Exploring the mechanism of Astragali radix for promoting osteogenic differentiation based on network pharmacology, molecular docking, and experimental validation.

The present study used network pharmacology and molecular docking to predict the active ingredients and mechanisms of action of Astragalus radix (AR) to promote osteogenic differentiation of bone marrow mesenchymal stem cells (BM-MSCs), and cell experiments were conducted for verification. First, network pharmacology was used to predict the effective components, targets, and mechanisms of action of AR to promote osteogenic differentiation. The effective components and corresponding target proteins of AR, and the target proteins of osteogenic differentiation were collected through the database. The intersection targets of the two were used for the construction and analysis of a protein-protein interaction (PPI) network. Gene Oncology (GO) and Kyoto Encyclopedia of Genes, and Genomes (KEGG) enrichment analyses were conducted. Next, molecular docking technology was carried out to verify the interaction between the active ingredient and the target protein, and to select the appropriate effective active ingredient. Finally, the results of network pharmacology analysis were verified by in vitro experiments. A total of 95 potential targets were retrieved by searching the intersection of AR and osteogenic differentiation targets. PPI network analysis indicated that RAC-α-serine-threonine-protein kinase (Akt1) was considered to be the most reliable target for AR to regulate osteogenic differentiation. GO enrichment analysis included 21 biological processes, 21 cellular components and 100 molecular functions. KEGG enrichment analysis indicated that the class I phosphatidylinositol-3 kinase (PI3K)-serine-threonine kinase (Akt) signaling pathway may play an important role in promoting osteogenic differentiation. The results of molecular docking showed that quercetin's performance was improved compared with that of kaempferol. In vitro experiments showed that quercetin promoted the expression of osteogenic marker proteins (including collagen I, Runt-related transcription factor 2 and osteopontin) in BMSCs and activated the PI3K/Akt signaling pathway. AR acted on Akt1 targets through its main active component quercetin, and promoted the osteogenic differentiation of BM-MSCs by activating the PI3K/Akt signaling pathway.

Mesenchymal stromal cells modulate YAP by verteporfin to mimic cartilage development and construct cartilage organoids based on decellularized matrix scaffolds.

The mechanical elasticity or stiffness of the ECM modulates YAP activity to regulate the differentiation of stem cells during the development and defect regeneration of cartilage tissue. However, the understanding of the scaffold-associated mechanobiology during the initiation of chondrogenesis and hyaline cartilaginous phenotype maintenance remains unclear. In order to elucidate such mechanisms to promote articular cartilage repair by producing more hyaline cartilage, we identify the relationship between YAP subcellular localization and variation of the cartilage structure and organization during the early postnatal explosive growth in incipient articular cartilage. Next, we prepared a decellularized cartilage scaffold with different stiffness (2-33 kPa) to investigate the effect of scaffold stiffness on the formation of hyaline cartilage by mesenchymal stem cells and the change of YAP activity. Furthermore, we simulated the decrease of cellular YAP activity during postnatal cartilage development by inhibiting YAP activity with verteporfin, and realized that the timing of drug incorporation was critical to regulate the differentiation of MSCs to hyaline chondrocytes and inhibit their hypertrophy and fibrosis. On this basis, we constructed hyaline cartilage organoids by decellularized matrix scaffolds. Collectively, the results herein demonstrate that YAP plays a critical role during in vitro chondrogenic differentiation which is tightly regulated by biochemical and mechanical regulation.

Xeno-free cultured mesenchymal stromal cells release extracellular vesicles with a "therapeutic" miRNA cargo ameliorating cartilage inflammation in vitro.

Rationale: Mesenchymal stromal cells (MSCs)-derived extracellular vesicles (EVs) emerged as an innovative strategy for the treatment of chronic disorders such as osteoarthritis (OA). Biological activity of EVs is generally driven by their cargo, which might be influenced by microenvironment. Therefore, pre-conditioning strategies, including modifications in culture conditions or oxygen tension could directly impact on MSCs paracrine activity. In this study we selected an appropriate preconditioning system to induce cells to perform the most suitable therapeutic response by EV-encapsulated bioactive factors. Methods: A xeno-free supplement (XFS) was used for isolation and expansion of MSCs and compared to conventional fetal bovine serum (FBS) culture. Bone Marrow-derived MSCs (BMSCs) were pre-conditioned under normoxia (20% O2) or under hypoxia (1% O2) and EVs production was evaluated. Anti-OA activity was evaluated by using an in vitro inflammatory model. miRNA content was also explored, to select putative miRNA that could be involved in a biological function. Results: Modulation of IL-6, IL-8, COX-2 and PGE2 was evaluated on hACs simultaneously treated with IL-1α and BMSC-derived EVs. FBS-sEVs exerted a blunt inhibitory effect, while a strong anti-inflammatory outcome was achieved by XFS-sEVs. Interestingly, in both cases hypoxia pre-conditioning allowed to increase EVs effectiveness. Analysis of miRNA content showed the upregulation in XFS-hBMSC-derived EVs of miRNA known to have a chondroprotective role, such as let-7b-5p, miR-17, miR-145, miR-21-5p, miR-214-3p, miR-30b-5p, miR-30c-5p. Activated pathways and target genes were investigated in silico and upregulated miRNAs functionally validated in target cells. MiR-145 and miR-214 were found to protect chondrocytes from IL-1α-induced inflammation and to reduce production of pro-inflammatory cytokines. Conclusions: XFS medium was found to be suitable for isolation and expansion of MSCs, secreting EVs with a therapeutic cargo. The application of cells cultured exclusively in XFS overcomes issues of safety associated with serum-containing media and makes ready-to-use clinical therapies more accessible.

Microbial Sterility Testing for Mesenchymal Progenitor Cells Derived from Human Osteoarthritis and Rheumatoid Arthritis Cartilage.

BACKGROUND: The goal is to evaluate the microbial sterility of mesenchymal progenitor cells (MPCs)-derived from osteoarthritis (OA) and rheumatoid arthritis (RA) articular cartilages. METHODS: Contaminants, including bacteria and fungi in MPC cultures were initially evaluated by inoculation culture methods and then affirmed through the amplification of 16S ribosomal DNA (rDNA) and internally transcribed spacer (ITS) regions, respectively, using polymerase chain reaction (PCR). Further, the mollicutes, if any, were identified by genus-specific 16S rDNA, and the positive samples were reamplified using species-specific primers. RESULTS: No bacteria or fungi were found to be compromising the sterility of MPCs (n = 20) assessed by both traditional culture methods and PCR. However, two in early passages and three in later passages of MPCs had the presence of mollicutes. Further rescreening for species of mollicutes indicated the presence of Mycoplasma hyorhinis, M. salivarium, and M. arginine. CONCLUSIONS: The PCR methods employed in this study could be beneficial as a rapid sterility testing of cell lines.

Exosomes from miR-374a-5p-modified mesenchymal stem cells inhibit the progression of renal fibrosis by regulating MAPK6/MK5/YAP axis.

Chronic kidney disease (CKD) in clinical is defined as a gradual loss of kidney function for more than 3 months. The pathologic course of CKD is characterized by extensive renal fibrosis; thus, preventing renal fibrosis is vital for the treatment of CKD. It has been reported that microRNA (miR)-374a-5p was under-expressed in renal venous blood samples from patients with CKD. In addition, it exhibited anti-apoptotic effects in renal tissues suggesting that miR-374a-5p may play an important role in CKD. However, it is not clear whether miR-374a-5p could be delivered to renal cells by exosomes and exerts anti-renal fibrosis effects. To mimic renal fibrosis in vitro, human renal tubular epithelial cell lines (HK-2 cells) were treated by transforming growth factor-ß (TGF-ß) 1. Reverse transcription-quantitative polymerase-chain reaction (RT-qPCR) or Western blot was carried out to evaluate the mechanism by which miR-374a-5p regulated the development of renal fibrosis. Next, exosomes were isolated using with ultracentrifugation method, and the relationship between miR-374a-5p and MAPK6 was evaluated using dual-Luciferase a reporter assay system. The results indicated TGF-ß1 significantly down-regulated the expression of miR-374a-5p in HK-2 cells and miR-374a-5p agomir remarkably inhibited the progression of fibrosis in vitro. In addition, exosomal miR-374a-5p could be internalized by HK-2 cells and obviously enhanced the level of miR-374a-5p in HK-2 cells. Furthermore, exosomal miR-374a-5p prevented the progression of renal fibrosis in vivo by regulating MAPK6/MK5/YAP axis. In conclusion, exosomal miR-374a-5p inhibited the progression of renal fibrosis by regulating MAPK6/MK5/YAP axis.

Nanoparticles functionalized with stem cell secretome and CXCR4-overexpressing endothelial membrane for targeted osteoporosis therapy.

BACKGROUND: Osteoporosis is a chronic condition affecting patients' morbidity and mortality and represents a big socioeconomic burden. Because stem cells can proliferate and differentiate into bone-forming cells, stem cell therapy for osteoporosis has been widely studied. However, cells as a live drug face multiple challenges because of their instability during preservation and transportation. In addition, cell therapy has potential adverse effects such as embolism, tumorigenicity, and immunogenicity. RESULTS: Herein, we sought to use cell-mimicking and targeted therapeutic nanoparticles to replace stem cells. We fabricated nanoparticles (NPs) using polylactic-co-glycolic acid (PLGA) loaded with the secretome (Sec) from mesenchymal stem cells (MSCs) to form MSC-Sec NPs. Furthermore, we cloaked the nanoparticles with the membranes from C-X-C chemokine receptor type 4 (CXCR4)-expressing human microvascular endothelial cells (HMECs) to generate MSC-Sec/CXCR4 NP. CXCR4 can target the nanoparticles to the bone microenvironment under osteoporosis based on the CXCR4/SDF-1 axis. CONCLUSIONS: In a rat model of osteoporosis, MSC-Sec/CXCR4 NP were found to accumulate in bone, and such treatment inhibited osteoclast differentiation while promoting osteogenic proliferation. In addition, our results showed that MSC-Sec/CXCR4 NPs reduce OVX-induced bone mass attenuation in OVX rats.

Local Elimination of Senescent Cells Promotes Bone Defect Repair during Aging.

Due to the declined function of bone marrow mesenchymal stem cells (BMSCs), the repair of bone defects in the elderly is retarded. Elimination of senescent cells emerges as a promising strategy for treating age-related diseases. However, whether the local elimination of senescent BMSCs can promote bone regeneration in the elderly remains elusive. To tackle the above issue, we first screened out the specific senolytics for BMSCs and confirmed their effect of eliminating senescent BMSCs in vitro. Treatment with quercetin, which is determined the best senolytics for senescent BMSCs, efficiently removed senescent cells in the population. Moreover, the self-renewal capacity was restored as well as osteogenic ability of BMSCs after treatment. We then designed a microenvironment-responsive hydrogel based on the MMPs secreted by senescent cells. This quercetin-encapsulated hydrogel exhibited a stable microstructure and responsively released quercetin in the presence of senescence in vitro. In vivo, the quercetin-loaded hydrogel effectively cleared the local senescent cells and reduced the secretion of MMPs in the bone. Due to the removal of local senescent cells, the hydrogel significantly accelerated the repair of bone defects in the femur and skull of old rats. Taken together, our study revealed the role of removing senescent cells in bone regeneration and provided a novel therapeutic approach for bone defects in aged individuals.

Bone mesenchymal stem cell-derived exosomal microRNA-7-5p inhibits progression of acute myeloid leukemia by targeting OSBPL11.

BACKGROUND: Acute myeloid leukemia (AML) is a malignant clonal disease of hematopoietic stem- and progenitor-cell origin. AML features massive proliferation of abnormal blasts and leukemia cells in the bone marrow and the inhibition of normal hematopoiesis at onset. Exosomes containing proteins or nucleic acids are secreted by cells; they participate in intercellular communication and serve as key modulators of hematopoiesis. The purpose of this study was to investigate the effects of exosomes derived from bone marrow mesenchymal stem cells (BMSCs) on the regulation of AML and the underlying mechanisms mediated by microRNA (miRNA). METHODS: Dysregulated miR-7-5p in AML patients was identified using qRT-PCR and its clinical significance was explored. Bioinformatic analysis revealed the target gene OSBPL11 that could be regulated by miR-7-5p. The findings were validated using a dual-luciferase reporter assay and western blotting. The functional genes of the PI3K/AKT/mTOR signaling pathway were identified, and the functional significance of miR-7-5p in AML cells was determined using a functional recovery assay. AML cells were co-cultured with exosomes originating from BMSCs overexpressing miR-7-5p to determine cell-cell regulation by Exo-miR-7-5p, as well as in vitro and in vivo functional validation via gain- and loss-of-function methods. RESULTS: Expression of miR-7-5p was decreased in AML patients and cells. Overexpression of miR-7-5p curbed cellular proliferation and promoted apoptosis. Overexpression of OSBPL11 reversed the tumorigenic properties of miR-7-5p in AML cells in vitro. Exo-miR-7-5p derived from BMSCs induced formation of AML cells prone to apoptosis and a low survival rate, with OSBPL11 expression inhibited through the PI3K/AKT/mTOR signaling pathway. Exo-miR-7-5p derived from BMSCs exhibited tumor homing effects in vitro and in vivo, and inhibited AML development. CONCLUSIONS: Exo-miR-7-5p derived from BMSCs negatively regulates OSBPL11 by suppressing the phosphorylation of the PI3K/AKT/mTOR signaling pathway, thereby inhibiting AML proliferation and promoting apoptosis. The data will inform the development of AML therapies based on BMSC-derived exosomes.

Exosomal microRNA⁃93⁃3p secreted by bone marrow mesenchymal stem cells downregulates apoptotic peptidase activating factor 1 to promote wound healing.

Wounds are soft tissue injuries, which are difficult to heal and can easily lead to other skin diseases. Bone marrow mesenchymal stem cells (BMSCs) and the secreted exosomes play a key role in skin wound healing. This study aims to clarify the effects and mechanisms of exosomes derived from BMSCs in wound healing. Exosomes were extracted from the supernatant of the BMSCs. The expression of the micro-RNA miR-93-3p was determined by qRT-PCR analysis. HaCaT cells were exposed to hydrogen peroxide (H2O2) to establish a skin lesion model. MTT, flow cytometry, and transwell assays were conducted to determine cellular functions. The binding relationship between miR-93-3p and apoptotic peptidase activating factor 1 (APAF1) was measured using a dual luciferase reporter gene assay. The results showed that BMSC-derived exosomes or BMSC-exos promoted proliferation and migration and suppressed apoptosis in HaCaT cells damaged by H2O2. However, the depletion of miR-93-3p in BMSC-exos antagonized the effects of BMSC-exos on HaCaT cells. In addition, APAF1 was identified as a target of miR-93-3p. Overexpression of APAF1 induced the dysfunction of HaCaT cells. Collectively, the results indicate that BMSC-derived exosomes promote skin wound healing via the miR-93-3p/APAF1 axis. This finding may help establish a new therapeutic strategy for skin wound healing.

Superferromagnetic Nanoparticles Enable Order-of-Magnitude Resolution & Sensitivity Gain in Magnetic Particle Imaging.

Magnetic nanoparticles have many advantages in medicine such as their use in non-invasive imaging as a Magnetic Particle Imaging (MPI) tracer or Magnetic Resonance Imaging contrast agent, the ability to be externally shifted or actuated and externally excited to generate heat or release drugs for therapy. Existing nanoparticles have a gentle sigmoidal magnetization response that limits resolution and sensitivity. Here it is shown that superferromagnetic iron oxide nanoparticle chains (SFMIOs) achieve an ideal step-like magnetization response to improve both image resolution & SNR by more than tenfold over conventional MPI. The underlying mechanism relies on dynamic magnetization with square-like hysteresis loops in response to 20 kHz, 15 kAm-1 MPI excitation, with nanoparticles assembling into a chain under an applied magnetic field. Experimental data shows a "1D avalanche" dipole reversal of every nanoparticle in the chain when the applied field overcomes the dynamic coercive threshold of dipole-dipole fields from adjacent nanoparticles in the chain. Intense inductive signal is produced from this event resulting in a sharp signal peak. Novel MPI imaging strategies are demonstrated to harness this behavior towards order-of-magnitude medical image improvements. SFMIOs can provide a breakthrough in noninvasive imaging of cancer, pulmonary embolism, gastrointestinal bleeds, stroke, and inflammation imaging.

Childhood Cartilage ECM Enhances the Chondrogenesis of Endogenous Cells and Subchondral Bone Repair of the Unidirectional Collagen-dECM Scaffolds in Combination with Microfracture.

Despite the formation of mechanically inferior fibrocartilage, microfracture (MF) still remains the gold standard to repair the articular cartilage defects in clinical settings. To date, although many tissue-engineering scaffolds have been developed to enhance the MF outcome, the clinical outcomes remain inconsistent. Decellularized extracellular matrix (dECM) is among the most promising scaffold for cartilage repair due to its inheritance of the natural cartilage components. However, the impact of dECM from different developmental stages on cellular chondrogenesis and therapeutic effect remains elusive, as the development of native cartilage involves the distinct temporal dependency of the ECM components and various growth factors. Herein, we hypothesized that the immature cartilage dECM at various developmental stages was inherently different, and would consequently impact the chondrogenic potential BMSCs. In this study, we fabricated three different unidirectional collagen-dECM scaffolds sourced from neonatal, childhood, and adolescent rabbit cartilage tissues, and identified the age-dependent biological variations, including DNA, cartilage-specific proteins, and growth factors; along with the mechanical and degradation differences. Consequently, the different local cellular microenvironments provided by these scaffolds led to the distinctive cell morphology, circularity, proliferation, chondrogenic genes expression, and chondrogenesis of BMSCs in vitro, and the different gross morphology, cartilage-specific protein production, and subchondral bone repair when in combination with microfracture in vivo. Together, this work highlights the immature cartilage dECM at different developmental stages that would result in the diversified effects to BMSCs, and childhood cartilage would be considered the optimal dECM source for the further development of dECM-based tissue engineering scaffolds in articular cartilage repair.

Restoring Cardiac Functions after Myocardial Infarction-Ischemia/Reperfusion via an Exosome Anchoring Conductive Hydrogel.

Both myocardial infarction (MI) and the follow-up reperfusion will lead to an inevitable injury to myocardial tissues, such as cardiac dysfunctions, fibrosis, and reduction of intercellular cell-to-cell interactions. Recently, exosomes (Exo) derived from stem cells have demonstrated a robust capability to promote angiogenesis and tissue repair. However, the short half-life of Exo and rapid clearance lead to insufficient therapeutic doses in the lesion area. Herein, an injectable conductive hydrogel is constructed to bind Exo derived from human umbilical cord mesenchymal stem cells to treat myocardial injuries after myocardial infarction-ischemia/reperfusion (MI-I/R). To this end, a hyperbranched epoxy macromer (EHBPE) grafted by an aniline tetramer (AT) was synthesized to cross-link thiolated hyaluronic acid (HA-SH) and thiolated Exo anchoring a CP05 peptide via an epoxy/thiol "click" reaction. The resulting Gel@Exo composite system possesses multiple features, such as controllable gelation kinetics, shear-thinning injectability, conductivity matching the native myocardium, soft and dynamic stability adapting to heartbeats, and excellent cytocompatibility. After being injected into injured hearts of rats, the hydrogel effectively prolongs the retention of Exo in the ischemic myocardium. The cardiac functions have been considerably improved by Gel@Exo administration, as indicated by the enhancing ejection fraction and fractional shortening, and reducing fibrosis area. Immunofluorescence staining and reverse transcription-polymerase chain reaction (RT-PCR) results demonstrate that the expression of cardiac-related proteins (Cx43, Ki67, CD31, and α-SMA) and genes (VEGF-A, VEGF-B, vWF, TGF-ß1, MMP-9, and Serca2a) are remarkably upregulated. The conductive Gel@Exo system can significantly improve cell-to-cell interactions, promote cell proliferation and angiogenesis, and result in a prominent therapeutic effect on MI-I/R, providing a promising therapeutic method for injured myocardial tissues.

Extruded Mesenchymal Stem Cell Nanovesicles Are Equally Potent to Natural Extracellular Vesicles in Cardiac Repair.

Mesenchymal stem cells (MSCs) repair injured tissues mainly through their paracrine actions. One of the important paracrine components of MSC secretomes is the extracellular vesicle (EV). The therapeutic potential of MSC-EVs has been established in various cardiac injury preclinical models. However, the large-scale production of EVs remains a challenge. We sought to develop a scale-up friendly method to generate a large number of therapeutic nanovesicles from MSCs by extrusion. Those extruded nanovesicles (NVs) are miniature versions of MSCs in terms of surface marker expression. The yield of NVs is 20-fold more than that of EVs. In vitro, cell-based assays demonstrated the myocardial protective effects and therapeutic potential of NVs. Intramyocardial delivery of NVs in the injured heart after ischemia-reperfusion led to a reduction in scar sizes and preservation of cardiac functions. Such therapeutic benefits are similar to those injected with natural EVs from the same MSC parental cells. In addition, NV therapy promoted angiogenesis and proliferation of cardiomyocytes in the post-injury heart. In summary, extrusion is a highly efficient method to generate a large quantity of therapeutic NVs that can potentially replace extracellular vesicles in regenerative medicine applications.

Hemin enhances the cardioprotective effects of mesenchymal stem cell-derived exosomes against infarction via amelioration of cardiomyocyte senescence.

BACKGROUND: Application of mesenchymal stem cell-derived exosomes (MSC-EXO) has emerged as a novel therapeutic strategy for myocardial infarction (MI). Our previous study showed that pretreatment with hemin, a potent heme oxygenase-1 (HO-1) inducer, enhanced the cardioprotective effects of MSCs in a mouse model of MI. This study aimed to investigate the therapeutic effects of EXO derived from hemin-pretreated MSCs (Hemin-MSC-EXO) in MI and explore the potential mechanisms. METHODS: MSC-EXO and Hemin-MSC-EXO were collected and characterized. MSC-EXO and Hemin-MSC-EXO were intramuscularly injected into the peri-infarct region in a mouse model of MI. Heart function of mice was assessed by echocardiography. The mitochondrial morphology of neonatal mice cardiomyocytes (NMCMs) under serum deprivation and hypoxic (SD/H) conditions was examined by Mitotracker staining. The cellular senescence of NMCMs was determined by senescence-associated-ß-galactosidase assay. A loss-of-function approach was adopted to determine the role of Hemin-MSC-exosomal-miR-183-5p in the regulation of cardiomyocyte senescence RESULTS: EXO were successfully isolated from the supernatant of MSCs and Hemin-pretreated MSCs. Compared with MSC-EXO, injection of Hemin-MSC-EXO significantly improved cardiac function and reduced fibrosis. Both MSC-EXO and Hemin-MSC-EXO ameliorated cardiomyocyte senescence and mitochondrial fission in vitro and in vivo, and the latter exhibited better protective effects. MicroRNA sequencing revealed a higher level of miR-183-5p in Hemin-MSC-EXO than in MSC-EXO. MiR-183-5p knockdown partially abrogated the protective effects of Hemin-MSC-EXO in attenuating mitochondrial fission and cellular senescence of cardiomyocytes induced by SD/H. High mobility group box-1 (HMGB1) abundance was lower in Hemin-MSC-EXO-treated than MSC-EXO-treated mouse hearts, and HMGB1 was identified as one of the potential target genes of miR-183-5p. Mechanistically, Hemin-MSC-EXO inhibited SD/H-induced cardiomyocyte senescence partially by delivering miR-183-5p into recipient cardiomyocytes via regulation of the HMGB1/ERK pathway. Furthermore, knockdown of miR-183-5p reduced the Hemin-MSC-EXO-mediated cardioprotective effects in a mouse model of MI. CONCLUSION: Our results reveal that Hemin-MSC-EXO are superior to MSC-EXO in treating MI. Exosomal miR-183-5p mediates, at least partially, the cardioprotective effects of Hemin-MSC-EXO by inhibiting cardiomyocyte senescence via regulation of the HMGB1/ERK pathway. This study highlights that MSC-EXO have high translational value in repairing cardiac dysfunction following infarction.

H19 Overexpression Improved Efficacy of Mesenchymal Stem Cells in Ulcerative Colitis by Modulating the miR-141/ICAM-1 and miR-139/CXCR4 Axes.

Overexpression of C-X-C motif chemokine receptor 4 (CXCR4) and intercellular cell adhesion molecule-1 (ICAM-1) may promote homing of mesenchymal stem cells (MSC). In this study, we treated ulcerative colitis animals with MSC preconditioned with or without H19 and compared the therapeutic effect of MSC and MSC-H19. We evaluated the regulatory relationship of H19 vs. miR-141/miR-139 and miR-141/miR-139 vs. ICAM-1/CXCR4. We established an ulcerative colitis mouse model to assess the effect of MSC and MSC-H19. H19 was found to bind to miR-141 and miR-139. The activity of H19 was strongly decreased in cells c-transfected with miR-141/miR-139 and WT H19. ICAM-1 was confirmed to be targeted by miR-141 and CXCR4 was targeted by miR-139. The H19 expression showed a negative regulatory relationship with the miR-141 and miR-139 expression but a positive regulatory relationship with the ICAM-1 and CXCR4 expression. In summary, the overexpression of H19 in MSC downregulated miR-139 and miR-141, thus increasing the activity of their targets ICAM-1 and CXCR4, respectively, to exhibit therapeutic effects in ulcerative colitis.

Mechanics-driven nuclear localization of YAP can be reversed by N-cadherin ligation in mesenchymal stem cells.

Mesenchymal stem cells adopt differentiation pathways based upon cumulative effects of mechanosensing. A cell's mechanical microenvironment changes substantially over the course of development, beginning from the early stages in which cells are typically surrounded by other cells and continuing through later stages in which cells are typically surrounded by extracellular matrix. How cells erase the memory of some of these mechanical microenvironments while locking in memory of others is unknown. Here, we develop a material and culture system for modifying and measuring the degree to which cells retain cumulative effects of mechanosensing. Using this system, we discover that effects of the RGD adhesive motif of fibronectin (representative of extracellular matrix), known to impart what is often termed "mechanical memory" in mesenchymal stem cells via nuclear YAP localization, are erased by the HAVDI adhesive motif of the N-cadherin (representative of cell-cell contacts). These effects can be explained by a motor clutch model that relates cellular traction force, nuclear deformation, and resulting nuclear YAP re-localization. Results demonstrate that controlled storage and removal of proteins associated with mechanical memory in mesenchymal stem cells is possible through defined and programmable material systems.

Development of Cell-Derived Matrices for Three-Dimensional In Vitro Cancer Cell Models.

Most morphogenetic and pathological processes are driven by cells responding to the surrounding matrix, such as its composition, architecture, and mechanical properties. Despite increasing evidence for the role of extracellular matrix (ECM) in tissue and disease development, many in vitro substitutes still fail to effectively mimic the native microenvironment. We established a novel method to produce macroscale (>1 cm) mesenchymal cell-derived matrices (CDMs) aimed to mimic the fibrotic tumor microenvironment surrounding epithelial cancer cells. CDMs are produced by human adipose mesenchymal stem cells cultured in sacrificial 3D scaffold templates of fibronectin-coated poly-lactic acid microcarriers (MCs) in the presence of macromolecular crowders. We showed that decellularized CDMs closely mimic the fibrillar protein composition, architecture, and mechanical properties of human fibrotic ECM from cancer masses. CDMs had highly reproducible composition made of collagen types I and III and fibronectin ECM with tunable mechanical properties. Moreover, decellularized and MC-free CDMs were successfully repopulated with cancer cells throughout their 3D structure, and following chemotherapeutic treatment, cancer cells showed greater doxorubicin resistance compared to 3D culture in collagen hydrogels. Collectively, these results support the use of CDMs as a reproducible and tunable tool for developing 3D in vitro cancer models.

Microrna analysis of human decidua mesenchymal stromal cells from preeclampsia patients.

INTRODUCTION: In preeclampsia (PE), human decidua mesenchymal stromal cells (hDMSCs) are exposed to abnormally high levels of oxidative stress and inflammatory factors circulating in the maternal blood. MicroRNAs (miRNAs) have been shown to have a significant impact on the differentiation, maturation and function of mesenchymal stromal cells (MSCs). Our aim in the present study is firstly to investigate differentially expressed miRNA levels to be used as a biomarker in the early detection of PE and secondly to investigate whether those differentially expressed miRNAs in hDMSCs have an effect on the pathogenesis of PE. METHODS: This study covers miRNA expression analysis of hDMSCs from 7 PE patient and 7 healthy pregnant women and is a preliminary study to investigate putative biomarkers. After cell culture and cell sorting, total RNA including miRNAs were isolated from hDMSCs. Let-7b-3p, let-7f-1-3p, miR-191-3p, miR-550a-5p, miR-33b-3p and miR-425-3p were used for miRNA analysis and U6 snRNA was used for normalization of the samples. MiRNA analysis was performed by droplet digital polymerase chain reaction (ddPCR) method and obtained results were evaluated statistically. RESULTS: As a result of the analysis, it was observed that the levels of hsa-miR-33b-3p significantly (AUC: 0.93, p = 0.04, fold change: 4.5) increased in hDMSC of PE patients compared to healthy controls. However, let-7b-3p, let-7f-1-3p, miR-191-3p, miR-550a-5p, and miR-425-3p were not considered as significant because they did not meet the p < 0,05 requirement. DISCUSSION: Within the scope of the study, it is predicted that miR-33b-3p (p = 0.004, AUC = 0.93) can be used as a biomarker in detecting PE.

Identification of intracellular glycosaminoglycan-interacting proteins by affinity purification mass spectrometry.

Glycosaminoglycans (GAGs) are essential functional components of the extracellular matrix (ECM). Artificial GAGs like sulfated hyaluronan (sHA) exhibit pro-osteogenic properties and boost healing processes. Hence, they are of high interest for supporting bone regeneration and wound healing. Although sulfated GAGs (sGAGs) appear intracellularly, the knowledge about intracellular effects and putative interaction partners is scarce. Here we used an affinity-purification mass spectrometry-based (AP-MS) approach to identify novel and particularly intracellular sGAG-interacting proteins in human bone marrow stromal cells (hBMSC). Overall, 477 proteins were found interacting with at least one of four distinct sGAGs. Enrichment analysis for protein localization showed that mainly intracellular and cell-associated interacting proteins were identified. The interaction of sGAG with α2-macroglobulin receptor-associated protein (LRPAP1), exportin-1 (XPO1), and serine protease HTRA1 (HTRA1) was confirmed in reverse assays. Consecutive pathway and cluster analysis led to the identification of biological processes, namely processes involving binding and processing of nucleic acids, LRP1-dependent endocytosis, and exosome formation. Respecting the preferentially intracellular localization of sGAG in vesicle-like structures, also the interaction data indicate sGAG-specific modulation of vesicle-based transport processes. By identifying many sGAG-specific interacting proteins, our data provide a resource for upcoming studies aimed at molecular mechanisms and understanding of sGAG cellular effects.

Facile extrusion 3D printing of gelatine methacrylate/Laponite nanocomposite hydrogel with high concentration nanoclay for bone tissue regeneration.

The extrusion 3D printing of hydrogels has evolved as a promising approach that can be applied for specific tissue repair. However, the printing process of hydrogel scaffolds with high shape fidelity is inseparable from the complex crosslinking strategy, which significantly increases the difficulty and complexity of printing. The aim of this study was to develop a printable hydrogel that can extrude at room temperature and print scaffolds with high shape fidelity without any auxiliary crosslinking during the printing process. To this end, a novel formulation consisting of a Laponite suspension with a high solid concentration and a gelatine methacrylate (GelMA) nanocomposite hydrogel was developed. A homogeneously dispersed high-concentration (up to 20% w/v) Laponite suspension was obtained by stirring at 0 °C. The addition of Laponite with high concentration improved the rheological properties, the degradation stability, and the mechanical strength of the hydrogel. The formulation of 15% (w/v) GelMA and 8% (w/v) Laponite nanocomposite hydrogel exhibited desirable printability and biocompatibility. The GelMA/Laponite hydrogels significantly promoted bone marrow mesenchymal stem cell (BMSC) proliferation and osteogenic differentiation. Both desirable printability under mild conditions and cyto-compatibility enable composite hydrogel a potential candidate as biomaterial inks to be applied for bone tissue regeneration.