Human Umbilical Cord Mesenchymal Stem Research Articles

Human mesenchymal stem cells derived exosomes improve ovarian function in chemotherapy-induced premature ovarian insufficiency mice by inhibiting ferroptosis through Nrf2/GPX4 pathway.

Chemotherapy exposure has become a main cause of premature ovarian insufficiency (POI). This study aimed to evaluate the role and molecular mechanism of human umbilical cord mesenchymal stem cell-derived exosomes (hUMSC-Exos) in ovarian function protection after chemotherapy. hUMSC-Exos were applied to cyclophosphamide-induced premature ovarian insufficiency mice and human ovarian granulosa tumor cells (KGN) to determine their effects on follicular development and granulosa cell apoptosis. Evaluation was done for iron ion and reactive oxygen species (ROS) production, lipid peroxidation levels, and changes in iron death-related molecules (nuclear factor (erythroid-derived 2)-like 2 (Nrf2), Glutathione Peroxidase enzyme 4 (GPX4), and Solute carrier family 7 member 11 cystine glutamate transporter (SLC7A11; xCT)). Furthermore, rescue experiments using an Nrf2 inhibitor were performed to assess the therapeutic effects of hUMSC-Exos on granulosa cells. hUMSC-Exos promoted ovarian hormone levels and primary follicle development in POI mice and reduced granulosa cell apoptosis. After hUMSC-Exos treatment, the ROS production, free iron ions and lipid peroxidation levels of granulosa cells decreased, and the iron death marker proteins Nrf2, xCT and GPX4 also decreased. Furthermore, the Nrf2 inhibitor ML385 significantly attenuated the effects of hUMSC-Exos on granulosa cells. hUMSC-Exos inhibit ferroptosis and protect against CTX-induced ovarian damage and granulosa cell apoptosis through the Nrf2/GPX4 signaling pathway, revealing a novel mechanism of hUMSC-Exos in POI therapy.

Fibroblast matrix implants - a better alternative for incisional hernia repair?

The standard surgical procedure for abdominal hernia repair with conventional prosthetic mesh still results in a high recurrence rate. In the present study, we propose a Fibroblast
Matrix Implant (FMI), which is a three-dimensional (3D) Poly-L-lactic acid (PLLA) scaffold coated with collagen (matrix) and seeded with fibroblasts, as an alternative mesh for hernia repair. The matrix was seeded with fibroblasts (cellularized) and treated with a Conditioned
Medium (CM) of human Umbilical Cord Mesenchymal Stem Cells (hUC-MSC). Fibroblast proliferation and function were assessed and compared between treated with CM hUC-MSC and untreated group, 24 hours after seeding onto the matrix (n=3). To study the matrices in vivo, the hernia was surgically created on male Sprague Dawley rats and repaired with four different grafts (n=3), including a commercial mesh (mesh group), a matrix without cells
(cell-free group), a matrix seeded with fibroblasts (FMI group), and a matrix seeded with fibroblasts and cultured in medium treated with 1 % CM hUC-MSC (FMI-CM group). In vitro examination showed that the fibroblasts' proliferation on the matrices (treated group) did not differ significantly compared to the untreated group. CM hUC-MSC was able to promote the collagen synthesis of the fibroblasts, resulting in a higher collagen concentration compared to the untreated group. Furthermore, the in vivo study showed that the matrices allowed fibroblast growth and supported cell functionality for at least 1 month after implantation. The highest number of fibroblasts was observed in the FMI group at the 14-day endpoint, but at the 28-day endpoint, the FMI-CM group had the highest. Collagen deposition area and neovascularization at the implantation site were observed in all groups without any significant difference between the groups. FMI combined with CM hUC-MSC may serve as a better option for hernia repair, providing additional reinforcement which in turn should reduce hernia recurrence.&#xD.

The impact of hUC MSC-derived exosome-nanoliposome hybrids on α-synuclein fibrillation and neurotoxicity.

Amyloid aggregation of α-synuclein (αSN) protein amplifies the pathogenesis of neurodegenerative diseases (NDs) such as Parkinson's disease (PD). Consequently, blocking aggregation or redirecting self-assembly to less toxic aggregates could be therapeutic. Here, we improve brain-specific nanocarriers using a hybrid of exosomes (Ex) from human umbilical cord mesenchymal stem cells (hUC MSCs) and nanoliposomes containing baicalein (Ex-NLP-Ba) and oleuropein (Ex-NLP-Ole). The hybrids contained both lipid membranes, Ex proteins, and baicalein or oleuropein. Fluorescence resonance energy transfer analysis confirmed their proper integration. The hybrids reduced the extent of αSN fibrillation and interfered with secondary nucleation and disaggregation. They not only reduced αSN pathogenicity but also enhanced drug internalization into cells, surpassing the efficacy of NLP alone, and also crossed the blood-brain barrier in a cellular model. We conclude that Ex can be successfully extracted and efficiently merged with NLPs while retaining its original properties, demonstrating great potential as a theranostic drug delivery vehicle against NDs like PD.

Human umbilical cord mesenchymal stem cell-derived exosomes ameliorate renal fibrosis in diabetic nephropathy by targeting Hedgehog/SMO signaling.

Diabetic nephropathy (DN) is the leading cause of end-stage renal disease globally. Currently, there are no effective drugs for the treatment of DN. Although several studies have reported the therapeutic potential of mesenchymal stem cells, the underlying mechanisms remain largely unknown. Here, we report that both human umbilical cord MSCs (UC-MSCs) and UC-MSC-derived exosomes (UC-MSC-exo) attenuate kidney damage, and inhibit epithelial-mesenchymal transition (EMT) and renal fibrosis in streptozotocin-induced DN rats. Strikingly, the Hedgehog receptor, smoothened (SMO), was significantly upregulated in the kidney tissues of DN patients and rats, and positively correlated with EMT and renal fibrosis. UC-MSC and UC-MSC-exo treatment resulted in decrease of SMO expression. Invitro co-culture experiments revealed that UC-MSC-exo reduced EMT of tubular epithelial cells through inhibiting Hedgehog/SMO pathway. Collectively, UC-MSCs inhibit EMT and renal fibrosis by delivering exosomes and targeting Hedgehog/SMO signaling, suggesting that UC-MSCs and their exosomes are novel anti-fibrotic therapeutics for treating DN.

Roles of Akirin1 in early prediction and treatment of graft kidney ischemia‒reperfusion injury

AbstractFerroptosis is a predominant contributor to graft kidney ischemia‒reperfusion injury (IRI), resulting in delayed graft function (DGF). However, much less is known about the early predicting biomarkers and therapeutic targets of DGF, especially aiming at ferroptosis. Here, we propose a precise predicting model for DGF, relying on the Akirin1 level in extracellular vesicles (EVs) derived from recipient urine 48 h after kidney transplant. In addition, we decipher a new molecular mechanism whereby Akirin1 induces ferroptosis by strengthening TP53‐mediated suppression of SLC7A11 during the graft kidney IRI process, that is, Akirin1 activates the EGR1/TP53 axis and inhibits MDM2‐mediated TP53 ubiquitination, accordingly upregulating TP53 in two ways. Meanwhile, we present the first evidence that miR‐136‐5p enriched in EVs secreted by human umbilical cord mesenchymal stem cells (UM‐EVs) confers robust protection against ferroptosis and graft kidney IRI by targeted inhibition of Akirin1 but knockout of miR‐136‐5p in UM sharply mitigates the protection of UM‐EVs. The functional and mechanistic regulation of Akirin1 is further corroborated in an allograft kidney transplant model in wild‐type and Akirin1‐knockout mice. In summary, these findings suggest that Akirin1, which prominently induces ferroptosis, is a pivotal biomarker and target for early diagnosis and treatment of graft kidney IRI and DGF after kidney transplant.

BMP7-Loaded Human Umbilical Cord Mesenchymal Stem Cell-Derived Small Extracellular Vesicles Ameliorate Liver Fibrosis by Targeting Activated Hepatic Stellate Cells.

Human umbilical cord mesenchymal stem cell (hucMSC)-derived small extracellular vesicles (sEVs) are natural nanocarriers with promising potential in treating liver fibrosis and have widespread applications in the fields of nanomedicine and regenerative medicine. However, the therapeutic efficacy of natural hucMSC-sEVs is currently limited owing to their non-specific distribution in vivo and partial removal by mononuclear macrophages following systemic delivery. Thus, the therapeutic efficacy can be improved through the development of engineered hucMSC-sEVs capable to overcome these limitations. To improve the anti-liver fibrosis efficacy of hucMSC-sEVs, we genetically engineered hucMSC-sEVs to overexpress the anti-fibrotic gene bone morphogenic protein 7 (BMP7) in parental cells. This was achieved using lentiviral transfection, following which BMP7-loaded hucMSC-sEVs were isolated through ultracentrifugation. First, the liver fibrosis was induced in C57BL/6J mice by intraperitoneal injection of 50% carbon tetrachloride (CCL4) twice a week for 8 weeks. These mice were subsequently treated with BMP7+sEVs via tail vein injection, and the anti-liver fibrosis effect of BMP7+sEVs was validated using small animal in vivo imaging, immunohistochemistry (IHC), tissue immunofluorescence, and enzyme-linked immunosorbent assay (ELISA). Finally, cell function studies were performed to confirm the in vivo results. Liver imaging and liver histopathology confirmed that the engineered hucMSC-sEVs could reach the liver of mice and aggregate around activated hepatic stellate cells (aHSCs) with a significantly stronger anti-liver fibrosis effect of BMP7-loaded hucMSC-sEVs compared to those of blank or negative control-transfected hucMSC-sEVs. In vitro, BMP7-loaded hucMSC-sEVs promoted the phenotypic reversal of aHSCs and inhibited their proliferation to enhance the anti-fibrotic effects. These engineered BMP7-loaded hucMSC-sEVs offer a novel and promising strategy for the clinical treatment of liver fibrosis.

Fibroblasts/three-dimensional scaffolds complexes promote wound healing in rats with skin defects

ABSTRACT We aim to construct a three-dimensional nano-skin scaffold material in vitro and study its promoting effect on wound healing in vivo. In this study, hybrid constructs of three-dimensional (3D) scaffolds were successfully fabricated by combination of type I collagen (COL-1) and polylactic-glycolic acid (PLGA). Fibroblasts and human umbilical cord mesenchymal stem cells (hUCMSCs) were used to implanted into 3D scaffolds and constructed into SD skin scaffolds in vitro. Finally, the fibroblasts/scaffolds complexes were inoculated on the surface of rat wound skin to study the promoting effect of the complex on wound healing. In our study, we successfully built a 3D scaffold, which had a certain porosity. Meanwhile, the content of COL-1 in the cell supernatant of fibroblast/scaffold complexes was increased. Furthermore, the expression of F-actin, CD105, integrin β, VEGF, and COL-1 was up-regulated in hUCMSC/scaffold complexes compared with the control group. In vivo, fibroblast/scaffold complexes promoted wound healing in rats. Our data suggested that the collagen Ⅳ and vimentin were elevated and collagen fibers were neatly arranged in the fibroblast/scaffold complex group was significantly higher than that in the scaffold group. Taken together, fibroblast/scaffold complexes were expected to be novel materials for treating skin defects.

The Combined Anti-Aging Effect of Hydrolyzed Collagen Oligopeptides and Exosomes Derived from Human Umbilical Cord Mesenchymal Stem Cells on Human Skin Fibroblasts.

Stem cell-derived exosomes (SC-Exos) are used as a source of regenerative medicine, but certain limitations hinder their uses. The effect of hydrolyzed collagen oligopeptides (HCOPs), a functional ingredient of SC-Exos is not widely known to the general public. We herein evaluated the combined anti-aging effects of HCOPs and exosomes derived from human umbilical cord mesenchymal stem cells (HucMSC-Exos) using a senescence model established on human skin fibroblasts (HSFs). This study discovered that cells treated with HucMSC-Exos + HCOPs enhanced their proliferative and migratory capabilities; reduced both reactive oxygen species production and senescence-associated β-galactosidase activity; augmented type I and type III collagen expression; attenuated the expression of matrix-degrading metalloproteinases (MMP-1, MMP-3, and MMP-9), interleukin 1 beta (IL-1β), and tumor necrosis factor-alpha (TNF-α); and decreased the expression of p16, p21, and p53 as compared with the cells treated with HucMSC-Exos or HCOPs alone. These results suggest a possible strategy for enhancing the skin anti-aging ability of HucMSC-Exos with HCOPs.

Unveiling the functional heterogeneity of cytokine-primed human umbilical cord mesenchymal stem cells through single-cell RNA sequencing

BackgroundMesenchymal stem cells (MSCs) hold immense promise for use in immunomodulation and regenerative medicine. However, their inherent heterogeneity makes it difficult to achieve optimal therapeutic outcomes for a specific clinical disease. Primed MSCs containing a certain cytokine can enhance their particular functions, thereby increasing their therapeutic potential for related diseases. Therefore, understanding the characteristic changes and underlying mechanisms of MSCs primed by various cytokines is highly important.ResultsIn this study, we aimed to reveal the cellular heterogeneity, functional subpopulations, and molecular mechanisms of MSCs primed with IFN-γ, TNF-α, IL-4, IL-6, IL-15, and IL-17 using single-cell RNA sequencing (scRNA-seq). Our results demonstrated that cytokine priming minimized the heterogeneity of the MSC transcriptome, while the expression of MSC surface markers exhibited only slight changes. Notably, compared to IL-6, IL-15, and IL-17; IFN-γ, TNF-α, and IL-4 priming, which stimulated a significantly greater number of differentially expressed genes (DEGs). Functional analysis, which included Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses, indicated that IFN-γ, TNF-α, and IL-4-primed hUC-MSCs are involved in interferon-mediated immune-related processes, leukocyte migration, chemotaxis potential, and extracellular matrix and cell adhesion, respectively. Moreover, an investigation of various biological function scores demonstrated that IFN-γ-primed hUC-MSCs exhibit strong immunomodulatory ability, TNF-α-primed hUC-MSCs exhibit high chemotaxis potential, and IL-4-primed hUC-MSCs express elevated amounts of collagen. Finally, we observed that cytokine priming alters the distribution of functional subpopulations of MSCs, and these subpopulations exhibit various potential biological functions. Taken together, our study revealed the distinct regulatory effects of cytokine priming on MSC heterogeneity, biological function, and functional subpopulations at the single-cell level.ConclusionsThese findings contribute to a comprehensive understanding of the inflammatory priming of MSCs, paving the way for their precise treatment in clinical applications.

Differential expression of circular RNAs in human umbilical cord mesenchymal stem cells treated with icariin.

Human umbilical cord mesenchymal stem cells (hUMSCs) belong to a multipotent stem cell population. Transplantation of icariin (ICA)-treated hUMSCs have better tissue repairing function in chronic liver injury. This study was to investigate whether the tissue-repairing effects and migration of hUMSCs after ICA treatment were regulated by circular RNAs (circRNAs). ICA was used to treat hUMSCs in vitro for 1 week and the expression profiles of circRNAs were generated using RNA sequencing. Differentially expressed circRNAs in hUMSCs after ICA intervention were screened. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analysis were carried out to predict the potential function of dysregulated circRNAs. There were 52 differentially expressed circRNAs (32 circRNAs up-regulated and 20 circRNAs down-regulated) with fold change ≥2.0 before and after ICA treatment. ADP-ribosylation factors were associated with the dysregulated circRNAs among Gene Ontology analysis. Kyoto Encyclopedia of Genes and Genomes analysis showed that only endocytosis pathway was associated with up-regulated circRNAs, whereas 4 pathways including homologous recombination, RNA transport, axon guidance, and proteoglycans in cancer were related to down-regulated circRNAs. Fifty-two differentially expressed circRNAs and 238 predicted microRNAs were included in circRNAs-microRNAs network. The mechanism of ICA inducing hUMSCs migration may be through regulating circRNAs expression which affects ADP-ribosylation factors protein signal pathways.

Targeted Repair of Spinal Cord Injury Based on miRNA-124-3p-Loaded Mesoporous Silica Camouflaged by Stem Cell Membrane Modified with Rabies Virus Glycoprotein.

Spinal cord injury (SCI) has no effective treatment modalities. It faces a significant global therapeutical challenge, given its features of poor axon regeneration, progressive local inflammation, and inefficient systemic drug delivery due to the blood-spinal cord barrier (BSCB). To address these challenges, a new nano complex that achieves targeted drug delivery to the damaged spinal cord is proposed, which contains a mesoporous silica nanoparticle core loaded with microRNA and a cloaking layer of human umbilical cord mesenchymal stem cell membrane modified with rabies virus glycoprotein (RVG). The nano complex more readily crosses the damaged BSCB with its exosome-resembling properties, including appropriate size and a low-immunogenic cell membrane disguise and accumulates in the injury center because of RVG, where it releases abundant microRNAs to elicit axon sprouting and rehabilitate the inflammatory microenvironment. Culturing with nano complexes promotes axonal growth in neurons and M2 polarization in microglia. Furthermore, it showed that SCI mice treated with this nano complex by tail vein injection display significant improvement in axon regrowth, microenvironment regulation, and functional restoration. The efficacy and biocompatibility of the targeted delivery of microRNA by nano complexes demonstrate their immense potential as a noninvasive treatment for SCI.

Extracellular vesicles secreted from mesenchymal stem cells ameliorate renal ischemia reperfusion injury by delivering miR-100-5p targeting FKBP5/AKT axis

The incidence of acute kidney injury (AKI) due to ischemia–reperfusion (IR) injury is increasing. There is no effective treatment for AKI, and because of this clinical challenge, AKI often progresses to chronic kidney disease, which is closely associated with poor patient outcomes and high mortality rates. Small extracellular vesicles from human umbilical cord mesenchymal stem cells (hUCMSC-sEVs) play increasingly vital roles in protecting tissue function from the effects of various harmful stimuli owing to their specific biological features. In this study, we found that miR-100-5p was enriched in hUCMSC-sEVs, and miR-100-5p targeted FKBP5 and inhibited HK-2 cell apoptosis by activating the AKT pathway. HK-2 cells that were exposed to IR injury were cocultured with hUCMSC-sEVs, leading to an increase in miR-100-5p levels, a decrease in FKBP5 levels, and an increase in AKT phosphorylation at Ser 473 (AKT-473 phosphorylation). Notably, these effects were significantly reversed by transfecting hUCMSCs with an miR-100-5p inhibitor. Moreover, miR-100-5p targeted FKBP5, as confirmed by a dual luciferase reporter assay. In vivo, intravenous infusion of hUCMSC-sEVs into mice suffering from IR injury resulted in significant apoptosis inhibition, functional maintenance and renal histological protection, which in turn decreased FKBP5 expression levels. Overall, this study revealed an effect of hUCMSC-sEVs on inhibiting apoptosis; hUCMSC-sEVs reduced renal IR injury by delivering miR-100-5p to HK-2 cells, targeting FKBP5 and thereby promoting AKT-473 phosphorylation to activate the AKT pathway. This study provides novel insights into the role of hUCMSC-sEVs in the treatment of AKI.

Isolation, Biological Characterization, and Quality Inspection of Human Umbilical Cord-derived Mesenchymal Stem Cells for Clinical Research

Background: Human umbilical cord mesenchymal stem cells (hUCMSCs) represent a valuable and versatile cell type derived from the umbilical cord tissue of newborns. These cells exhibit diverse characteristics, including multipotent differentiation potential and low immunogenicity. hUCMSCs hold promise for clinical applications, yet concerns persist regarding their chromosomal stability. This study employs short tandem repeat (STR) analysis to assess genomic integrity. By focusing on chromosomal stability, the study aims to enhance confidence in the clinical viability of hUCMSCs, contributing to their safe and effective use in regenerative medicine. hUCMSCs were extracted from healthy newborn umbilical cords using tissue block adherence and enzyme digestion. The third generation of hUCMSCs undergoes biological characterization and quality checks, including examinations of cell morphology, viability, growth curves, surface markers, cell cycle, and multi-lineage differentiation potential. The result of the present study shows that hUCMSCs exhibit mesenchymal stem cell characteristics, with robust growth and high viability. Positive surface markers are expressed at rates exceeding 95%, while negative markers are ≤2%. Cell karyotyping analysis, STR spectrum identification, and chromosomal microarray analysis confirm genetic stability. Microbial testing shows the absence of contaminants, and immunological studies demonstrate the immunomodulatory capabilities of hUCMSCs. Specific residues, such as trypsin, were not detected. In conclusion, hUCMSCs produced under strict GLP and GMP conditions meet the quality guidelines for clinical application in various stem cell therapies. The findings support the clinical application of hUCMSCs in various medical contexts.

Intrauterine injection of bioengineered hydrogel loaded exosomes derived from HUCM stem cells and spermidine prominently augments the pregnancy rate in thin endometrium rats

The endometrium is essential to the development of embryos and pregnancy. Human umbilical cord mesenchymal stem cells (HUCMSCs) are promising stem cell sources. HUCMSCs self-renew quickly and are painless to collect. Spermidine is an inherent polyamine needed for cellular and molecular processes that regulate physiology and function. HUCMSCs and spermidine (SN) may heal intrauterine adhesions. HUCMSCs were investigated for endometrial repair in rats. Composite hydrogels are used for medical exosome implantation, including their materials, properties, and embedding procedures. This study examined whether bioengineered hydrogel-loaded exosomes from HUCMSCs and spermidine prenatally improved conception rates in mice with poor endometrial lining. The data show that HUCMSC and SN provide a good experimental base for HUCMSC safety and intrauterine treatment in rats. Western blots, exosome structural analysis, pregnancy outcomes, flow cytometry, H&E staining, immunohistochemistry, and immunofluorescence labelling found and recovered the aberrant area. HUCM-derived stem cells and spermidine-derived exosomes biophysically match. These traits strengthen and prolong endometrial function. Pregnant rats with HUCMSC and SN had thinner endometrium. Hydrogel-incorporated HEHUCMSC and SN exosomes may improve IUI in rats with thin endometrium.

Stimulated Human Umbilical Cord Mesenchymal Stem Cells Enhance the Osteogenesis and Cranial Bone Regeneration through IL-32 Mediated P38 Signaling Pathway.

Our previous study found that it could significantly increase the expression of IL32 after stimulating the human umbilical cord mesenchymal stem cells (S-HuMSCs). However, its role on the osteogenesis and cranial bone regeneration is still largely unknown. Here, we investigated the possible mechanism of this effect. Material and Methods. A series of experiments, including single-cell sequencing, flow cytometry, quantitative real-time polymerase chain reaction, and western blotting, were carried out to evaluate the characteristic and adipogenic-osteogenic differentiation potential of IL-32 overexpression HuMSCs (IL-32highHuMSCs) through mediating the P38 signaling pathway. Moreover, a rat skull bone defect model was established and treated by directly injecting the IL-32highHuMSCs to conduct its role on the cranial bone regeneration. In total, it found that compared to HuMSCs, IL32 was significantly increased and promoted the osteogenic differentiation (lower expressions of PPARγ, Adiponectin, and C/EBPα, and increased expressions of RUNX2, ALP, BMP2, OPN, SP7, OCN, and DLX5) in the S-HuMSCs (P < 0.05). Meanwhile, the enhanced osteogenic differentiation of HuMSCs was recovered by IL-32 overexpression (IL-32highHuMSCs) through activating the P38 signaling pathway, like as the S-HuMSCs (P < 0.05). However, the osteogenic differentiation potential of IL-32highHuMSCs was significantly reversed by the P38 signaling pathway inhibitor SB203580 (P < 0.05). Additionally, the HuMSCs, S-HuMSCs, and IL-32highHuMSCs all presented adipogenic-osteogenic differentiation potential, with higher levels of CD73, CD90, and CD105, and lower CD14, CD34, and CD45 (P > 0.05). Furthermore, these findings were confirmed by the rat skull bone defect model, in which the cranial bone regeneration was more pronounced in the IL-32highHuMSCs treated group compared to those in the HuMSCs group, with higher expressions of RUNX2, ALP, BMP2, and DLX5 (P < 0.05). We have confirmed that S-HuMSCs can enhance the osteogenesis and cranial bone regeneration through promoting IL-32-mediated P38 signaling pathway, which is proved that IL-32 may be a therapeutic target, or a biomarker for the treatment of cranial bone injuries.

METTL3 promotes osteogenic differentiation of human umbilical cord mesenchymal stem cells by up-regulating m6A modification of circCTTN.

Human umbilical cord mesenchymal stem cells (hUCMSCs) are promising seed cells in bone tissue engineering. circRNA and N6-methyladenosine (m6A) RNA methylation play important roles in osteogenic differentiation. Here, we investigated the potential relevance of a critical circRNA, hsa_circ_0003376 (circCTTN), and methyltransferase-like 3 (METTL3) in osteogenic differentiation of hUCMSCs. Expression of circCTTN after hUCMSC osteogenic induction was detected by qRT-PCR. Three databases (RMBase v2.0, BERMP, and SRAMP) were used to predict m6A sites of circCTTN. RNA was enriched by methylated RNA immunoprecipitation (MeRIP), followed by quantitative real-time polymerase chain reaction to detect m6A level of circCTTN after METTL3 overexpression and osteogenic induction. RNA pull-down, Western blotting, and protein mass spectrometry were performed to investigate the potential mechanisms by which METTL3 promoted m6A modification of circCTTN. Bioinformatic analyses based on database (STRING) search and co-immunoprecipitation were used to analyze the proteins that interacted with METTL3. Overexpression of METTL3 promoted osteogenic differentiation of hUCMSCs and increased m6A level of circCTTN. Two potential m6A modification sites of circCTTN were predicted. No direct interaction between METTL3 and circCTTN was observed. Thirty-one proteins were pulled down by probes specific for circCTTN, including NOP2, and two m6A reading proteins, EIF3A and SND1. Bioinformatics analysis and co-immunoprecipitation showed that METTL3 interacted with EIF3A indirectly through NOP2. METTL3 promotes the osteogenic differentiation of hUCMSCs by increasing the m6A level of circCTTN. However, METTL3 does not bind directly to circCTTN. METTL3 interacts with circCTTN indirectly through NOP2 and EIF3A.

Proteomic characterization of hUC-MSC extracellular vesicles and evaluation of its therapeutic potential to treat Alzheimer’s disease

In recent years, human umbilical cord mesenchymal stem cell (hUC-MSC) extracellular vesicles (EVs) have been used as a cell replacement therapy and have been shown to effectively overcome some of the disadvantages of cell therapy. However, the specific mechanism of action of EVs is still unclear, and there is no appropriate system for characterizing the differences in the molecular active substances of EVs produced by cells in different physiological states. We used a data-independent acquisition (DIA) quantitative proteomics method to identify and quantify the protein composition of two generations EVs from three different donors and analysed the function and possible mechanism of action of the proteins in EVs of hUC-MSCs via bioinformatics. By comparative proteomic analysis, we characterized the different passages EVs. Furthermore, we found that adaptor-related protein complex 2 subunit alpha 1 (AP2A1) and adaptor-related protein complex 2 subunit beta 1 (AP2B1) in hUC-MSC-derived EVs may play a significant role in the treatment of Alzheimer's disease (AD) by regulating the synaptic vesicle cycle signalling pathway. Our work provides a direction for batch-to-batch quality control of hUC-MSC-derived EVs and their application in AD treatment.

Recombinant human collagen I/carboxymethyl chitosan hydrogel loaded with long-term released hUCMSCs derived exosomes promotes skin wound repair

BackgroundStem cell exosomes are beneficial in accelerating wound repair. However, the therapeutic function is limited due to its rapid clearance in vivo. To improve the functionality of exosomes in cutaneous wound healing, a novel hydrogel was designed and fabricated by recombinant human collagen I and carboxymethyl chitosan loaded with exosomes derived from human umbilical cord mesenchymal stem cells (hUCMSCs), named as the rhCol I/CMC-Exos hydrogel. MethodsExosomes were extracted from hUCMSCs and were characterizated by TEM (Transmission Electron Microscopy), and biomarker detection. The rhCol I hydrogel, rhCol I/carboxymethyl chitosan (rhCol I/CMC) hydrogel and the rhCol I/CMC-Exos hydrogel composites were cross-linked by genipin. These materials were assessed and compared for their physical characteristics, including cross-sectional morphology, porosity, pore distribution, and hydrophilicity. Cell biocompatibility on biomaterials was investigated using scanning electron microscopy and CFDA staining, as well as assessed in vivo through histological examination of major organs in mice. Effects of the hydrogel composite on wound healing were further evaluated by using the full-thickness skin defect mice model. ResultsSuccessful extraction of hUCMSCs-derived exosomes was confirmed by TEM，Western Blotting and flow cytometry. The synthesized rhCol I/CMC-Exos hydrogel composite exhibited cytocompatibility and promoted cell growth in vitro. The rhCol I/CMC-Exos hydrogel showed sustained release of exosomes. In the mice full skin-defects model, the rhCol I/CMC-Exos-treated group showed superior wound healing efficiency, with 15 % faster wound closure compared to controls. Histological examinations revealed thicker dermis formation and more balanced collagen deposition in wounds treated with rhCol I/CMC-Exos hydrogel. Mechanistically, the application of rhCol I/CMC-Exos hydrogel increased fibroblasts proliferation, alleviated inflammation responses as well as promoted angiogenesis, thereby was beneficial in promoting skin wound healing and regeneration. ConclusionOur study, for the first time, introduced recombinant human Collagen I in fabricating a novel hydrogel loaded with hUCMSCs-derived exosomes, which effectively promoted skin wound closure and regeneration, demonstrating a great potential in severe skin wound healing treatment.

Identifying key antioxidative stress factors regulating Nrf2 in the genioglossus with human umbilical cord mesenchymal stem-cell therapy

Intermittent hypoxia in patients with obstructive sleep apnea (OSA) hypopnea syndrome (OSAHS) is associated with pharyngeal cavity collapse during sleep. The effect of human umbilical cord mesenchymal stem cells (HUCMSCs) on OSA-induced oxidative damage in the genioglossus and whether nuclear factor erythroid 2-related factor 2 (Nrf2) or its upstream genes play a key role in this process remains unclear. This study aimed to identify the key factors responsible for oxidative damage during OSAHS through Nrf2 analysis and hypothesize the mechanism of HUCMSC therapy. We simulated OSA using an intermittent hypoxia model, observed the oxidative damage in the genioglossus and changes in Nrf2 expression during intermittent hypoxia, and administered HUCMSCs therapy. Nrf2 initially increased, then decreased, aggravating the oxidative damage in the genioglossus; Nrf2 protein content decreased during hypoxia. Using transcriptomics, we identified seven possible factors in HUCMSCs involved in ameliorating oxidative stress by Nrf2, of which DJ-1 and MEF2A, showing trends similar to Nrf2, were selected by polymerase chain reaction. HUCMSCs may reduce oxidative stress induced by intermittent hypoxia through Nrf2, and the possible upstream target genes in this process are MEF2A and DJ-1. Further studies are needed to verify these findings.

Exosomes derived from human umbilical cord mesenchymal stem cells decrease neuroinflammation and facilitate the restoration of nerve function in rats suffering from intracerebral hemorrhage.

Exosomes derived from human umbilical cord mesenchymal stem cells (hUCMSC-ex) have become a hopeful substitute for whole-cell therapy due to their minimal immunogenicity and tumorigenicity. The present study aimed to investigate the hypothesis that hUCMSC-ex can alleviate excessive inflammation resulting from intracerebral hemorrhage (ICH) and facilitate the rehabilitation of the nervous system in rats. In vivo, hemorrhagic stroke was induced by injecting collagenase IV into the striatum of rats using stereotactic techniques. hUCMSC-ex were injected via the tail vein at 6h after ICH model establishment at a dosage of 200µg. In vitro, astrocytes were pretreated with hUCMSC-ex and then stimulated with hemin (20μmol/mL) to establish an ICH cell model. The expression of TLR4/NF-κB signaling pathway proteins and inflammatory factors, including TNF-α, IL-1β, and IL-10, was assessed both in vivo and in vitro to investigate the impact of hUCMSC-ex on inflammation. The neurological function of the ICH rats was evaluated using the corner turn test, forelimb placement test, Longa score, and Bederson score on the 1st, 3rd, and 5th day. Additionally, RT-PCR was employed to examine the mRNA expression of TLR4 following hUCMSC-ex treatment. The findings demonstrated that hUCMSC-ex downregulated the protein expression of TLR4, NF-κB/P65, and p-P65, reduced the levels of pro-inflammatory cytokines TNF-α and IL-1β, and increased the expression of the anti-inflammatory cytokine IL-10. Ultimately, the administration of hUCMSC-ex improved the behavioral performance of the ICH rats. However, the results of PT-PCR indicated that hUCMSC-ex did not affect the expression of TLR4 mRNA induced by ICH, suggesting that hUCMSCs-ex may inhibit TLR4 translation rather than transcription, thereby suppressing the TLR4/NF-κB signaling pathway. We can conclude that hUCMSC-ex mitigates hyperinflammation following ICH by inhibiting the TLR4/NF-κB signaling pathway. This study provides preclinical evidence for the potential future application of hUCMSC-ex in the treatment of cerebral injury.