwork for developing therapies targeting the tumor microenvironment. Our results suggest a paradigm shift in understanding glioblastoma resistance, emphasizing the therapeutic potential of disrupting EV-mediated communication in the tumor microenvironment.

Background: Diabetic kidney disease (DKD) is the leading cause of end-stage renal disease (ESRD) globally, presenting a significant therapeutic challenge. Extracellular vesicles (EVs) from mesenchymal stem cells (MSCs) have emerged as promising therapeutic agents. This study explored the therapeutic effects and mechanisms of EVs derived from human placental mesenchymal stem cells (hP-MSCs) on DKD.

Methods: EVs were isolated from cultured hP-MSCs and administered to streptozotocin (STZ)-induced diabetic mice and high glucose-treated glomerular mesangial cells. The therapeutic impact of EVs was assessed through histological analysis and biochemical assays. miR-99b-5p expression in EVs and its role in modulating the mechanistic target of rapamycin (mTOR)/autophagy pathway were examined via western blotting and RT‒qPCR.

Results: Treatment with hP-MSC-derived EVs significantly alleviated renal fibrosis and improved renal function in DKD models. These EVs were enriched with miR-99b-5p, which targeted and inhibited mTOR signaling, thereby increasing autophagic activity and reducing cellular proliferation and extracellular matrix accumulation in renal tissues.

Conclusions: hP-MSC-derived EVs can mitigate renal injury in DKD by modulating the miR-99b-5p/mTOR/autophagy pathway. These findings suggest a potential cell-free therapeutic strategy for managing DKD.

Background: Human bronchial epithelial cell-derived extracellular vesicles have demonstrated the ability to attenuate fibroblasts activation. However, the specific key effector cell populations mediating this inhibitory effect remain unidentified. Airway basal stem cells (BSCs), which serve as progenitor cells for bronchial epithelial cells, play a critical role in fibrotic remodeling processes and possess significant therapeutic potential. This study aimed to characterize BSC-derived extracellular vesicles (BSC-EVs) and investigate their regulatory influence on fibroblasts behavior.

Methods: Airway BSCs were collected through bronchoscopic brushing and differential centrifugation. Fibroblasts were subsequently treated with BSC-EVs at various concentrations to evaluate their dose- and time-dependent effects in vitro. The proteomic composition of BSC-EVs was analyzed using four-dimensional data-independent acquisition quantitative mass spectrometry (4D-DIA). Moreover, a bleomycin-induced pulmonary fibrosis model was established to evaluate the safety and preliminary efficacy of BSC-EVs.

Results: We successfully isolated and identified BSC-EVs, which expressed the nucleus-specific marker TP63, indicative of BSCs, but lacked the BSC marker KRT5. Our findings demonstrated that BSC-EVs enhanced fibroblasts proliferation and migration in a dose-dependent manner. Importantly, BSC-EVs significantly attenuated fibroblasts activation and promoted fibroblasts senescence. Utilizing 4D-DIA quantitative proteomics, we revealed that BSC-EVs modulate extracellular matrix remodeling processes and regulate the expression of key proteins, including collagen I/III and matrix metalloproteinases. Animal models utilizing intratracheal administration of BSC-EVs demonstrate efficient reduction of collagen deposition.

Conclusion: This study offers an extensive characterization of BSC-EVs, adhering to the guidelines set forth by MISEV2023. The findings underscore the significant therapeutic potential of BSC-EVs in the management of fibrotic diseases.

Background: In premature newborn infants, preterm white matter injury (PWMI) causes motor and cognitive disabilities. Accumulating evidence suggests that PWMI may result from defected differentiation of oligodendrocyte precursor cells (OPCs) and impaired maturation of oligodendrocytes. However, the underlying mechanisms remain unclear.

Methods: Using RNAscope, we analyzed the expression level of RNA-binding protein LIN28A in individual OPCs. Knockout of one or both alleles of Lin28a in OPCs was achieved by administrating tamoxifen to NG2^CreER::Ai14::Lin28a^flox/+ or NG2^CreER::Ai14::Lin28a^flox/flox mice. Lentivirus expressing FLEX-Lin28a was used in NG2^CreER mice to overexpress LIN28A in OPCs. A series of behavioral tests were performed to assess the cognitive functions of mice. Two-tailed unpaired t-tests was carried out for statistical analysis between groups.

Results: We found that the expression of Lin28a was decreased in OPCs in a PWMI mouse model. Knockout of one or both alleles of Lin28a in OPCs postnatally resulted in reduced OPC differentiation, decreased myelinogenesis and impaired cognitive functions. Supplementing LIN28A in OPCs postnatally was able to promote OPC differentiation and enhance myelinogenesis, thus rescuing the cognitive functions in PWMI mice.

Conclusion: Our study reveals that LIN28A is critical in regulating postnatal myelinogenesis. Overexpression of LIN28A in OPCs rescues cognitive deficits in PWMI mice by promoting myelinogenesis, thus providing a potential strategy for the treatment of PWMI.

Background: RNA-binding proteins (RBPs) are essential in cardiac development. However, a large of them have not been characterized during the process.

Methods: We applied the human embryonic stem cells (hESCs) differentiated into cardiomyocytes model and constructed SAMD4A-knockdown/overexpression hESCs to investigate the role of SAMD4A in cardiomyocyte lineage specification.

Results: SAMD4A, an RBP, exhibits increased expression during early heart development. Suppression of SAMD4A inhibits the proliferation of hESCs, impedes cardiac mesoderm differentiation, and impairs the function of hESC-derived cardiomyocytes. Correspondingly, forced expression of SAMD4A enhances proliferation and promotes cardiomyogenesis. Mechanistically, SAMD4A specifically binds to FGF2 via a specific CNGG/CNGGN motif, stabilizing its mRNA and enhancing translation, thereby upregulating FGF2 expression, which subsequently modulates the AKT signaling pathway and regulates cardiomyocyte lineage differentiation. Additionally, supplementation of FGF2 can rescue the proliferation defect of hESCs in the absence of SAMD4A.

Conclusions: Our study demonstrates that SAMD4A orchestrates cardiomyocyte lineage commitment through the post-transcriptional regulation of FGF2 and modulation of AKT signaling. These findings not only underscore the essential role of SAMD4A in cardiac organogenesis, but also provide critical insights into the molecular mechanisms underlying heart development, thereby informing potential therapeutic strategies for congenital heart disease.