Development of an alcoholic liver disease model for drug evaluation from human induced pluripotent stem cell-derived liver organoids.

Alcoholic liver disease (ALD) poses a significant health challenge, so comprehensive research efforts to improve our understanding and treatment strategies are needed. However, the development of effective treatments is hindered by the limitation of existing liver disease models. Liver organoids, characterized by their cellular complexity and three-dimensional (3D) tissue structure closely resembling the human liver, hold promise as ideal models for liver disease research. In this study, we use a meticulously designed protocol involving the differentiation of human induced pluripotent stem cells (hiPSCs) into liver organoids. This process incorporates a precise combination of cytokines and small molecule compounds within a 3D culture system to guide the differentiation process. Subsequently, these differentiated liver organoids are subject to ethanol treatment to induce ALD, thus establishing a disease model. A rigorous assessment through a series of experiments reveals that this model partially recapitulates key pathological features observed in clinical ALD, including cellular mitochondrial damage, elevated cellular reactive oxygen species (ROS) levels, fatty liver, and hepatocyte necrosis. In addition, this model offers potential use in screening drugs for ALD treatment. Overall, the liver organoid model of ALD, which is derived from hiPSC differentiation, has emerged as an invaluable platform for advancing our understanding and management of ALD in clinical settings.

Bone mesenchymal stem cells improve cholestatic liver fibrosis by targeting ULK1 to regulate autophagy through PI3K/AKT/mTOR pathway.

Cholestatic liver disease (CLD) is a severe disease, which can progress to liver cirrhosis, even liver cancer. Hepatic stellate cells (HSCs) activation plays a crucial role in CLD development. Bone mesenchymal stem cells (BMSCs) treatment was demonstrated to be beneficial in liver diseases. However, the therapeutic effect and mechanism of BMSCs on CLD are poorly known. In the present study, we investigated the therapeutic effects and underlying mechanisms of BMSCs transplantation in mouse models of bile duct ligation-induced cholestatic liver fibrosis (CLF). The results revealed that BMSCs significantly improved liver function and reduced the formation of fibrosis after portal vein transplantation. Mechanistically, after coculturing BMSCs and HSCs, we identified that BMSCs alleviated starvation-induced HSCs activation. Further, BMSCs inhibited HSCs activation by decreasing autophagy, and PI3K/AKT/mTOR pathway was involved in the regulation. More importantly, ULK1 is identified as the main autophagy-related gene regulated by BMSCs in HSCs autophagy. Overexpression of ULK1 reversed the suppression of HSCs autophagy by BMSCs. Collectively, our results provide a theoretical basis for BMSCs targeting ULK1 to attenuate HSCs autophagy and activation and suggest that BMSCs or ULK1 may be an alternative therapeutic approach/target for the treatment of CLF.

Development of alginate-collagen interpenetrating network for osteoarthritic cartilage by in situ softening.

This study presents an alginate-collagen interpenetrating network (IPN) matrix of incorporating collagen fibrils into an alginate hydrogel by physical mixing and controlled gelation. The resulting matrix closely mimics the physiological and pathological stiffness range of the chondrocyte pericellular matrix (PCM). Chondrocytes were cultured within three-dimensional (3D) alginate-collagen IPN matrices with varying stiffness, namely Firm, Medium, and Soft. Alginate lyase was introduced to study the effects of the changes in stiffness of the Firm on chondrocyte response by in situ softening. The developed alginate-collagen IPN matrix displayed good cell-biocompatibility. Compared with stiffer tissue culture plastic (TCP), chondrocytes grown within Firm displayed a stabilized differentiated phenotype characterized by higher expression levels of aggrecan, collagen II, and SOX-9. Moreover, the developed alginate-collagen IPN matrix exhibited a gradually increased percentage of propidium iodide (PI)-positive dead cells with decreasing stiffness. Softer matrices directed cells towards higher proliferation rates and spherical morphologies while stimulating chondrocyte cluster formation. Furthermore, reducing Firm stiffness by in situ softening decreased aggrecan expression, contributing to matrix degradation similar to that seen in osteoarthritis (OA). Hence, the 3D alginate-collagen IPN constructs hold significant potential for in vitro replicating PCM stiffness changes observed in OA cartilage.

Enhancement of therapeutic potential of mesenchymal stem cell by IGF-1 delivery in PLGA microspheres for tissue regeneration.

The use of stem cell-based treatment systems is prevalent in regenerative medicine. To enhance the regenerative capabilities of stem cells, growth factors are typically incorporated into the treatment system. Nonetheless, traditional hydrogel-encapsulated or heparinized scaffolds that bind factors have limitations. In this study, we prepared a biomaterial strategy using uniform poly(lactic-co-glycolic) acid (PLGA) microspheres (uPLGA-Ms) fabricated by microfluidic to sustain delivery of insulin-like growth factor 1 (IGF-1), a critical protein for hMSCs biological functions. The uPLGA-Ms loaded IGF-1 were highly monodispersed through precise manipulation of the flow rate of the two-phase of the flow-focusing microchannle. The results showed that the uPLGA-Ms stabilize IGF-1 and provide a more efficient sustained delivery and cost-effective of growth factor. Gene expression analysis demonstrated the uPLGA delivery of IGF-1 results in a (enhanced) supported hMSCs expansion, survival, stemness, and secretion abilities comparable with the conventional soluble IGF-1 group. In summary, this material-based strategy to stabilize and sustain delivery of growth factor has broad potential to regeneration of various tissues and organs.

Incorporation of Decidual Stromal Cells Derived Exosomes in Sodium Alginate Hydrogel as an Innovative Therapeutic Strategy for Advancing Endometrial Regeneration and Reinstating Fertility.

Intrauterine adhesion (IUA) stands as a prevalent medical condition characterized by endometrial fibrosis and scar tissue formation within the uterine cavity, resulting in infertility and, in severe cases, recurrent miscarriages. Cell therapy, especially with stem cells, offers an alternative to surgery, but concerns about uncontrolled differentiation and tumorigenicity limit its use. Exosomes, more stable and immunogenicity-reduced than parent cells, have emerged as a promising avenue for IUA treatment. In this study, a novel approach has been proposed wherein exosomes originating from decidual stromal cells (DSCs) are encapsulated within sodium alginate hydrogel (SAH) scaffolds to repair endometrial damage and restore fertility in a mouse IUA model. Current results demonstrate that in situ injection of DSC-derived exosomes (DSC-exos)/SAH into the uterine cavity has the capability to induce uterine angiogenesis, initiate mesenchymal-to-epithelial transformation (MET), facilitate collagen fiber remodeling and dissolution, promote endometrial regeneration, enhance endometrial receptivity, and contribute to the recovery of fertility. RNA sequencing and advanced bioinformatics analysis reveal miRNA enrichment in exosomes, potentially supporting endometrial repair. This finding elucidates how DSC-exos/SAH mechanistically fosters collagen ablation, endometrium regeneration, and fertility recovery, holding the potential to introduce a novel IUA treatment and offering invaluable insights into the realm of regenerative medicine.

Melatonin alleviates valproic acid-induced neural tube defects by modulating Src/PI3K/ERK signaling and oxidative stress.

Neural tube defects (NTDs) represent a developmental disorder of the nervous system that can lead to significant disability in children and impose substantial social burdens. Valproic acid (VPA), a widely prescribed first-line antiepileptic drug for epilepsy and various neurological conditions, has been associated with a 4-fold increase in the risk of NTDs when used during pregnancy. Consequently, urgent efforts are required to identify innovative prevention and treatment approaches for VPA-induced NTDs. Studies have demonstrated that the disruption in the delicate balance between cell proliferation and apoptosis is a crucial factor contributing to NTDs induced by VPA. Encouragingly, our current data reveal that melatonin (MT) significantly inhibits apoptosis while promoting the restoration of neuroepithelial cell proliferation impaired by VPA. Moreover, further investigations demonstrate that MT substantially reduces the incidence of neural tube malformations resulted from VPA exposure, primarily by suppressing apoptosis through the modulation of intracellular reactive oxygen species levels. In addition, the Src/PI3K/ERK signaling pathway appears to play a pivotal role in VPA-induced NTDs, with significant inhibition observed in the affected samples. Notably, MT treatment successfully reinstates Src/PI3K/ERK signaling, thereby offering a potential underlying mechanism for the protective effects of MT against VPA-induced NTDs. In summary, our current study substantiates the considerable protective potential of MT in mitigating VPA-triggered NTDs, thereby offering valuable strategies for the clinical management of VPA-related birth defects.

Sodium alginate hydrogel integrated with type III collagen and mesenchymal stem cell to promote endometrium regeneration and fertility restoration.

A thinner endometrium has been linked to implantation failure, and various therapeutic strategies have been attempted to improve endometrial regeneration, including the use of mesenchymal stem cells (MSCs). However, low survival and retention rates of transplanted stem cells are main obstacles to efficient stem cell therapy in thin endometrium. Collagen type III is a key component of the extracellular matrix, plays a crucial role in promoting cell proliferation and differentiation, and has been identified as the major collagen expressed at the implantation site. Herein, composite alginate hydrogel containing recombinant type III collagen (rCo III) and umbilical cord mesenchymal stem cells are developed. rCo III serves as favorable bioactive molecule, displaying that rCo III administration promotes MSCs proliferation, stemness maintenance and migration. Moreover, rCo III administration enhances cell viability and migration of mouse endometrial stromal cells (ESCs). In a mouse model of thin endometrium, the Alg-rCo III hydrogel loaded with MSCs (MSC/Alg-rCo III) significantly induces endometrial regeneration and fertility enhancement in vivo. Further studies demonstrate that the MSC/Alg-rCo III hydrogel promoted endometrial function recovery partly by regulating mesenchymal-epithelial transition of ESCs. Taken together, the combination of Alg-rCo III hydrogel and MSCs has shown promising results in promoting endometrium regeneration and fertility restoration, and may provide new therapeutic options for endometrial disease.

Radionuclide labeled nanocarrier for imaging guided combined radionuclide, sonodynamic, and photothermal therapy of pancreatic tumours.

Radionuclide therapy (RNT) is an effective method for the clinical precise treatment of cancer. However, the uneven dose distribution and rapid metabolism of nuclides limit the effective killing of tumors. To overcome the limitations of radionuclide therapeutic approaches, combining different therapeutic strategies to treat cancer has manifested great promise in basic and clinical research. Here, a new combination therapy strategy was developed to combine radionuclide therapy, sonodynamic therapy and photothermal therapy (RNT-SDT-PTT) under radionuclide imaging guided achieve highly effective combination therapy. We prepared a polydopamine-modified Au nanostar (AN), then loaded with the acoustic sensitizer protoporphyrin (IX) and labeled with diagnostic (99mTc) or therapeutic (131I) radionuclides (131I/99mTc-AN@D/IX) for the precise diagnosis and treatment of pancreatic cancer. After intratumor administration, single photon emission computed tomography imaging showed that the nanocarriers were mostly retained in the tumor compared to free radionuclide. As well as using near-infrared light to trigger PTT and ultrasound with high penetration depth to activate IX to generate reactive oxygen species achieved SDT of tumor. The ultimate significantly improved the inhibitory effects by the RNT-SDT-PTT combined therapy for pancreatic cancer. Therefore, this study proposes an effective radionuclide combination therapy regimen consisting of three widely used treatments, offering promising prospects for the future of oncology.

1-Nitropyrene exposure impairs embryo implantation through disrupting endometrial receptivity genes expression and producing excessive ROS.

Haze problem is an important factor threatening human health. PM2.5 is the main culprit haze. 1-Nitropyrene (1-NP) is the main nitrated polycyclic aromatic hydrocarbon, the toxic component of PM2.5 particles. The effects of 1-NP on various organs and reproductive health have been extensively and deeply studied, but the effects of 1-NP on embryo implantation and endometrial receptivity remain to be determined. The purpose of this study was to investigate the adverse effects of 1-NP on mouse embryo implantation and human endometrial receptivity. In early pregnancy, CD1 mice were given 2 mg/kg 1-NP by oral gavage, which resulted in a decreased embryo implantation number on day 5, inhibited leukemic inhibitory factor (LIF)/STAT3 pathway, decreased expression of estrogen receptor and progesterone receptor, and disrupted regulation of uterine cell proliferation. In addition, in a human in vitro implantation model, 1-NP was found to significantly inhibit the adhesion rate between trophoblast spheroids and endometrial epithelial cells, possibly by inhibiting the expression of receptivity molecules in Ishikawa cells. Promoting reactive oxygen species (ROS) production may be an additional mechanism by which it inhibits trophoblast spheroid adhesion. In this study, we used an in vivo mouse pregnancy model and an in vitro human embryo implantation model to demonstrate that 1-NP can impair endometrial receptivity and compromise embryo implantation.

VE-cadherin fusion protein substrate enhanced the vasculogenic mimicry capability of hepatocellular carcinoma cells.

To investigate the VE-cadherin-based intercellular crosstalk in tumor cells, a fusion protein consisting of a human VE-cadherin extracellular domain and an immunoglobulin G Fc region (hVE-cad-Fc) was prepared as an artificial extracellular matrix (ECM) for the culture of hepatocellular carcinoma cells (Bel7402 cells). Compared with cells cultured on TCPS and collagen coated plates, the Bel7402 cells cultured on a series concentration of hVE-cad-Fc coated plates showed elongated morphology, inhibited proliferation, and significantly enhanced migration and secretion of ECM compounds and cytokines in a concentration-dependent manner. When the concentration of hVE-cad-Fc reached 15 µg mL-1, the Bel7402 cells formed a patterned network with positive-staining of PAS. The high-density hVE-cad-Fc substrate markedly elevated the expression of endogenous VE-cadherin and EphA2, and subsequently activated the intracellular signal transduction pathways including VE-cadherin/PI3K/MMPs and VE-cadherin/EphA2/FAK/p-VE-cadherin axes as well as epithelial-mesenchymal transition (EMT). Therefore, the high-density hVE-cad-Fc substrate was able to induce the vasculogenic mimicry (VM) formation of Bel7402 cells, and exhibit the potential for the construction of an in vitro tumor model of VM. Moreover, hVE-cad-Fc appeared to be a promising candidate for the investigation of VE-cadherin mediated cell-cell interactions in tumor development.

DOX-loaded peptide dendritic copolymer nanoparticles for combating multidrug resistance by regulating the lysosomal pathway of apoptosis in breast cancer cells.

Multidrug resistance (MDR) is a common phenomenon in clinical oncology and is a major obstacle to cancer chemotherapy. Many nanoparticle (NP)-based drug delivery systems have been developed to overcome MDR depending on increasing intracellular drug concentrations via increased cellular uptake and rapid drug release. The objective of this work was to investigate the performance and possible mechanisms of enzyme-sensitive mPEGylated dendron-GFLG-DOX conjugate based nanoparticles for blockading the MDR phenotype of MCF-7/ADR. In vitro, mPEGylated dendron-GFLG-DOX conjugate based nanoparticles could significantly promote cellular uptake and accumulation, potent cytotoxicity and apoptosis compared to free DOX in resistant cells. mPEGylated dendron-GFLG-DOX conjugate based nanoparticles were found to translocate across the membranes of resistant cells via active endocytic pathways leading to more DOX accumulating in the nuclei of MCF-7/ADR cells. Importantly, we found that mPEGylated dendron-GFLG-DOX conjugate based nanoparticles could induce cathepsin B in the cytoplasm and enhance lysosomal-mediated cell death compared to free DOX. Furthermore, mPEGylated dendron-GFLG-DOX conjugate based nanoparticles enhanced the drug's penetration, toxicity, and growth inhibition compared to free DOX in the three-dimensional multicellular tumor spheroid model. In vivo, mPEGylated dendron-GFLG-DOX conjugate based nanoparticles significantly improved the therapeutic efficacy against MDR xenograft tumors, and showed better biocompatibility than free DOX. These results indicated that mPEGylated dendron-GFLG-DOX conjugate based nanoparticles could be used as an alternative drug delivery system for MDR tumor treatment through initiating the lysosomal apoptosis pathway.

Construction of multicellular aggregate by E-cadherin coated microparticles enhancing the hepatic specific differentiation of mesenchymal stem cells.

The differentiation of human mesenchymal stem cells (hMSCs) into hepatocyte-like cells in vitroprovides a promising candidate for cell therapy of liver diseases, and cell aggregates have been proposed to improve the efficiency of expansion and differentiation. Previously, we engineered multicellular aggregates incorporating human E-cadherin fusion protein (hE-cad-Fc)-coated poly(lactic-co-glycolic acid) (PLGA) microparticles (hE-cad-PLGAs), and a significant improvement was obtained in both cellular proliferation of and cytokine secretion by hMSCs. In this study, hepatic differentiation of hMSCs was induced by a biomimetic microenvironment consisting of these engineered aggregates and a cocktail of specific cytokines. The ratio of hE-cad-PLGAs to hMSCs in engineered hMSCs aggregates was optimized to 1:3 for hepatic differentiation. The expressions of hepatic-specific markers were significantly promoted, and cell polarity and activated drug metabolism enzymes were established in MSC/hE-cad-PLGA aggregates compared with MSC and MSC/PLGA aggregates. Moreover, the expressions of stemness and definitive endoderm markers confirmed effectively induced endoderm differentiation in MSC/hE-cad-PLGA aggregates, which was consistent with the pattern of embryonic development. After pre-differentiation for 1 week, the MSC/hE-cad-PLGA aggregates continuously progressed the hepatic phenotype expression in healthy rat peritoneum. Therefore, the biomimetic microenvironment constructed by hE-cad-PLGAs in engineered multicellular aggregates was able to promote the process of endoderm differentiation and the subsequent hepatic differentiation of hMSCs. It would be appropriate for applied research in hepatotoxic drug screening and cell-based treatment of liver diseases. By optimizing with other cytokine cocktail, the engineered multicellular aggregates can be applied to the construction of other endoderm-derived organs. STATEMENT OF SIGNIFICANCE: The differentiation of mesenchymal stem cells (MSCs) into hepatocyte-like cells in vitroprovides a promising for cell therapy for liver diseases, and cell aggregates have been proposed to improve the expansion and differentiation efficiency. Here, engineered multicellular aggregates were constructed by E-cadherin modified microparticles (hE-cad-PLGAs) construct a biomimetic microenvironment to promote the process of endoderm differentiation and the subsequent hepatic differentiation of hMSCs. Furthermore, after pre-differentiation for 1 week, the MSC/hE-cad-PLGA aggregates continuously progressed the hepatic phenotype expression in healthy rat peritoneum. Therefore, engineered multicellular aggregates with hE-cad-PLGAs would be appropriate for applied research in hepatotoxic drug screening and cell-based treatment of liver diseases, and provide a promising method in the construction of other endoderm-derived organs.

Enhanced Biological Functions of Human Mesenchymal Stem-Cell Aggregates Incorporating E-Cadherin-Modified PLGA Microparticles.

Mesenchymal stem cells (MSCs) have emerged as a promising source of multipotent cells for various cell-based therapies due to their unique properties, and formation of 3D MSC aggregates has been explored as a potential strategy to enhance therapeutic efficacy. In this study, poly(lactic-co-glycolic acid) (PLGA) microparticles modified with human E-cadherin fusion protein (hE-cad-PLGA microparticles) have been fabricated and integrated with human MSCs to form 3D cell aggregates. The results show that, compared with the plain PLGA, the hE-cad-PLGA microparticles distribute within the aggregates more evenly and further result in a more significant improvement of cellular proliferation and secretion of a series of bioactive factors due to the synergistic effects from the bioactive E-cadherin fragments and the PLGA microparticles. Meanwhile, the hE-cad-PLGA microparticles incorporated in the aggregates upregulate the phosphorylation of epidermal growth factor receptors and activate the AKT and ERK1/2 signaling pathways in the MSCs. Additionally, the E-cadherin/ß-catenin cellular membrane complex in the MSCs is markedly stimulated by the hE-cad-PLGA microparticles. Therefore, engineering 3D cell aggregates with hE-cad-PLGA microparticles can be a promising method for ex vivo multipotent stem-cell expansion with enhanced biological functions and may offer a novel route to expand multipotent stem-cell-based clinical applications.

Human VE-Cadherin Fusion Protein as an Artificial Extracellular Matrix Enhancing the Proliferation and Differentiation Functions of Endothelial Cell.

In an attempt to enhance endothelial cell capture and promote the vascularization of engineered tissue, we biosynthesized and characterized the recombinant fusion protein consisting of human vascular endothelial-cadherin extracellular domain and immunoglobulin IgG Fc region (hVE-cad-Fc) to serve as a bioartificial extracellular matrix. The hVE-cad-Fc protein naturally formed homodimers and was used to construct hVE-cad-Fc matrix by stably adsorbing on polystyrene plates. Atomic force microscop assay showed uniform hVE-cad-Fc distribution with nanorod topography. The hVE-cad-Fc matrix markedly promoted human umbilical vein endothelial cells (HUVECs) adhesion and proliferation with fibroblastoid morphology. Additionally, the hVE-cad-Fc matrix improved HUVECs migration, vWF expression, and NO release, which are closely related to vascularization. Furthermore, the hVE-cad-Fc matrix activated endogenous VE-cadherin/ß-catenin proteins and effectively triggered the intracellular signals such as F-actin stress fiber, p-FAK, AKT, and Bcl-2. Taken together, hVE-cad-Fc could be a promising bioartificial matrix to promote vascularization in tissue engineering.

Surface modification with E-cadherin fusion protein for mesenchymal stem cell culture.

To effectively expand human mesenchymal stem cells (hMSCs) in vitro without affecting their innate biological properties, a fusion protein (hE-cad-Fc) consisting of a human E-cadherin extracellular domain and an immunoglobulin G Fc region was fabricated and used as a biomimetic matrix for MSC culture surface modification. The results showed that cells cultured on hE-cad-Fc-modified polystyrene surfaces exhibited improved proliferation and paracrine functions compared with cells cultured on unmodified and collagen-modified polystyrene surfaces. Meanwhile, surfaces modified with hE-cad-Fc effectively inhibited cell apoptosis even under the serum deprivation conditions. Additionally, the hE-cad-Fc not only up-regulated the expression of ß-catenin in MSCs and stimulated the cellular membrane complex of E-cadherin/ß-catenin, but also effectively activated the intracellular signals such as EGFR, AKT and ERK phosphorylation. Therefore, hE-cad-Fc appeared to be a promising candidate for biological surface modification and stem cell culture.