HGF secreted by hUC-MSCs mitigates neuronal apoptosis to repair the injured spinal cord via phosphorylation of Akt/FoxO3a pathway.

Spinal cord injury (SCI) can cause severe and permanent neurological damage, and neuronal apoptosis could inhibit functional recovery of damaged spinal cord greatly. Human umbilical cord mesenchymal stem cells (hUC-MSCs) have great potential to repair SCI because of a series of advantages, including inhibition of neuronal apoptosis and multiple differentiation. The former may play an important role. However, the detailed regulatory mechanism associated with the inhibition of neuronal apoptosis after hUC-MSCs administration has not been elucidated. In this study, proteomics analysis of precious human cerebrospinal fluid (CSF) samples collected from SCI subjects receiving hUC-MSCs delivery indicated that hepatocyte growth factor (HGF) is largely involved in SCI repair. Furthermore, overexpression of HGF derived from hUC-MSCs could decrease reactive oxygen species to prevent neuron apoptosis to the maximum, and thus lead to significant recovery of spinal cord dysfunction. Moreover, HGF could promote phosphorylation of Akt/FoxO3a pathway to decrease reactive oxygen species to reduce neuron apoptosis. For the first time, our research revealed that HGF secreted by hUC-MSCs inhibits neuron apoptosis by phosphorylation of Akt/FoxO3a to repair SCI. This study provides important clues associated with drug selection for the effective treatment of SCI in humans.

Neuroprotection of Human Umbilical Cord-Derived Mesenchymal Stem Cells (hUC-MSCs) in Alleviating Ischemic Stroke-Induced Brain Injury by Regulating Inflammation and Oxidative Stress.

Brain injury caused by stroke has a high rate of mortality and remains a major medical challenge worldwide. In recent years, there has been significant attention given to the use of human Umbilical cord-derived Mesenchymal Stem Cells (hUC-MSCs) for the treatment of stroke in different adult and neonate animal models of stroke. However, using hUC-MSCs by systemic administration to treat ischemic stroke has not been investigated sufficiently. In this study, we conducted various experiments to explore the neuroprotection of hUC-MSCs in rats. Our findings demonstrate that an intravenous injection of a high dose of hUC-MSCs at 2 × 10^7 cells/kg markedly ameliorated brain injury resulting from ischemic stroke. This improvement was observed one day after inducing transient middle cerebral artery occlusion (MCAO) and subsequent reperfusion in rats. Notably, the efficacy of this single administration of hUC-MSCs surpassed that of edaravone, even when the latter was used continuously over three days. Mechanistically, secretory factors derived from hUC-MSCs, such as HGF, BDNF, and TNFR1, ameliorated the levels of MDA and T-SOD to regulate oxidative stress. In particular, TNFR1 also improved the expression of NQO-1 and HO-1, important proteins associated with oxidative stress. More importantly, TNFR1 played a significant role in reducing inflammation by modulating IL-6 levels in the blood. Furthermore, TNFR1 was observed to influence the permeability of the blood-brain barrier (BBB) as demonstrated in the evan's blue experiment and protein expression of ZO-1. This study represented a breakthrough in traditional methods and provided a novel strategy for clinical medication and trials.

The proliferation/migration ability mediated by CD151/PI3K/AKT pathway determines the therapeutic effect of hUC-MSCs transplantation on rheumatoid arthritis.

OBJECTIVE: To elucidate the underlying mechanism by which the proliferation and migration abilities of human umbilical cord mesenchymal stem cells (hUC-MSCs) determine their therapeutic efficacy in rheumatoid arthritis treatment. METHODS: The DBA/1J mice were utilized to establish a collagen-induced RA (CIA) mouse model and to validate the therapeutic efficacy of hUC-MSCs transfected with CD151 siRNA. RNA-seq, QT-PCR and western blotting were utilized to evaluate the mRNA and protein levels of the PI3K/AKT pathway, respectively. RESULTS: IFN-γ significantly enhanced the proliferation and migration abilities of hUC-MSCs, up-regulating the expression of CD151, a gene related to cell proliferation and migration. Effective inhibition of this effect was achieved through CD151 siRNA treatment. However, IFN-γ did not affect hUC-MSCs differentiation or changes in cell surface markers. Additionally, transplantation of CD151-interfered hUC-MSCs (siRNA-CD151-hUC-MSCs) resulted in decreased colonization in the toes of CIA mice and worse therapeutic effects compared to empty vector treatment (siRNA-NC-hUC-MSCs). CONCLUSION: IFN-γ facilitates the proliferation and migration of hUC-MSCs through the CD151/PI3K/AKT pathway. The therapeutic efficacy of siRNA-CD151-hUC-MSCs was found to be inferior to that of siRNA-NC-hUC-MSCs.

VCAM-1+ hUC-MSCs Exert Considerable Neuroprotection Against Cerebral Infarction in Rats by Suppression of NLRP3-Induced Pyroptosis.

Mesenchymal stem/stromal cells (MSCs) are spindle-like heterogeneous cell populations with advantageous bidirectional immunomodulatory and hematopoietic support effects. Vascular cellular adhesion molecule-1 (VCAM-1)+ MSCs have been reported to exhibit immunoregulatory and proangiogenic capacities. Here, we studied the effects of VCAM-1+ human umbilical cord (hUC)-MSCs on neuroprotection against cerebral infarction. Sprague-Dawley rats were subjected to middle cerebral artery occlusion (MCAO), and VCAM-1- and VCAM-1+ hUC-MSCs were intravenously injected into the rat 4 h post-MCAO surgery. Thereafter, modified neurological severity scores (mNSS) were determined, and the Morris water maze test, 2,3,5-triphenyltetrazolium chloride (TTC), hematoxylin and eosin (H&E), Nissl, TUNEL staining, and qRT-PCR were conducted. Following induction of oxygen-glucose deprivation/reoxygenation (OGD/R), SH-SY5Y cells were co-cultured with VCAM-1- and VCAM-1+ hUC-MSCs. CCK-8, flow cytometry, ELISA, and western blot analyses were performed in vitro. Compared with VCAM-1- hUC-MSCs, administration of VCAM-1+ hUC-MSCs revealed improved therapeutic efficacy against cerebral infarction in rats, as confirmed by lower mNSS scores and infarct volumes, as well as improved learning and memory capacities. In addition, VCAM-1+ hUC-MSCs exhibited improved efficacy against neurological defects in rats with cerebral infarction, accompanied by inhibition of the NLRP3-mediated inflammatory response. VCAM-1+ hUC-MSC co-culture improved the viability and diminished NLRP3-mediated inflammatory response in OGD/R-treated SH-SY5Y cells. Moreover, NLRP3 overexpression in SH-SY5Y cells prevented the beneficial effects of VCAM-1+ hUC-MSC co-culture. Overall, our findings demonstrated the relevance of VCAM-1+ hUC-MSC-based cytotherapy for preclinical neuroprotection against cerebral infarction.

Huc-MSC-derived exosomes modified with the targeting peptide of aHSCs for liver fibrosis therapy.

BACKGROUND: Effective therapeutics to stop or reverse liver fibrosis have not emerged, because these potential agents cannot specifically target activated hepatic stellate cells (aHSCs) or are frequently toxic to parenchymal cells. Human umbilical cord mesenchymal stem cell (Huc-MSC)-derived exosomes show promise in nanomedicine for the treatment of liver fibrosis. However, systemic injection showed that unmodified exosomes were mainly taken up by the mononuclear phagocyte system. The discovery of ligands that selectively bind to a specific target plays a crucial role in clinically relevant diagnostics and therapeutics. Herein, we aimed to identify the targeting peptide of aHSCs by screening a phage-displayed peptide library, and modify Huc-MSC-derived exosomes with the targeting peptide. RESULTS: In this study, we screened a phage-displayed peptide library by biopanning for peptides preferentially bound to HSC-T6 cells. The identified peptide, HSTP1, also exhibited better targeting ability to aHSCs in pathological sections of fibrotic liver tissues. Then, HSTP1 was fused with exosomal enriched membrane protein (Lamp2b) and was displayed on the surface of exosomes through genetic engineering technology. The engineered exosomes (HSTP1-Exos) could be more efficiently internalized by HSC-T6 cells and outperformed both unmodified exosomes (Blank-Exos) and Lamp2b protein overexpressed exosomes (Lamp2b + Exos) in enhancing the ability of exosomes to promote HSC-T6 reversion to a quiescent phenotype. In vivo results showed HSTP1-Exos could specifically target to the aHSC region after intravenous administration, as demonstrated by coimmunofluorescence with the typical aHSCs marker α-SMA, and enhance the therapeutic effect on liver fibrosis. CONCLUSION: These results suggest that HSTP1 is a reliable targeting peptide that can specifically bind to aHSCs and that HSTP1-modified exosomes realize the precise treatment for aHSCs in complex liver tissue. We provide a novel strategy for clinical liver fibrosis therapy.

Huc-MSCs-derived exosomes attenuate inflammatory pain by regulating microglia pyroptosis and autophagy via the miR-146a-5p/TRAF6 axis.

BACKGROUND: Chronic inflammatory pain significantly reduces the quality of life and lacks effective interventions. In recent years, human umbilical cord mesenchymal stem cells (huc-MSCs)-derived exosomes have been used to relieve neuropathic pain and other inflammatory diseases as a promising cell-free therapeutic strategy. However, the therapeutic value of huc-MSCs-derived exosomes in complete Freund's adjuvant (CFA)-induced inflammatory pain remains to be confirmed. In this study, we investigated the therapeutic effect and related mechanisms of huc-MSCs-derived exosomes in a chronic inflammatory pain model. METHODS: C57BL/6J male mice were used to establish a CFA-induced inflammatory pain model, and huc-MSCs-derived exosomes were intrathecally injected for 4 consecutive days. BV2 microglia cells were stimulated with lipopolysaccharide (LPS) plus adenosine triphosphate (ATP) to investigate the effect of huc-MSCs-derived exosomes on pyroptosis and autophagy. Bioinformatic analysis and rescue experiments were used to demonstrate the role of miR-146a-5p/ TRAF6 in regulating pyroptosis and autophagy. Western blotting, RT-qPCR, small interfering RNA and Yo-Pro-1 dye staining were performed to investigate the related mechanisms. RESULTS: Huc-MSCs-derived exosomes alleviated mechanical allodynia and thermal hyperalgesia in CFA-induced inflammatory pain. Furthermore, huc-MSCs-derived exosomes attenuated neuroinflammation by increasing the expression of autophagy-related proteins (LC3-II and beclin1) and inhibiting the activation of NLRP3 inflammasomes in the spinal cord dorsal horn. In vitro, NLRP3 inflammasome components (NLRP3, caspase1-p20, ASC) and gasdermin D (GSDMD-F, GSDMD-N) were inhibited in BV2 cells pretreated with huc-MSCs-derived exosomes. Western blot and Yo-Pro-1 dye staining demonstrated that 3-MA, an autophagy inhibitor, weakened the protective effect of huc-MSCs-derived exosomes on BV2 cell pyroptosis. Importantly, huc-MSCs-derived exosomes transfected with miR-146a-5p mimic promoted autophagy and inhibited BV2 cell pyroptosis. TRAF6, as a target gene of miR-146a-5p, was knocked down via small-interfering RNA, which increased pyroptosis and inhibited autophagy. CONCLUSION: Huc-MSCs-derived exosomes attenuated inflammatory pain via miR-146a-5p/TRAF6, which increased the level of autophagy and inhibited pyroptosis.

hUC-MSCs exosomal miR-451 alleviated acute lung injury by modulating macrophage M2 polarization via regulating MIF-PI3K-AKT signaling pathway.

In the previous study, we have proved that exosomal miR-451 from human umbilical cord mesenchymal stem cells (hUC-MSCs) attenuated burn-induced acute lung injury (ALI). However, the mechanism of exosomal miR-451 in ALI remains unclear. Therefore, this study aimed to study the molecular mechanism of hUC-MSCs-derived exosomal miR-451 on ALI by regulating macrophage polarization. Exosomes were isolated and identified by transmission electron microscope (TEM) and nanoparticle tracking analysis (NTA). The expression of miR-451, macrophage migration inhibitory factor (MIF) and PI3K/AKT signaling pathway proteins were detected by qRT-PCR and western blot. Flow cytometry was used to detect the CD80 and CD206 positive cells. Severe burn rat model was established and HE was used to detect the inflammatory cell infiltration and inflammatory injury. Dual luciferase reporter system was used to detect the regulation of miR-451 to MIF. The contents of cytokines were detected by ELISA. The results showed that hUC-MSCs exosomes promoted macrophage M1 to M2 polarization. Furthermore, hUC-MSCs-derived exosomal miR-451 alleviated ALI development and promoted macrophage M1 to M2 polarization. Moreover, MIF was a direct target of miR-451. Downregulation of MIF regulated by miR-451 alleviated ALI development promoted macrophage M1 to M2 polarization. In addition, we found that MIF and hUC-MSCs-derived exosomal miR-451 participated in ALI by regulating PI3K/AKT signaling pathway. In conclusion, we indicated that hUC-MSCs-derived exosomal miR-451 alleviated ALI by modulating macrophage M2 polarization via regulating MIF-PI3K-AKT signaling pathway, which provided great scientific significance and clinical application value for the treatment of burn-induced ALI.

Induction of PLXNA4 Gene during Neural Differentiation in Human Umbilical-Cord-Derived Mesenchymal Stem Cells by Low-Intensity Sub-Sonic Vibration.

Human umbilical-cord-derived mesenchymal stem cells (hUC-MSC) are a type of mesenchymal stem cells and are more primitive than other MSCs. In this study, we identify novel genes and signal-activating proteins involved in the neural differentiation of hUC-MSCs induced by Low-Intensity Sub-Sonic Vibration (LISSV). RNA sequencing was used to find genes involved in the differentiation process by LISSV. The changes in hUC-MSCs caused by LISSV were confirmed by PLXNA4 overexpression and gene knockdown through small interfering RNA experiments. The six genes were increased among genes related to neurons and the nervous system. One of them, the PLXNA4 gene, is known to play a role as a guide for axons in the development of the nervous system. When the PLXNA4 recombinant protein was added, neuron-related genes were increased. In the PLXNA4 gene knockdown experiment, the expression of neuron-related genes was not changed by LISSV exposure. The PLXNA4 gene is activated by sema family ligands. The expression of SEMA3A was increased by LISSV, and its downstream signaling molecule, FYN, was also activated. We suggest that the PLXNA4 gene plays an important role in hUC-MSC neuronal differentiation through exposure to LISSV. The differentiation process depends on SEMA3A-PLXNA4-dependent FYN activation in hUC-MSCs.

Identification of differentially expressed genes by single-cell transcriptional profiling of umbilical cord and synovial fluid mesenchymal stem cells.

The purpose of this study was to measure the heterogeneity in human umbilical cord-derived mesenchymal stem cells (hUC-MSCs) and human synovial fluid-derived mesenchymal stem cells (hSF-MSCs) by single-cell RNA-sequencing (scRNA-seq). Using Chromium™ technology, scRNA-seq was performed on hUC-MSCs and hSF-MSCs from samples that passed our quality control checks. In order to identify subgroups and activated pathways, several bioinformatics tools were used to analyse the transcriptomic profiles, including clustering, principle components analysis (PCA), t-Distributed Stochastic Neighbor Embedding (t-SNE), gene set enrichment analysis, as well as Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses. scRNA-seq was performed on the two sample sets. In total, there were 104 761 163 reads for the hUC-MSCs and 6 577 715 for the hSF-MSCs, with >60% mapping rate. Based on PCA and t-SNE analyses, we identified 11 subsets within hUC-MSCs and seven subsets within hSF-MSCs. Gene set enrichment analysis determined that there were 533, 57, 32, 44, 10, 319, 731, 1037, 90, 25 and 230 differentially expressed genes (DEGs) in the 11 subsets of hUC-MSCs and 204, 577, 30, 577, 16, 57 and 35 DEGs in the seven subsets of hSF-MSCs. scRNA-seq was not only able to identify subpopulations of hUC-MSCs and hSF-MSCs within the sample sets, but also provided a digital transcript count of hUC-MSCs and hSF-MSCs within a single patient. scRNA-seq analysis may elucidate some of the biological characteristics of MSCs and allow for a better understanding of the multi-directional differentiation, immunomodulatory properties and tissue repair capabilities of MSCs.

Repair of alveolar cleft bone defects by bone collagen particles combined with human umbilical cord mesenchymal stem cells in rabbit.

BACKGROUND: Alveolar cleft is a type of cleft lip and palate that seriously affects the physical and mental health of patients. In this study, a model of the alveolar cleft phenotype was established in rabbits to evaluate the effect of bone collagen particles combined with human umbilical cord mesenchymal stem cells (HUC-MSCs) on the repair of alveolar cleft bone defects. METHODS: A model of alveolar clefts in rabbits was established by removing the incisors on the left side of the upper jaw bone collagen particles combined with HUC-MSCs that were then implanted in the defect area. Blood biochemical analysis was performed 3 months after surgery. Skull tissues were harvested for gross observation, and micro-focus computerised tomography (micro-CT) analysis. Tissues were harvested for histological and immunohistochemical staining. The experiments were repeated 6 months after surgery. RESULTS: Bone collagen particles and HUC-MSCs showed good biocompatibility. Bone collagen particles combined with HUC-MSCs were markedly better at inducing bone repair and regeneration than bone collagen particles alone. CONCLUSIONS: Combining HUC-MSCs with bone collagen particles provides a simple, rapid and suitable method to fill a bone defect site and treat of alveolar cleft bone defects.

Human umbilical cord-derived mesenchymal stem cells enhanced HK-2 cell autophagy through MicroRNA-145 by inhibiting the PI3K/AKT/mTOR signaling pathway.

Recent studies have shown that autophagy exhibits a protective role in acute kidney injury (AKI), and the accumulation of advanced oxidation protein products (AOPP) participates in the progression of kidney diseases. Our previous study indicated that AOPP induced injury in renal tubular epithelial cells (RTECs) through autophagy inhibition. Besides, we found that human umbilical cord-derived mesenchymal stem cells (hUC-MSCs) enhanced autophagy in AOPP-treated RTECs, but the underlying mechanism remains unclear. We regulated microRNA-145 (miR-145) expression in HK-2 cells (a cell line of RTECs), or co-cultured hUC-MSCs with HK-2 cells and studied the autophagic activity in HK-2 cells to explore the underlying mechanism. Our data demonstrated that upregulated miR-145 increased LC3 II and Beclin 1 levels, decreased p62 level, three autophagy related proteins, inhibited the phosphorylation of PI3K/AKT/mTOR, and increased LC3B-positive staining and the autophagosome number. Furthermore, hUC-MSCs enhanced autophagy and inhibited phosphorylation of PI3K/AKT/mTOR in AOPP-treated HK-2 cells, which was then partially rescued using miR-145 knockdown in the hUC-MSCs co-culture system. In conclusion, our study showed that hUC-MSCs enhanced autophagy in AOPP-treated HK-2 cells mediated by miR-145 via inhibition of the PI3K/AKT/mTOR pathway, which indicated that hUC-MSCs might serve as a prospective therapy for AKI.

Decline of p300 contributes to cell senescence and growth inhibition of hUC-MSCs through p53/p21 signaling pathway.

Human umbilical cord-derived mesenchymal stromal cells (hUC-MSCs) in vitro expansion for long term may undergo epigenetic and genetic alterations that subsequently induce cellular senescence and associated growth inhibition. Increasing evidence implicated that aberrant histone acetylation modulates gene expression responsible for MSCs aging. Whether the dysregulation of p300 and its KAT activity is involved in the aging process of MSCs was still unexplored. In this study, we found a significant decrease of p300 but elevated p53/p21 levels in senescent hUC-MSCs at late-passage. Then we used two different approaches: (i) downregulation of p300 by siRNA and (ii) inhibition of the acetyltransferase(KAT) activity by C646 to determine the role of p300 in regulating MSCs senescence. We showed that inhibition of p300 induce premature senescence and decrease proliferation potential in hUC-MSCs. Moreover, upregulations of p53 and p21 expressions were confirmed in p300 knockdown and C646-treated hUC-MSCs. Taken together, these results suggest that p300 plays an important role in aging process of MSCs associated with activation of p53/p21 signaling pathway.

Human Umbilical Cord Mesenchymal Stem Cells Overexpressing Nerve Growth Factor Ameliorate Diabetic Cystopathy in Rats.

Diabetic cystopathy is a common complication of voiding disorders in diabetes mellitus. Neuropathy and bladder remodeling underlie the lack of efficacy of pharmacological and surgical treatments. Previous studies have shown that decreased levels of nerve growth factor (NGF) are closely associated with disease progression. Besides, application of human umbilical cord mesenchymal stem cells (hUC-MSCs) is also considered a promising therapeutic strategy for treatment of diabetic neuropathy. In our study, we determine the therapeutic efficacy and mechanisms of hUC-MSCs which transfected with NGF geen in ameliorating diabetic cystopathy for the first time. We transducted hUC-MSCs with NGF-expressing lentivirus so that the hUC-MSCs can express NGF efficiently, then the NGF-expressing hUC-MSCs were intrathecally administrated in L6-S1 spinal cord of diabetic rats 3 days after induced by streptozotocin. Nine weeks later, the level of neurotrophins and voiding function of bladder were detected. Results show that improvements in voiding function were related to the neurotrophins and cytokines released by the intrathecally transplanted hUC-MSCs. In addition, the hUC-MSCs also differentiated into neurons and astrocytes within the spinal cord in rats. These two mechanisms play a combined role in neural regeneration and the amelioration of the symptoms of diabetic cystopathy.

[Protective Effects of Intraventricular Transplanted Human Umbilical Cord-derived Mesenchymal Stem Cells on Hypoxic Ischemic Brain Damages in Rats].

OBJECTIVES: To determine the therapeutic effect and protective mechanism of human umbilical cord-derived mesenchymal stem cells (hUC-MSCs) on newborn rats with hypoxia ischemic brain damage (HIBD). METHODS: Umbilical cord (3-4 cm) was collected from a healthy male infant for preparation of hUC-MSCs using explants technique. The hUC-MSCs were cultured and labeled with BrdU. The differentiation function of MSCs was identified. Healthy SPF grade neonatal SD rats were randomly divided into sham (n =30), HIBD (n =36) and hUC-MSCs treated HIBD (n =32) groups. BrdU-labeled hUC-MSCs were injected into the right ventricle of the rats in the hUC-MSCs treatment group 24 h after successful induction of HIBD. The growth and development of the rats were recorded. The neurological behavior of the rats were evaluated with Longa score method 3 weeks after hUC-MSCs transplantation. The survival, migration, differentiation and pro-differentiation of the transplanted hUC-MSCs were measured using immunological fluorescence method. RESULTS: Rats in the hUC-MSCs treatment group had significant higher weight gain and lower Longa scores (at the second and the third week post transplantation) than those in the HIBD group (P<0.05). BrdU positive cells were found in brain tissues 3 weeks after transplantation, and they were mainly distributed in the damaged hippocampus and cerebral cortex. Three weeks after transplantation, the total signal strength of glial fibrillary acidic protein (GFAP) or neuron-specific enclase (NSE) gradually increased. CONCLUSION: Transplanted hUC-MSCs can migrate brain damages through differentiating into neuron-like cells and promoting endogenous neurological differentiations.

Human umbilical cord derived mesenchymal stem cells promote interleukin-17 production from human peripheral blood mononuclear cells of healthy donors and systemic lupus erythematosus patients.

Inflammation instigated by interleukin (IL)-17-producing cells is central to the development and pathogenesis of several human autoimmune diseases and animal models of autoimmunity. The expansion of IL-17-producing cells from healthy donors is reportedly promoted by mesenchymal stem cells derived from fetal bone marrow. In the present study, human umbilical cord-derived mesenchymal stem cells (hUC-MSCs) were examined for their effects on lymphocytes from healthy donors and from patients with systemic lupus erythematosus (SLE). Significantly higher levels of IL-17 were produced when CD4(+) T cells from healthy donors were co-cultured with hUC-MSCs than those that were cultured alone. Blocking experiments identified that this effect might be mediated partially through prostaglandin E2 (PGE2 ) and IL-1ß, without IL-23 involvement. We then co-cultured hUC-MSCs with human CD4(+) T cells from systemic lupus erythematosus patients. Ex-vivo inductions of IL-17 by hUC-MSCs in stimulated lymphocytes were significantly higher in SLE patients than in healthy donors. This effect was not observed for IL-23. Taken together, our results represent that hUC-MSCs can promote the IL-17 production from CD4(+) T cells in both healthy donor and SLE patients. PGE2 and IL-1ß might also be partially involved in the promotive effect of hUC-MSCs.

In vitro and in vivo evaluation of the developed PLGA/HAp/Zein scaffolds for bone-cartilage interface regeneration.

OBJECTIVE: To investigate the effect of electronspun PLGA/HAp/Zein scaffolds on the repair of cartilage defects. METHODS: The PLGA/HAp/Zein composite scaffolds were fabricated by electrospinning method. The physiochemical properties and biocompatibility of the scaffolds were separately characterized by scanning electron microscope (SEM), transmission electron microscope (TEM), and fourier transform infrared spectroscopy (FTIR), human umbilical cord mesenchymal stem cells (hUC-MSCs) culture and animal experiments. RESULTS: The prepared PLGA/HAp/Zein scaffolds showed fibrous structure with homogenous distribution. hUC-MSCs could attach to and grow well on PLGA/HAp/Zein scaffolds, and there was no significant difference between cell proliferation on scaffolds and that without scaffolds (P>0.05). The PLGA/HAp/Zein scaffolds possessed excellent ability to promote in vivo cartilage formation. Moreover, there was a large amount of immature chondrocytes and matrix with cartilage lacuna on PLGA/HAp/Zein scaffolds. CONCLUSION: The data suggest that the PLGA/HAp/Zein scaffolds possess good biocompatibility, which are anticipated to be potentially applied in cartilage tissue engineering and reconstruction.

Reprogramming human umbilical cord mesenchymal stromal cells to islet-like cells with the use of in vitro-synthesized pancreatic-duodenal homebox 1 messenger RNA.

BACKGROUND AIMS: Human umbilical cord mesenchymal stromal cells (hUC-MSCs) hold great potential as a therapeutic candidate to treat diabetes, owing to their unlimited source and ready availability. METHODS: In this study, we differentiated hUC-MSCs with in vitro-synthesized pancreatic-duodenal homebox 1 (PDX1) messenger (m)RNA into islet-like cell clusters. hUC-MSCs were confirmed by both biomarker detection and functional differentiation. In vitro-synthesized PDX1 messenger RNA can be transfected into hUC-MSCs efficiently. The upregulated expression of PDX1 protein can be detected 4 h after transfection and remains detectable for 36 h. RESULTS: The induction of islet-like structures was confirmed by means of morphology and dithizone staining. Reverse transcriptase-polymerase chain reaction results revealed the expression of some key pancreatic transcription factors, such as PDX1, NeuroD, NKX6.1, Glut-2 and insulin in islet-like cell clusters. Immunofluorescence analysis showed that differentiated cells express both insulin and C-peptide. Enzyme-linked immunosorbent assay analysis validated the insulin secretion of islet-like cell clusters in response to the glucose stimulation. CONCLUSIONS: Our results demonstrate the use of in vitro-synthesized PDX1 messenger RNA to differentiate hUC-MSCs into islet-like cells and pave the way toward the development of reprogramming and directed-differentiation methods for the expression of encoded proteins.

Anticancer effect of hUC-MSC-derived exosome-mediated delivery of PMO-miR-146b-5p in colorectal cancer.

Antisense oligonucleotide (ASO) is a novel therapeutic platform for targeted cancer therapy. Previously, we have demonstrated that miR-146b-5p plays an important role in colorectal cancer progression. However, a safe and effective strategy for delivery of an ASO to its targeted RNA remains as a major hurdle in translational advances. Human umbilical cord mesenchymal cell (hUC-MSC)-derived exosomes were used as vehicles to deliver an anti-miR-146b-5p ASO (PMO-146b). PMO-146b was assembled onto the surface of exosomes (e) through covalent conjugation to an anchor peptide CP05 (P) that recognized an exosomal surface marker, CD63, forming a complex named ePPMO-146b. After ePPMO-146b treatment, cell proliferation, uptake ability, and migration assays were performed, and epithelial-mesenchymal transition progression was evaluated in vitro. A mouse xenograft model was used to determine the antitumor effect and distribution of ePPMO-146b in vivo. ePPMO-146b was taken up by SW620 cells and effectively inhibited cell proliferation and migration. The conjugate also exerted antitumor efficacy in a xenograft mouse model of colon cancer by systematic administration, where PPMO-146b was enriched in tumor tissue. Our study highlights the potential of hUC-MSC-derived exosomes anchored with PPMO-146b as a novel safe and effective approach for PMO backboned ASO delivery.

Intravenous application of human umbilical cord mesenchymal stem cells alleviate neuropathic pain by suppressing microglia activation in rats.

Objective: Neuropathic pain has been considered as one of the most serious chronic pain subtypes and causes intolerable suffering to patients physically and mentally. This study aimed to verify the analgesic effect of intravenous administration of human umbilical cord mesenchymal stem cells (HUC-MSCs) upon rats with chronic constriction injury (CCI)-induced neuropathic pain and the concomitant mechanism via modulating microglia. Methods: 30 male SD rats were randomized divided into three groups (n = 10 per group): Sham + Saline group (S&S group), CCI + Saline group (C&S group) and CCI + HUC-MSCs group (C&U group). Rats were injected with either saline or HUC-MSCs via the caudal vein on the 7th day after modelling. The paw mechanical withdrawal threshold (PMWT) and thermal withdrawal latency (TWL) of the ligation side were measured before (day 0) and after (day 1, 3, 5, 7, 9, 11, 13, and 15) modelling. On day 15 after modelling, western-blotting and immunofluorescent staining were used to assess the expressive abundance of Iba-1 (a typical biomarker of activated microglia) in the ligation side of the spinal cord dorsal horn, and ultrastructural changes of the ligation of sciatic nerve were evaluated by transmission electron microscope (TEM). Results: Compared with the S&S group, PMWT and TWL in the C&S group were significantly decreased on day 5 and then persisted to day 15 after modelling (C&S vs S&S, P < 0.05), while a significant amelioration of mechanical hyperalgesia (day 13, day 15) and thermal allodynia (day 9, day 11, day 15) was observed in the C&U group (C&U vs C&S, P < 0.05). Meanwhile, the expression of Iba-1 was significantly suppressed by systemic infusion of HUC-MSCs in the C&U group according to western-blotting and immunofluorescent staining analyses (P < 0.05). With the aid of TEM detection, we intuitively noticed the efficacious reconstruction of the laminate structure of the sciatic nerve ligation, elimination of mitochondrial swelling, and formation of new myelination were noted on day 15 after modelling in the C&U group. Conclusions: Overall, intravenous administration of HUC-MSCs systemically revealed an ameliorative effect upon CCI-induced neuropathic pain in SD rats by inhibiting microglia activation in the dorsal horn of the impaired spinal cord and alleviating sciatic nerve injury. Our findings supply new references for the further development of HUC-MSCs-based cytotherapy for neuropathic pain administration.

ROS-responsive nanoparticle delivery of ferroptosis inhibitor prodrug to facilitate mesenchymal stem cell-mediated spinal cord injury repair.

Spinal cord injury (SCI) is a traumatic condition that results in impaired motor and sensory function. Ferroptosis is one of the main causes of neural cell death and loss of neurological function in the spinal cord, and ferroptosis inhibitors are effective in reducing inflammation and repairing SCI. Although human umbilical cord mesenchymal stem cells (Huc-MSCs) can ameliorate inflammatory microenvironments and promote neural regeneration in SCI, their efficacy is greatly limited by the local microenvironment after SCI. Therefore, in this study, we constructed a drug-release nanoparticle system with synergistic Huc-MSCs and ferroptosis inhibitor, in which we anchored Huc-MSCs by a Tz-A6 peptide based on the CD44-targeting sequence, and combined with the reactive oxygen species (ROS)-responsive drug nanocarrier mPEG-b-Lys-BECI-TCO at the other end for SCI repair. Meanwhile, we also modified the classic ferroptosis inhibitor Ferrostatin-1 (Fer-1) and synthesized a new prodrug Feborastatin-1 (Feb-1). The results showed that this treatment regimen significantly inhibited the ferroptosis and inflammatory response after SCI, and promoted the recovery of neurological function in rats with SCI. This study developed a combination therapy for the treatment of SCI and also provides a new strategy for the construction of a drug-coordinated cell therapy system.