Cellular and molecular bioengineering最新文献

Purpose: The gut microbiome interacts with many systems throughout the human body. Microbiome disruption reduces bone tissue mechanics but paradoxically slows osteoarthritis progression. The microbiome also mediates inflammatory and immune responses, including serum cytokines. Towards our long-term goal of studying how the gut microbiome interacts with synovial joint health and disease, we examined how antibiotics-induced changes to microbial taxa abundance associated to serum cytokine levels.

Methods: Mice (n = 5 + ) were provided ad libitum access to water containing antibiotics (1 g/L neomycin, 1 g/L ampicillin, or 1 g/L ampicillin with 0.5 g/L neomycin) or control water from 5- to 16-weeks old, corresponding in skeletal development to ~ 10 to ~ 25 years in humans. At humane euthanasia, we collected cecum contents for 16S metagenomics and blood for serum cytokine quantification for comparison to control and among antibiotic groups. We used dimensional reduction techniques, multiomic integration, and correlation to discriminate antibiotic groups and identify specific relationships between high-abundance taxa and serum cytokines.

Results: Antibiotic treatment significantly lowered diversity, altered phylum relative abundance, and resulted in significant association with specific taxa. Dimensional reduction techniques and multiomic integration revealed distinct antibiotic-associated clusters based on genera relative abundance and cytokine serum concentration. Cytokines IL-6, MIP-1B, and IL-10 significantly contributed to antibiotic discrimination, significantly different among antibiotic treatments, and had significant correlations with specific taxa.

Conclusions: Antibiotic treatment resulted in heterogenous response in gut microbiome and serum cytokines, allowing significant microbe-cytokine links to emerge. The relationships identified here will enable further investigation of the gut microbiome's role in modifying joint health and disease.

Supplementary information: The online version contains supplementary material available at 10.1007/s12195-025-00861-2.

Purpose: Myosteatosis and muscle atrophy are key pathological features of skeletal muscle degeneration in chronic injuries, degenerative myopathies, and aging. While recombinant WNT7A has shown promise in stimulating muscle hypertrophy and reducing fatty infiltration, its clinical translation is limited by challenges in delivery, scalability, and cost. The objective of this study was to evaluate the feasibility of lipid nanoparticle (LNP)-mediated mRNA delivery of WNT7A (W7a-LNP) as an alternative strategy for mitigating muscle degeneration.

Methods: W7a-LNP efficacy was assessed in vitro and in vivo using primary murine fibro-adipogenic progenitors (FAPs), C2C12 myoblasts, and mouse models of muscle injury. FAP adipogenesis and myofiber size were quantified following W7a-LNP treatment. In vivo, W7a-LNP was administered via intramuscular injection in uninjured and glycerol-injured muscles, and its effects on myofiber size and intramuscular adipose tissue (IMAT) formation were analyzed.

Results: W7a-LNP inhibited adipogenesis and increased myofiber size in vitro. In uninjured muscle, multiple W7a-LNP injections significantly increased myofiber size without inducing fibrosis, confirming its safety and efficacy in promoting muscle hypertrophy. However, in the glycerol injury model, W7a-LNP treatment showed variable effects on IMAT reduction when delivered early post-injury, likely due to the absence of viable myofibers needed for mRNA uptake and protein production. Delayed delivery at 4 days post-injury significantly reduced fatty infiltration, supporting the importance of timing and target cell availability for therapeutic efficacy.

Conclusions: These findings provide proof-of-concept that W7a-LNP enhances myofiber hypertrophy and modulates fatty infiltration, supporting mRNA LNP technology as a scalable and localized alternative to recombinant protein therapy for combating muscle degeneration. Further optimization of dose, delivery frequency, and biodistribution will be critical for clinical translation.

Purpose: Mitochondrial dysfunction contributes to endothelial injury in vascular diseases and interventions. While mitochondrial transplantation offers a promising therapeutic strategy, current approaches lack target specificity, efficient uptake, and long-term retention. This study presents a surface-engineering approach to enhance mitochondria delivery to the vascular endothelium as a step toward novel endothelial repair strategies.

Methods: Mitochondria were isolated from healthy induced pluripotent stem cell-derived mesenchymal stem cells (iPSC-MSCs) and surface functionalized with a phospholipid-based coating platform (DSPE-PEG) to enable peptide functionalization. DSPE-PEG was conjugated to either VCAM-1-binding peptide and collagen-binding peptide to enable targeting to dysfunctional and injured endothelium. Mitochondria particle characteristics were measured using flow cytometry, dynamic light scattering and Seahorse. Mitochondrial uptake, retention, and function were assessed in human diabetic aortic endothelial cells (DAECs) using confocal microscopy, flow cytometry, JC-1 staining, and Seahorse metabolic analysis.

Results: iPSC-MSCs provided bioenergetically competent mitochondria suitable for therapeutic delivery. DSPE-PEG surface functionalization significantly enhanced mitochondrial uptake in DAECs, compared to uncoated mitochondria. Confocal imaging and quantitative analysis revealed increased cytoplasmic retention and greater colocalization with the endogenous mitochondrial network after 24 h. Functional assays demonstrated improved mitochondrial membrane potential and sustained oxygen consumption in recipient cells, indicating enhanced host mitochondrial function following treatment with surface-engineered mitochondria.

Conclusions: This study establishes a proof-of-concept for mitochondria surface engineering to enhance mitochondria transplantation to damaged endothelium, demonstrating improved cellular uptake and bioenergetic restoration. These findings provide a foundation for developing adaptable, cell-free therapeutics for vascular disease.

Supplementary information: The online version contains supplementary material available at 10.1007/s12195-025-00862-1.

Purpose: Human adipose tissue-derived stem cells (hADSCs) have emerged as a promising source of cells for tissue engineering and regenerative medicine. However, their differentiation potential is restricted and requires enhancements. This study explores the reprogramming of hADSCs through exogenous induction of the miR-302/367 cluster.

Methods: Human ADSCs were transfected with the mock or miR-302/367 cluster-expressing vectors. One week after transfection, expression levels of several pluripotency-related genes, epithelial-to-mesenchymal (EMT) markers, and mechanistic target of rapamycin kinase (mTOR) signaling factors were assessed by qPCR and western blot. Additionally, the influence of miR-302/367 cluster overexpression on the proliferation and adipogenic differentiation of the ADSCs was evaluated.

Results: One week after transfection, the expression of several pluripotency-related genes and epithelial markers was significantly upregulated, while mesenchymal markers were downregulated in the miR-302/367-transfected cells compared with the mock group. Additionally, the levels of several mTOR signaling factors were reduced in the miR-302/367-transfected ADSCs. Flow cytometry analysis showed a decrease in the abundance of ADSCs in the S phase and an increase in the population of cells in the G1 phase of the cell cycle. Moreover, the adipogenic differentiation of the miR-302/367-transfected cells was significantly enhanced.

Conclusion: The overexpression of the miR-302/367 cluster directed the ADSCs towards a more pluripotent state and promoted their adipogenic potential. However, miR-302/367 overexpression diminished the proliferative capacity of hADSCs, which warrants a comprehensive investigation. Further evaluations are needed to fully elucidate the differentiation potential and regenerative capacity of the ADSCs reprogrammed by the miR-302/367 cluster before any clinical application.

Supplementary information: The online version contains supplementary material available at 10.1007/s12195-025-00856-z.

Purpose: Developing non-invasive delivery platforms with a high level of structural and/or functional similarity to biological membranes is highly desirable to reduce toxicity and improve targeting capacity of nanoparticles. Numerous studies have investigated the impacts of physicochemical properties of engineered biomimetic nanoparticles on their interaction with cells, yet technical difficulties have led to the search for better biomimetics. To overcome such challenges, we aimed to develop a novel method using cell-derived giant plasma membrane vesicles (GPMVs) to encapsulate a variety of engineered nanoparticles, then use these core-shell, nanoparticle-GPMV vesicle structures to deliver cargo to other cells.

Methods: GPMVs were generated by chemically inducing vesiculation in A549 cells, a model human alveolar epithelial line. To evaluate the ability of GPMVs to encapsulate intracellular content, plain, carboxy-modified, or amine-modified silica nanoparticles (all, ~ 50 nm diameter) were loaded into the parent cells prior to vesiculation. GPMVs with or without nanoparticles were subsequently evaluated for stability, membrane protein and lipid constituents, and uptake into cells, and compared to relevant controls.

Results: Cell-derived GPMVs retained encapsulated silica nanoparticles for at least 48 hours at 37 °C. GPMVs showed nearly identical lipid and protein membrane profiles as the parental cell plasma membrane, with or without encapsulation of nanoparticles. Notably, GPMVs were readily endocytosed in the parental A549 cell line as well as the human monocytic THP-1 cell line. Higher cellular uptake levels were observed for GPMV-encapsulated nanoparticles compared to control groups, including free nanoparticles. Further, GPMVs delivered a variety of nanoparticles to parental cells with reduced cytotoxicity compared to free nanoparticles at concentrations that were otherwise significantly toxic.

Conclusions: We have introduced a novel technique to load nanoparticles within the cell plasma membrane during the GPMV vesiculation process. These GPMVs are capable of (a) encapsulating different types of nanoparticles (including larger and not highly-positively charged bodies that have been technically challenging cargoes) using a parental cell uptake technique, and (b) improving delivery of nanoparticles to cells without significant cytotoxicity. Ultimately, the use of GPMVs or other complex vesicles with endogenous cell surface membrane proteins and lipids can lead to highly effective cell membrane-based nanoparticle/drug delivery systems.

Purpose: Cells sense the mechanical properties of their environment through physical engagement and spreading, with high stiffness driving nuclear translocation of the mechanosensitive transcription factor YAP. Restriction of cell spread area or environmental stiffness both inhibit YAP activation and nuclear translocation. The Arp2/3 complex plays a critical role in polymerization of branched actin networks that drive cell spreading, protrusion, and migration. While YAP activation has been closely linked to cellular spreading, the specific role of actin branching in force buildup and YAP activation is unclear.

Methods: To assess the role of actin branching in this process, we measured cell spreading, YAP nuclear translocation, force on the adhesion adaptor protein Talin (FRET tension sensor), and extracellular forces (traction force microscopy, TFM) in 3T3 cells with and without inhibition of actin branching.

Results: The results indicate that YAP activation still occurs when actin branching and cell spreading is reduced. Interestingly, while actin de-branching resulted in decreased force on talin, relatively little change in average traction stress was observed, highlighting the distinct difference between molecular level and cellular level force regulation of YAP.

Conclusions: While cell spreading is a driver of YAP nuclear translocation, this is likely through indirect effects. Changes in cell spreading induced by actin branching inhibition do not significantly perturb YAP activation. Additionally, this work provides evidence that focal adhesion molecular forces are not a direct regulator of YAP activation.

Supplementary information: The online version contains supplementary material available at 10.1007/s12195-025-00852-3.

Background: The global rise of obesity has contributed to an increase in the incidence of endometrial cancer, the most common gynecologic malignancy. This obesity-driven increase, alongside limited therapeutic options, presents a growing public health concern. Our previous research indicated that adipose stem cells (ASCs), shed from fat depots, infiltrate the endometrium via the circulation in endometrial cancer patients with obesity. Furthermore, ASCs elicited the malignant transformation of endometrial epithelial cells (EECs) and fostered an oncogenic microenvironment driven by the plasminogen activator inhibitor 1 (PAI-1).

Objective: To develop a nanoparticle-based system to deliver PAI-1 siRNA targeting the microenvironment of obesity-driven endometrial tumors.

Methods: We developed 2D and 3D spheroid in vitro systems modeling the effects of endometrial microenvironment on ASCs to identify ASC integrin targeting markers. We also analyzed gonadal fat and uterine tissue from obese (ob/ob) mice, validating these ASC integrin markers in vivo. For targeted delivery, we engineered lipid-coated mesoporous silica nanoparticles (LCMSNs) loaded with PAI-1 siRNA. These nanoparticles were administered to ob/ob mice via intraperitoneal injection to evaluate targeting and therapeutic efficiency.

Results: ASCs exposed to an oncogenic endometrial microenvironment showed increased integrin alpha 7 (ITGA7) and PAI-1 expression in vitro. Analysis of gonadal fat and uterine tissue from obese mice confirmed ITGA7 as a promising ASC targeting marker within the endometrial cancer microenvironment. LCMSNs conjugated with anti-ITGA7 antibody exhibited targeting capability toward ITGA7-positive ASCs. In obese mice, these LCMSNs showed strong uterine retention and effective PAI-1 silencing.

Conclusion: Our findings demonstrate the potential of ITGA7-targeted LCMSNs as a PAI-1 siRNA delivery system to therapeutically target ASC-mediated oncogenesis in the endometrial tumor microenvironment. Future studies will evaluate the efficacy of PAI-1 silencing in inhibiting obesity-driven endometrial cancer growth, using in vivo models.

Purpose: Stem cell-derived models offer traceable cell sources for studying tissue development and disease mechanisms. However, many such models have inherently immature or fetal-like phenotypes, limiting their relevance for mechanistic studies of specialized adult tissues. Clinical observations suggest a potential link between epithelial cells and their transit-amplifying progenitors in disease onset and viral tropism, but experimental validation is needed. This study aimed to develop mature visceral epithelial cells (podocytes) from human induced pluripotent stem (iPS) cells using a developmental approach and model severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection in a vascularized microfluidic kidney-on-a-chip platform exhibiting in vivo-like tissue structure and function.

Methods: Mature podocytes and vascular endothelial cells were differentiated from patient-specific human iPS cells by transitioning through distinct lineages that mimic human development. A personalized vascularized microphysiological platform containing the stem cell-derived kidney cells was engineered to model glomerular tissue and the kidney's blood filtration barrier. SARS-CoV-2 entry mechanisms and cell lineage marker expression were assessed at the transcriptome and proteome levels in the developing and mature cells and tissues.

Results: The vascularized kidney-on-a-chip model revealed that susceptibility to SARS-CoV-2 particles was significantly higher in mature glomerular epithelium compared to less specialized derivatives and progenitor cells. The infection with SARS-CoV-2 also induced altered expression of cell lineage markers, with mature podocytes exhibiting distinct transcriptional responses linked to viral interacting epitopes and entry pathways.

Conclusions: This study underscores the importance of using developmentally appropriate preclinical models to investigate disease mechanisms and potential therapeutic responses. These findings highlight the maturation-dependent susceptibility of specialized epithelial cells to viral infections, providing insights into organ-specific disease mechanisms and potential therapeutic strategies. These insights reinforce the need to refine preclinical model systems to closely align with human physiology and ensure the translational relevance of biomedical research.

Supplementary information: The online version contains supplementary material available at 10.1007/s12195-025-00851-4.

Purpose: Tissue dissociation is a critical but often overlooked step in single-cell analysis, impacting data quality, reproducibility, and biological insights. Conventional enzymatic and mechanical dissociation methods introduce variability, damage cells, and alter transcriptomic profiles, compromising downstream applications. While the initial innovation in electrical dissociation was published, this work introduces expanded characterization, including bulk RNA sequencing, diverse tissue types, and improved flow cytometry.

Methods: Here, we present a fully automated, enzyme-free method that integrates electric field-based dissociation with purification and centrifugation, providing a standardized, scalable alternative. A square wave oscillating electric field at 100 V/cm was used for dissociating tissue samples in 5 minutes or less.

Results: The system rapidly and gently dissociated glioblastoma spheroids and mouse spleen tissue, achieving a 10 × increase in live cell yield compared to automated enzymatic and mechanical dissociation (gentleMACS) and a 96 ± 2% single-cell recovery rate in glioblastoma spheroids. Transcriptomic analysis revealed minimal gene expression changes post-dissociation, with an R² value of 0.997 between conditions, indicating high consistency. Flow cytometry confirmed that key immune cell populations (B, T, NK cells) were preserved, with comparable distributions between manual and electrical dissociation.

Conclusions: By reducing operator variability, improving scalability, and maintaining cellular integrity, this technology offers a robust solution for high-throughput single-cell applications in diagnostics, drug discovery, and precision medicine.

Supplementary information: The online version contains supplementary material available at 10.1007/s12195-025-00850-5.