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Uncovering mechanisms of endogenous regeneration and repair through resident stem cell activation will allow us to develop specific therapies for injuries and diseases by targeting resident stem cell lineages. Sox9 + stem cells have been reported to play an essential role in acute kidney injury (AKI). However, a complete view of the Sox9 + lineage was not well investigated to accurately elucidate the functional end state and the choice of cell fate during tissue repair after AKI. To identify the mechanisms of fate determination of Sox9 + stem cells, we set up an AKI model with prostaglandin E2 (PGE 2 ) treatment in a Sox9 lineage tracing mouse model. Single-cell RNA sequencing (scRNA-seq) was performed to analyse the transcriptomic profile of the Sox9 + lineage. Our results revealed that PGE 2 could activate renal Sox9 + cells and promote the differentiation of Sox9 + cells into renal proximal tubular epithelial cells and inhibit the development of fibrosis. Furthermore, single-cell transcriptome analysis demonstrated that PGE 2 could regulate the restoration of lipid metabolism homeostasis in proximal tubular epithelial cells by participating in communication with different cell types. Our results highlight the prospects for the activation of endogenous renal Sox9 + stem cells with PGE 2 for the regenerative therapy of AKI., (© 2024 The Author(s). Cell Proliferation published by Beijing Institute for Stem Cell and Regenerative Medicine and John Wiley & Sons Ltd.)

PACS (phosphofurin acidic cluster sorting) proteins are known for their roles in sorting cargo proteins to organelles and can physically interact with WD40 repeat-containing protein WDR37. PACS1, PACS2, and WDR37 variants are associated with multisystemic syndromes and neurodevelopmental disorders characterized by intellectual disability, seizures, developmental delays, craniofacial abnormalities, and autism spectrum disorder. However, the functional effects of syndromic variants at the cellular level remain unknown. Here, we report the expression pattern of Caenorhabditis elegans orthologs of PACS and WDR37 and their interaction. We show that cePACS-1 and ceWDR-37 colocalize to somatic cytoplasm of many types of cells and are mutually required for expression, supporting a conclusion that the intermolecular dependence of PACS1/PACS2/PACS-1 and WDR37/WDR-37 is evolutionarily conserved. We further show that editing in PACS1 and PACS2 variants in cePACS-1 changes protein localization in multiple cell types, including neurons. Moreover, expression of human PACS1 can functionally complement C. elegans PACS-1 in neurons, demonstrating conserved functions of the PACS-WDR37 axis in an invertebrate model system. Our findings reveal effects of human variants and suggest potential strategies to identify regulatory network components that may contribute to understanding molecular underpinnings of PACS/WDR37 syndromes., Competing Interests: Conflicts of interest The author(s) declare no conflicts of interest., (© The Author(s) 2024. Published by Oxford University Press on behalf of The Genetics Society of America.)

Much effort has been devoted to improving treatment efficiency for osteosarcoma (OS). However, most current approaches result in poor therapeutic responses, thus indicating the need for the development of other therapeutic options. This study developed a multifunctional nanoparticle, PDA-MOF-E-M, an aggregation of OS targeting, programmed death targeting, and near-infrared (NIR)-aided targeting. At the same time, a multifunctional nanoparticle that utilises Fe-MOFs to create a cellular iron-rich environment and erastin as a ferroptosis inducer while ensuring targeted delivery to OS cells through cell membrane encapsulation is presented. The combination of PDA-MOF-E-M and PTT increased intracellular ROS and LPO levels and induced ferroptosis-related protein expression. A PDA-based PTT combined with erastin showed significant synergistic therapeutic improvement in the anti-tumour efficiency of the nanoparticle in vitro and vivo. The multifunctional nanoparticle efficiently prevents the osteoclasia progression of OS xenograft bone tumors in vivo. Finally, this study provides guidance and a point of reference for clinical approaches to treating OS., Competing Interests: We declare that we have no financial and personal relationships with other people or organizations that can inappropriately influence our work, there is no professional or other personal interest of any nature or kind in any product, service and/or company that could be construed as influencing the position presented in, or the review of, the manuscript entitled, ‘A multifunctional biomimetic nanoplatform for image-guideded photothermal-ferroptotic synergistic osteosarcoma therapy’., (© 2024 The Authors.)

Nitric oxide (NO), as a gaseous therapeutic agent, shows great potential for the treatment of many kinds of diseases. Although various NO delivery systems have emerged, the immunogenicity and long-term toxicity of artificial carriers hinder the potential clinical translation of these gas therapeutics. Mesenchymal stem cells (MSCs), with the capacities of self-renewal, differentiation, and low immunogenicity, have been used as living carriers. However, MSCs as gaseous signaling molecule (GSM) carriers have not been reported. In this study, human MSCs were genetically modified to produce mutant β-galactosidase (β-GAL H363A ). Furthermore, a new NO prodrug, 6-methyl-galactose-benzyl-oxy NONOate (MGP), was designed. MGP can enter cells and selectively trigger NO release from genetically engineered MSCs (eMSCs) in the presence of β-GAL H363A . Moreover, our results revealed that eMSCs can release NO when MGP is systemically administered in a mouse model of acute kidney injury (AKI), which can achieve NO release in a precise spatiotemporal manner and augment the therapeutic efficiency of MSCs. This eMSC and NO prodrug system provides a unique and tunable platform for GSM delivery and holds promise for regenerative therapy by enhancing the therapeutic efficiency of stem cells., Competing Interests: HH, MQ, YL, SC, HL, XC, QZ, ZL No competing interests declared, ZH is an employee of AmCellGene Co., Ltd. The author has no further interests to declare, ZH is an employee of Health & Biotech Co and AmCellGene Co., Ltd. The author has no further interests to declare, (© 2023, Huang, Qian, Liu et al.)

Background: Mesenchymal stem cells (MSCs) have demonstrated remarkable therapeutic promise for acute lung injury (ALI) and its severe form, acute respiratory distress syndrome (ARDS). MSC secretomes contain various immunoregulatory mediators that modulate both innate and adaptive immune responses. Priming MSCs has been widely considered to boost their therapeutic efficacy for a variety of diseases. Prostaglandin E2 (PGE2) plays a vital role in physiological processes that mediate the regeneration of injured organs., Methods: This work utilized PGE2 to prime MSCs and investigated their therapeutic potential in ALI models. MSCs were obtained from human placental tissue. MSCs were transduced with firefly luciferase (Fluc)/eGFP fusion protein for real-time monitoring of MSC migration. Comprehensive genomic analyses explored the therapeutic effects and molecular mechanisms of PGE2-primed MSCs in LPS-induced ALI models., Results: Our results demonstrated that PGE2-MSCs effectively ameliorated lung injury and decreased total cell numbers, neutrophils, macrophages, and protein levels in bronchoalveolar lavage fluid (BALF). Meanwhile, treating ALI mice with PGE2-MSCs dramatically reduced histopathological changes and proinflammatory cytokines while increasing anti-inflammatory cytokines. Furthermore, our findings supported that PGE2 priming improved the therapeutic efficacy of MSCs through M2 macrophage polarization., Conclusion: PGE2-MSC therapy significantly reduced the severity of LPS-induced ALI in mice by modulating macrophage polarization and cytokine production. This strategy boosts the therapeutic efficacy of MSCs in cell-based ALI therapy., (© 2023. The Author(s).)

Endothelial cell injury plays a critical part in ischemic acute kidney injury (AKI) and participates in the progression of AKI. Targeting renal endothelial cell therapy may ameliorate vascular injury and further improve the prognosis of ischemic AKI. Here, P-selectin as a biomarker of ischemic AKI in endothelial cells is identified and P-selectin binding peptide (PBP)-engineered extracellular vesicles (PBP-EVs) with imaging and therapeutic functions are developed. The results show that PBP-EVs exhibit a selective targeting tendency to injured kidneys, while providing spatiotemporal information for the early diagnosis of AKI by quantifying the expression of P-selectin in the kidneys by molecular imaging. Meanwhile, PBP-EVs reveal superior nephroprotective functions in the promotion of renal repair and inhibition of fibrosis by alleviating inflammatory infiltration, improving reparative angiogenesis, and ameliorating maladaptive repair of the renal parenchyma. In conclusion, PBP-EVs, as an ischemic AKI theranostic system that is designed in this study, provide a spatiotemporal diagnosis in the early stages of AKI to help guide personalized therapy and exhibit superior nephroprotective effects, offering proof-of-concept data to design EV-based theranostic strategies to promote renal recovery and further improve long-term outcomes following AKI., (© 2022 The Authors. Advanced Science published by Wiley-VCH GmbH.)

Background: Intravenous administration of mesenchymal stromal cells (MSCs) has an acknowledged competence of cardiac repair, despite a lack of systematic description of the underlying biological mechanisms. The lung, but not the heart, is the main trapped site for intravenously transplanted MSCs, which leaves a spatial gap between intravenously transplanted MSCs and the injured myocardium. How lung-trapped MSCs after intravenous transplantation rejuvenate the injured myocardium remains unknown., Methods: MSCs were isolated from human placenta tissue, and DF-MSCs or Gluc-MSCs were generated by transduced with firefly luciferase (Fluc)/enhanced green fluorescence protein (eGFP) or Gaussia luciferase (Gluc) lactadherin fusion protein. The therapeutic efficiency of intravenously transplanted MSCs was investigated in a murine model of doxorubicin (Dox)-induced cardiotoxicity. Trans-organ communication from the lung to the heart with the delivery of blood was investigated by testing the release of MSC-derived extracellular vesicles (MSC-EVs), and the potential miRNA inner MSC-EVs were screened out and verified. The potential therapeutic miRNA inner MSC-EVs were then upregulated or downregulated to assess the further therapeutic efficiency RESULTS: Dox-induced cardiotoxicity, characterized by cardiac atrophy, left ventricular dysfunction, and injured myocardium, was alleviated by consecutive doses of MSCs. These cardioprotective effects might be attributed to suppressing GRP78 triggering endoplasmic reticulum (ER) stress-induced apoptosis in cardiomyocytes. Our results confirmed that miR-181a-5p from MSCs-derived EVs (MSC-EVs) inhibited GRP78. Intravenous DF-MSCs were trapped in lung vasculature, secreted a certain number of EVs into serum, which could be confirmed by the detection of eGFP + EVs. GLuc activity was increased in serum EVs from mice administrated with GLuc-MSCs. MiR-181a-5p, inhibiting GRP78 with high efficacy, was highly expressed in serum EVs and myocardium after injecting consecutive doses of MSCs into mice treated with Dox. Finally, upregulation or downregulation of miR-181a-5p levels in MSC-EVs enhanced or weakened therapeutic effects on Dox-induced cardiotoxicity through modulating ER stress-induced apoptosis., Conclusions: This study identifies intravenously transplanted MSCs, as an endocrine reservoir, to secrete cardioprotective EVs into blood continuously and gradually to confer the trans-organ communication that relieves Dox-induced cardiotoxicity., (© 2022. The Author(s).)

Metal clusters, such as iron-sulfur clusters, play key roles in sustaining life and are intimately involved in the functions of metalloproteins. Herein we report the formation and crystal structure of a planar square tetranuclear silver cluster when silver ions were mixed with human copper chaperone Atox1. Quantum chemical studies reveal that two Ag 5s 1 electrons in the tetranuclear silver cluster fully occupy the one bonding molecular orbital, with the assumption that this Ag 4 cluster is Ag 4 2+ , leading to extensive electron delocalization over the planar square and significant stabilization. This bonding pattern of the tetranuclear silver cluster represents an aromatic all-metal structure that follows a 4 n + 2 electron counting rule ( n = 0). This is the first time an all-metal aromatic silver cluster was observed in a protein., Competing Interests: There are no conflicts to declare., (This journal is © The Royal Society of Chemistry.)

Background: The promising therapeutic strategy for the treatment of peripheral artery disease (PAD) is to restore blood supply and promote regeneration of skeletal muscle regeneration. Increasing evidence revealed that prostaglandin E 2 (PGE 2 ), a lipid signaling molecule, has significant therapeutic potential for tissue repair and regeneration. Though PGE 2 has been well reported in tissue regeneration, the application of PGE 2 is hampered by its short half-life in vivo and the lack of a viable system for sustained release of PGE 2 ., Results: In this study, we designed and synthesized a new PGE 2 release matrix by chemically bonding PGE 2 to collagen. Our results revealed that the PGE 2 matrix effectively extends the half-life of PGE 2 in vitro and in vivo. Moreover, the PGE 2 matrix markedly improved neovascularization by increasing angiogenesis, as confirmed by bioluminescence imaging (BLI). Furthermore, the PGE 2 matrix exhibits superior therapeutic efficacy in the hindlimb ischemia model through the activation of MyoD1-mediated muscle stem cells, which is consistent with accelerated structural recovery of skeletal muscle, as evidenced by histological analysis., Conclusions: Our findings highlight the chemical bonding strategy of chemical bonding PGE 2 to collagen for sustained release and may facilitate the development of PGE 2 -based therapies to significantly improve tissue regeneration., (© 2022. The Author(s).)

Aim: To evaluate therapeutic outcomes of human umbilical cord-derived mesenchymal stem cells (HUC-MSCs) treatment in patients with refractory uveitis., Methods: A retrospective and noncomparative review was performed on four patients with refractory uveitis from December 2013 to December 2017. HUC-MSCs were administered intravenously at a dose of 1×10 6 cells/kg. Clinical response, relapse rate, change of visual acuity, and other metrics were evaluated., Results: All four patients presented with responses to HUC-MSCs treatment, with three males and one female. The numbers of uveitis attacks per year after the HUC-MSCs treatment (0, 2, 0, 0 respectively) all decreased compared with the numbers before the treatment (3, 6, 4, 4 respectively). The oral steroid and immunosuppressive agents were tapered in all patients without recrudescence of ocular inflammation, and three patients discontinued their oral medicine at the last visit. The best corrected visual acuity (BCVA) of 3 patients was improved to varying degrees, and the BCVA of 1 patient remained at 20/20 (Snellen chart) from the first to the last consultation., Conclusion: The study provides an effective therapy of HUC-MSCs in maintaining remission in patients affected by uveitis refractory to previous immunosuppressant treatments., (International Journal of Ophthalmology Press.)

Prostaglandin E 2 (PGE 2 ) has recently been recognized to play a role in immune regulation and tissue regeneration. However, the short half-life of PGE 2 limits its clinical application. Improving the delivery of PGE 2 specifically to the target organ with a prolonged release method is highly desirable. Taking advantage of the adequate space and proximity of the renal parenchyma, renal subcapsular delivery allows minimally invasive and effective delivery to the entire kidney. Here, we report that by covalently cross-linking it to a collagen matrix, PGE 2 exhibits an adequate long-term presence in the kidney with extensive intraparenchymal penetration through renal subcapsular delivery and significantly improves kidney function. Sox9 cell lineage tracing with intravital microscopy revealed that PGE 2 could activate the endogenous renal progenitor Sox9 + cells through the Yap signaling pathway. Our results highlight the prospects of utilizing renal subcapsular-based drug delivery and facilitate new applications of PGE 2 -releasing matrices for regenerative therapy., Competing Interests: The authors declare no competing interests., (© 2021 The Author(s).)

The ongoing outbreak of coronavirus disease 2019 (COVID-19) caused by the novel severe acute respiratory syndrome coronavirus 2 has become a sudden public emergency of international concern and seriously threatens millions of people's life health. Two current studies have indicated a favorable role for mesenchymal stem/stromal cells (MSCs) in clinical remission of COVID-19 associated pulmonary diseases, yet the systematical elaboration of the therapeutics and underlying mechanism is far from satisfaction. In the present review, we summarize the therapeutic potential of MSCs in COVID-19 associated pulmonary diseases such as pneumonia induced acute lung injury, acute respiratory distress syndrome, and pulmonary fibrosis. Furthermore, we review the underlying mechanism of MSCs including direct- and trans-differentiation, autocrine and paracrine anti-inflammatory effects, homing, and neovascularization, as well as constitutive microenvironment. Finally, we discuss the prospects and supervision of MSC-based cytotherapy for COVID-19 management before large-scale application in clinical practice. Collectively, this review supplies overwhelming new references for understanding the landscapes of MSCs in the remission of COVID-19 associated pulmonary diseases., Competing Interests: Conflict-of-interest statement: The authors have no conflicts of interest to disclose., (©The Author(s) 2021. Published by Baishideng Publishing Group Inc. All rights reserved.)

Fructose-1,6-biphosphate aldolase (FBA) is a multifunctional enzyme in plants, which participates in the process of Calvin-Benson cycle, glycolysis and gluconeogenesis. Despite the importance of FBA genes in regulating plant growth, development and abiotic stress responses, little is known about their roles in cotton. In the present study, we performed a genome-wide identification and characterization of FBAs in Gossypium hirsutum . Totally seventeen GhFBA genes were identified. According to the analysis of functional domain, phylogenetic relationship, and gene structure, GhFBA genes were classified into two subgroups. Furthermore, nine GhFBAs were predicted to be in chloroplast and eight were located in cytoplasm. Moreover, the promoter prediction showed a variety of abiotic stresses and phytohormone related cis -acting elements exist in the 2k up-stream region of GhFBA . And the evolutionary characteristics of cotton FBA genes were clearly presented by synteny analysis. Moreover, the results of transcriptome and qRT-PCR analysis showed that the expression of GhFBAs were related to the tissue distribution, and further analysis suggested that GhFBAs could respond to various abiotic stress and phytohormonal treatments. Overall, our systematic analysis of GhFBA genes would not only provide a basis for the understanding of the evolution of GhFBAs , but also found a foundation for the further function analysis of GhFBAs to improve cotton yield and environmental adaptability., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Li, Zhang, Li, Su, Liu, Han, Wang and Zhu.)

Objective: This study investigated the effect of salvia miltiorrhiza-asarum ointment (SMAO) plus Chinese medical massage on knee osteoarthritis in a rat model., Methods: Hulth's method was used to establish a Sprague-Dawley rat model of knee osteoarthritis (OA). The levels of matrix metalloproteinase-13 (MMP-13), collagen-II, aggrecan, interleukin (IL)-1β, tumor necrosis factor-α (TNF-α), and IL-6 were measured by enzyme-linked immunosorbent assays. The joint space was assessed by a Perlove X-ray system. Histopathology was examined by hematoxylin-eosin and Safranin O staining. The mRNA and protein expression levels of Notch1, MMP-13, collagen-II, and aggrecan were measured by quantitative reverse transcription-polymerase chain reaction and Western blotting, respectively., Results: SMAO plus Chinese medical massage significantly decreased the levels of MMP-13, IL-1β, TNF-α, and IL-6, and increased serum collagen-II and aggrecan levels. Pathological injury of the knee joint was improved by SMAO treatment. mRNA and protein expression of Notch1 and MMP-13 was remarkably downregulated, but collagen-II and aggrecan levels were significantly upregulated in cartilage tissues., Conclusion: SMAO combined with Chinese medical massage effectively relieves OA symptoms, which may involve inhibiting inflammation through the Notch1/MMP-13 signaling pathway.

Background: The senescence of dermal fibroblasts (DFLs) leads to an imbalance in the synthesis and degradation of extracellular matrix (ECM) proteins, presenting so-called senescence-associated secretory phenotype (SASP), which ultimately leads to skin aging. Recently, mesenchymal stem cell (MSC)-derived extracellular vesicles (EVs) have been recognized as a promising cell-free therapy for degenerative diseases, which opens a new avenue for skin aging treatment., Methods: In this study, we utilized chitosan (CS) hydrogel for effective loading and sustained release of EVs. In vitro, we explored the rejuvenation effects of CS hydrogel-incorporated EVs (CS-EVs) on replicative senescence DFLs through a series of experiments such as senescence-associated β-galactosidase (SA-β-gal) staining, RT-PCR, and Western blot analysis. Besides, we employed local multi-site subcutaneous injection to treat skin aging of naturally aged mice with CS-EVs and DiI fluorescent dye was used to label EVs to achieve in vivo real-time tracking., Results: CS-EVs can significantly improve the biological functions of senescent fibroblasts, including promoting their proliferation, enhancing the synthesis of ECM proteins, and inhibiting the overexpression of matrix metalloproteinases (MMPs). Moreover, CS hydrogel could prolong the release of EVs and significantly increase the retention of EVs in vivo. After CS-EVs subcutaneous injection treatment, the aging skin tissues showed a rejuvenation state, manifested explicitly as the enhanced expression of collagen, the decreased expression of SASP-related factors, and the restoration of tissue structures., Conclusions: CS hydrogel-encapsulated EVs could delay the skin aging processes by ameliorating the function of aging DFLs. Our results also highlight the potential of CS hydrogel-encapsulated EVs as a novel therapeutic strategy for improving aging skin to rejuvenation.

Perinatal-related tissues, such as the placenta, umbilical cord, and amniotic membrane, are generally discarded after delivery and are increasingly attracting attention as alternative sources for decellularized extracellular matrix (dECM) isolation. Recent studies indicate that glycosaminoglycans (GAGs) in the dECM play key roles during tissue regeneration. However, the dECM is organ specific, and the glycosaminoglycanomics of dECMs from perinatal tissues and the regulatory function of GAGs have been poorly investigated. In this study, we explored the glycosaminoglycanomics of dECMs from the placenta, umbilical cord and amniotic membrane. We hypothesized that the therapeutic effects of dECMs are related to the detailed composition of GAGs. Hydrogels of dECM derived from perinatal tissues were generated, and glycosaminoglycanomics analysis was employed to identify the cues that promote tissue repair and regeneration in a murine cutaneous wound-healing model. We utilized highly sensitive liquid chromatography-tandem mass spectrometry for glycosaminoglycanomics analysis. Our results revealed that placenta-derived dECM (PL-dECM) hydrogel has higher contents of chondroitin sulfate (CS) and heparan sulfate (HS). In addition, molecular imaging showed that the PL-dECM hydrogel exerted the best anti-inflammatory and proangiogenic effects in the skin wound healing model. Further in vitro analyses demonstrated that CS with 6-O-sulfo group (CS-6S) has an anti-inflammatory effect, while HS with 6-O-sulfo group (HS-6S) plays a crucial role in angiogenesis. In conclusion, this study highlights the critical roles of GAGs in perinatal tissue-derived dECMs by promoting angiogenesis and inhibiting inflammation and indicates that it is feasible to utilize 6-sulfated GAG-enriched placental dECM hydrogel as an attractive candidate for tissue engineering and drug delivery., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021. Published by Elsevier Ltd.)

Background: Chemotherapy is an effective anti-tumor treatment. Mesenchymal stem cells (MSCs), exerting therapy effect on injured tissues during chemotherapy, may be damaged in the process. The possibility of self-healing through long-range paracrine and the mechanisms are unclear., Methods: Doxorubicin, a commonly used chemotherapy drug, was to treat human umbilical cord-derived mesenchymal stem cells (hUC-MSCs) for 6 h as an in vitro cell model of chemotherapy-induced damage. Then we use extracellular vesicles derived from placental mesenchymal stem cells (hP-MSCs) to investigate the therapeutic potential of MSCs-EVs for chemotherapy injury. The mechanism was explored using microRNA sequencing., Results: MSC-derived extracellular vesicles significantly alleviated the chemotherapy-induced apoptosis. Using microRNA sequencing, we identified hsa-miR-11401, which was downregulated in the Dox group but upregulated in the EV group. The upregulation of hsa-miR-11401 reduced the expression of SCOTIN, thereby inhibiting p53-dependent cell apoptosis., Conclusions: Hsa-miR-11401 expressed by MSCs can be transported to chemotherapy-damaged cells by EVs, reducing the high expression of SCOTIN in damaged cells, thereby inhibiting SCOTIN-mediated apoptosis.

Mesenchymal stem cells (MSCs) are pluripotent cells that can be applied to the treatment of immune disorders, including inflammatory bowel disease (IBD). The therapeutic effects of MSCs have been mostly attributed to the secretion of soluble factors with paracrine actions, such as extracellular vesicles (EVs), which may play a relevant role in the repair of damaged tissues. In the present study, a mouse model of colitis was induced with the use of trinitrobenzene sulfonic acid (TNBS). EVs derived from human placental mesenchymal stem cells (hP‑MSCs) were used for the treatment of colitis by in situ injection. Clinical scores were applied to verify the therapeutic effects of EVs on mice with colitis. Inflammation in the colon was evaluated by measuring the levels of various inflammatory cytokines. The content of reactive oxygen species (ROS) was detected by the use of molecular imaging methods for real‑time tracking and the therapeutic effects of EVs on mucosal healing in mice with colitis were evaluated. The results revealed that the injection of EVs regulated the balance of pro‑inflammatory and anti‑inflammatory cytokines in colon tissue. Treatment with EVs also suppressed oxidative stress by decreasing the activity of myeloperoxidase (MPO) and ROS. Histological analysis further confirmed that the EVs significantly promoted mucosal healing, as reflected by the promotion of the proliferation of colonic epithelial cells and the maintenance of tight junctions. Taken together, the findings of the present study demonstrated that EVs derived from hP‑MSCs alleviated TNBS‑induced colitis by inhibiting inflammation and oxidative stress. These findings may provide a novel theoretical basis for the EV‑based treatment of IBD.

Therapeutic angiogenesis with mesenchymal stem cells (MSCs) is promising for the clinical treatment of peripheral artery disease (PAD). However, the heterogeneous proangiogenic nature of MSCs is a key challenge in developing more effective treatments with MSCs for therapeutic angiogenesis purposes. Here, we propose to enhance the therapeutic function of human placenta-derived MSCs (hP-MSCs) in hindlimb ischemia therapy by using nitric oxide (NO)-releasing chitosan hydrogel (CS-NO). Our data showed that the co-transplantation of CS-NO hydrogel with hP-MSCs remarkably improved the grafting of hP-MSCs and ameliorated the functional recovery of ischemic hindlimbs. Moreover, we found that the neovascularization of damaged hindlimbs was significantly increased after co-transplanting CS-NO hydrogel and hP-MSCs, as confirmed by bioluminescence imaging (BLI). Further analysis revealed an endothelial-like status transformation of hP-MSCs in the presence of NO, which was identified as a potential mechanism contributing to the enhanced endothelium-protective and proangiogenic capacities of hP-MSCs that promote angiogenesis in mouse models of hindlimb ischemia. In conclusion, this study provides a promising approach for using NO hydrogel to improve the proangiogenic potency of MSCs in ischemic diseases, and the strategy used here facilitates the development of controlled-release scaffolds for enhancing the therapeutic efficiency of MSCs in angiogenic therapy. STATEMENT OF SIGNIFICANCE: The heterogeneous proangiogenic nature of mesenchymal stem cells (MSCs) is a key challenge in developing more effective treatments with MSCs for therapeutic angiogenesis purposes. In this study, we investigated whether nitric oxide (NO)-releasing chitosan hydrogel (CS-NO) could improve the proangiogenic potency of MSCs in ischemic diseases. Our results revealed an endothelial-like status transformation of human placenta-derived MSCs (hP-MSCs) in the presence of NO, which was identified as a potential mechanism contributing to the enhanced endothelium-protective and proangiogenic capacities of hP-MSCs that promote angiogenesis in mouse models of hindlimb ischemia. The strategy for enhancing the pro-angiogenic activity of MSCs with biomaterials provides a practical idea for overcoming the challenges associated with the clinical application of MSCs in therapeutic angiogenesis., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

Mesenchymal stem cell (MSC)-derived extracellular vesicles (EVs) have been shown to stimulate regeneration in the treatment of kidney injury. Renal regeneration is also thought to be stimulated by the activation of Sox9 + cells. However, whether and how the activation mechanisms underlying EV treatment and Sox9 + cell-dependent regeneration intersect is unclear. We reasoned that a high-resolution imaging platform in living animals could help to untangle this system. To test this idea, we first applied EVs derived from human placenta-derived MSCs (hP-MSCs) to a Sox9-Cre ERT2 ; R26 mTmG transgenic mouse model of acute kidney injury (AKI). Then, we developed an abdominal imaging window in the mouse and tracked the Sox9 + cells in the inducible Sox9-Cre transgenic mice via in vivo lineage tracing with two-photon intravital microscopy. Our results demonstrated that EVs can travel to the injured kidneys post intravenous injection as visualized by Gaussia luciferase imaging and markedly increase the activation of Sox9 + cells. Moreover, the two-photon living imaging of lineage-labeled Sox9 + cells showed that the EVs promoted the expansion of Sox9 + cells in kidneys post AKI. Histological staining results confirmed that the descendants of Sox9 + cells contributed to nephric tubule regeneration which significantly ameliorated the renal function after AKI. In summary, intravital lineage tracing with two-photon microscopy through an embedded abdominal imaging window provides a practical strategy to investigate the beneficial functions and to clarify the mechanisms of regenerative therapies in AKI., Competing Interests: Conflict of interest—The authors declare that they have no conflicts of interest with the contents of this article., (© 2020 Zhang et al.)

Background: Complete regeneration after skin injury remains a critical clinical challenge. Hydrogels, modified with growth factors or mimicking peptides, have been applied for functional tissue regeneration by increasing the bioactivity of engineered matrices. Methodology & results:  We synthesized an injectable biological hydrogel, C domain of IGF-1 (IGF-1C)-modified chitosan (CS-IGF-1C) hydrogel. Mouse model of cutaneous wound healing was established to investigate whether this hydrogel could promote wound healing. Our results demonstrated that CS-IGF-1C hydrogel exhibited superior proangiogenic effects, resulting in accelerated wound closure and improved extracellular matrix remodeling. Bioluminescence imaging and histology analysis confirmed the proangiogenic role of CS-IGF-1C hydrogel. Conclusion: CS-IGF-1C hydrogel could accelerate cutaneous wound healing by stimulating angiogenesis.

Background: Mesenchymal stem cell (MSC)-based therapies hold great promise for the treatment of inflammatory bowel disease (IBD). In order to optimize and maximize the therapeutic benefits of MSCs, we investigated whether cotransplantation of a chitosan (CS)-based injectable hydrogel with immobilized IGF-1 C domain peptide (CS-IGF-1C) and human placenta-derived MSCs (hP-MSCs) could ameliorate colitis in mice. Methods: IGF-1C hydrogel was generated by immobilizing IGF-1C to CS hydrogel. Colitis was induced by 2,4,6-trinitrobenzene sulfonic acid (TNBS) in mice. We initially applied hP-MSCs and CS-IGF-1C hydrogel for the treatment of colitis by in situ injection, and molecular imaging methods were used for real-time imaging of reactive oxygen species (ROS) and tracking of transplanted hP-MSCs by bioluminescence imaging (BLI). Furthermore, the effects of CS-IGF-1C hydrogel on prostaglandin E 2 (PGE 2 ) secretion of hP-MSCs and polarization of M2 macrophages were investigated as well. Results: The CS-IGF-1C hydrogel significantly increased hP-MSC proliferation and promoted the production of PGE 2 from hP-MSCs in vitro . Moreover, in vivo studies indicated that the CS-IGF-1C hydrogel promoted hP-MSC survival as visualized by BLI and markedly alleviated mouse colitis, which was possibly mediated by hP-MSC production of PGE 2 and interleukin-10 (IL-10) production by polarized M2 macrophages. Conclusions: The CS-IGF-1C hydrogel improved the engraftment of transplanted hP-MSCs, ameliorated inflammatory responses, and further promoted the functional and structural recovery of colitis through PGE 2 -mediated M2 macrophage polarization. Molecular imaging approaches and therapeutic strategies for hydrogel application provide a versatile platform for exploring the promising therapeutic potential of MSCs in the treatment of IBD., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

Background: Mesenchymal stem cell (MSC)-derived extracellular vesicles (EVs) have been shown to have therapeutic potential for ischemic diseases and are considered an alternative to cell therapy. However, the low retention and poor stability of EVs post-transplantation in vivo remain obstacle prior to the clinical application of EVs., Methods: This study was designed to investigate whether collagen matrix could increase the retention and stability of EVs and further improve the therapeutic effects in murine acute kidney injury (AKI) model. EVs were isolated from human placental MSCs (hP-MSC-EVs) and encapsulated in a collagen matrix. Then, we investigated whether collagen matrix can prolong the retention of EVs in vivo, further enhancing the therapeutic efficiency of EVs in AKI., Results: Our results indicated that collagen matrix could effectively encapsulate EVs, significantly increase the stability of EVs, and promote the sustained release of EVs. Collagen matrix has improved the retention of EVs in the AKI model, which was proved by Gaussia luciferase (Gluc) imaging. The application of collagen matrix remarkably facilitated the proliferation of renal tubular epithelial cells in AKI compared with EVs alone. Moreover, collagen matrix could further augment the therapeutic effects of hP-MSC-EVs as revealed by angiogenesis, fibrosis and apoptosis, and functional analysis. Finally, we found that EVs play a therapeutic role by inhibiting endoplasmic reticulum (ER) stress., Conclusions: Collagen matrix markedly enhanced the retention of EVs and further augmented the therapeutic effects of EVs for AKI. This strategy for improving the efficacy of EVs therapy provides a new direction for cell-free therapy.

With the immunoregulation potential, mesenchymal stem cells (MSCs) have been used for tissue regeneration by relieving inflammation in the injured tissues. When this repair process is interfered by immune disorders or pathological angiogenesis, the delays in corneal epithelial wound healing can lead to a persistent epithelial defect. Stem cell-derived extracellular vesicles (EVs), which carry abundant bioactive molecules from stem cells, have provided an alternative to regeneration therapy. In this study, we aimed to investigate if EVs from human placenta-derived MSCs (hP-MSCs) could ameliorate alkali injury of the cornea in the mouse model. 33.33  μ g/ μ L EVs in 10  μ L PBS were applied to the cornea. Repeat application three times, and 100  μ g EVs (in 30  μ L PBS) in total were administrated per day for two weeks. Our results revealed that EVs from hP-MSCs had preferable functions including enhancing proliferation and anti-inflammation and suppressing apoptosis of corneal epithelial cells. Furthermore, hP-MSC-derived EVs ameliorated mouse corneal wound healing by inhibiting angiogenesis and inflammation. Taken together, our current data suggested that hP-MSC-derived EVs have the beneficial effects of corneal wound healing, which provide alternative cell-free therapy with great practical value., Competing Interests: The authors declare that there is no conflict of interest regarding the publication of this paper., (Copyright © 2019 Hongyan Tao et al.)

Background: Poor cell engraftment and survival after transplantation limited the application of stem cell therapy. Synthetic biomaterials could provide an artificial microenvironment for stem cells, thereby improve cell survival and enhance the therapeutic efficiency of stem cells., Methods: We synthesized a hydrogel by conjugating C domain peptide of insulin-like growth factor-1 (IGF-1C) onto chitosan (CS-IGF-1C hydrogel). Human placenta-derived mesenchymal stem cells (hP-MSCs), which constitutively express a red fluorescent protein (RFP) and renilla luciferase (Rluc), were co-transplanted with CS-IGF-1C hydrogel into a murine hindlimb ischemia model. Transgenic mice expressing firefly luciferase (Fluc) under the promoter of vascular endothelial growth factor receptor 2 (VEGFR2-Luc) were used. Dual bioluminescence imaging (BLI) was applied for tracking the survival of hP-MSCs by Rluc imaging and the VEGFR2 signal pathway activation by Fluc imaging. To investigate the therapeutic mechanism of CS-IGF-1C hydrogel, angiographic, real-time PCR, and histological analysis were carried out., Results: CS-IGF-1C hydrogel could improve hP-MSCs survival as well as promote angiogenesis as confirmed by dual BLI. These results were consistent with accelerated skeletal muscle structural and functional recovery. Histology analysis confirmed that CS-IGF-1C hydrogel robustly prevented fibrosis as shown by reduced collagen deposition, along with increased angiogenesis. In addition, the protective effects of CS-IGF-1C hydrogel, such as inhibiting H 2 O 2 -induced apoptosis and reducing inflammatory responses, were proved by in vitro experiments., Conclusions: Taken together, IGF-1Cs provides a conducive niche for hP-MSCs to exert pro-mitogenic, anti-apoptotic, and pro-angiogenic effects, as well as to inhibit fibrosis. Thus, the incorporation of functional peptide into bioscaffolds represents a safe and feasible approach to augment the therapeutic efficacy of stem cells.

Wound healing is regulated by a complex series of events and overlapping phases. A delicate balance of cytokines and mediators in tissue repair is required for optimal therapy in clinical applications. Molecular imaging technologies, with their versatility in monitoring cellular and molecular events in living organisms, offer tangible options to better guide tissue repair by regulating the balance of cytokines and mediators at injured sites. Methods: A murine cutaneous wound healing model was developed to investigate if incorporation of prostaglandin E 2 (PGE 2 ) into chitosan (CS) hydrogel (CS+PGE 2 hydrogel) could enhance its therapeutic effects. Bioluminescence imaging (BLI) was used to noninvasively monitor the inflammation and angiogenesis processes at injured sites during wound healing. We also investigated the M1 and M2 paradigm of macrophage activation during wound healing. Results: CS hydrogel could prolong the release of PGE 2 , thereby improving its tissue repair and regeneration capabilities. Molecular imaging results showed that the prolonged release of PGE 2 could ameliorate inflammation by promoting the M2 phenotypic transformation of macrophages. Also, CS+PGE 2 hydrogel could augment angiogenesis at the injured sites during the early phase of tissue repair, as revealed by BLI. Furthermore, our results demonstrated that CS+PGE 2 hydrogel could regulate the balance among the three overlapping phases-inflammation, regeneration (angiogenesis), and remodeling (fibrosis)-during cutaneous wound healing. Conclusion: Our findings highlight the potential of the CS+PGE 2 hydrogel as a novel therapeutic strategy for promoting tissue regeneration via M2 macrophage polarization. Moreover, molecular imaging provides a platform for monitoring cellular and molecular events in real-time during tissue repair and facilitates the discovery of optimal therapeutics for injury repair by regulating the balance of cytokines and mediators at injured sites., Competing Interests: Competing Interests: The authors have declared that no competing interest exists.

Mesenchymal stem cells (MSC) are able to differentiate into cells of multiple lineage, and additionally act to modulate the immune response. Interleukin (IL)-9 is primarily produced by cluster of differentiation (CD)4 + T cells to regulate the immune response. The present study aimed to investigate the effect of human umbilical cord derived-MSC (UC-MSC) on IL-9 production of human CD4 + T cells. It was demonstrated that the addition of UC-MSC to the culture of CD4 + T cells significantly enhanced IL-9 production by CD4 + T cells. Transwell experiments suggested that UC-MSC promotion of IL-9 production by CD4 + T cells was dependent on cell-cell contact. Upregulated expression of CD106 was observed in UC-MSC co-cultured with CD4 + T cells, and the addition of a blocking antibody of CD106 significantly impaired the ability of UC-MSC to promote IL-9 production by CD4 + T cells. Therefore, the results of the present study demonstrated that UC-MSC promoted the generation of IL-9 producing cells, which may be mediated, in part by CD106. The findings may act to expand understanding and knowledge of the immune modulatory role of UC-MSC.

Neovasculogenesis induced by stem cell therapy is an innovative approach to improve critical limb ischemia (CLI) in diabetes. Mesenchymal stem cells (MSCs) are ideal candidates due to their angiogenic and immunomodulatory features. The aim of this study is to determine the therapeutic effects of human placenta-derived MSCs (P-MSCs) on diabetic CLI, with or without exogenous insulin administration, and the underlying mechanism of any effect. A series of in vitro experiments were performed to assess the stemness and vasculogenic activity of P-MSCs. P-MSCs were intramuscularly injected at two different doses with and without the administration of insulin. The efficacy of P-MSC transplantation was evaluated by ischemia damage score, ambulatory score, laser Doppler perfusion image (LDPI), capillary, and vascular density. In vivo imaging was applied to track the implanted P-MSCs. In vivo differentiation and in situ secretion of angiogenic cytokines were determined. In vitro experimental outcomes showed the differentiation potential and potent paracrine effect of P-MSCs. P-MSCs survived in vivo for at least 3 weeks and led to the acceleration of ischemia recovery, due to newly formed capillaries, increased arterioles, and secretion of various proangiogenic factors. P-MSCs participate in angiogenesis and vascularization directly through differentiation and cytokine expression.

Background: Mesenchymal stem cells (MSCs) have been widely proven effective for therapeutic angiogenesis in ischemia animal models as well as clinical vascular diseases. Because of the invasive method, limited resources, and aging problems of adult tissue-derived MSCs, more perinatal tissue-derived MSCs have been isolated and studied as promising substitutable MSCs for cell transplantation. However, fewer studies have comparatively studied the angiogenic efficacy of MSCs derived from different tissues sources. Here, we evaluated whether the in-situ environment would affect the angiogenic potential of MSCs., Methods: We harvested MSCs from adult bone marrow (BMSCs), adipose tissue (AMSCs), perinatal umbilical cord (UMSCs), and placental chorionic villi (PMSCs), and studied their "MSC identity" by flow cytometry and in-vitro trilineage differentiation assay. Then we comparatively studied their endothelial differentiation capabilities and paracrine actions side by side in vitro., Results: Our data showed that UMSCs and PMSCs fitted well with the minimum standard of MSCs as well as BMSCs and AMSCs. Interestingly, we found that MSCs regardless of their tissue origins could develop similar endothelial-relevant functions in vitro, including producing eNOS and uptaking ac-LDL during endothelial differentiation in spite of their feeble expression of endothelial-related genes and proteins. Additionally, we surprisingly found that BMSCs and PMSCs could directly form tubular structures in vitro on Matrigel and their conditioned medium showed significant proangiogenic bioactivities on endothelial cells in vitro compared with those of AMSCs and UMSCs. Besides, several angiogenic genes were upregulated in BMSCs and PMSCs in comparison with AMSCs and UMSCs. Moreover, enzyme-linked immunosorbent assay further confirmed that BMSCs secreted much more VEGF, and PMSCs secreted much more HGF and PGE2., Conclusions: Our study demonstrated the heterogeneous proangiogenic properties of MSCs derived from different tissue origins, and the in vivo isolated environment might contribute to these differences. Our study suggested that MSCs derived from bone marrow and placental chorionic villi might be preferred in clinical application for therapeutic angiogenesis.

Introduction: Mesenchymal stem cells (MSCs) represent a heterogeneous cell population that is promising for regenerative medicine. The present study was designed to assess whether VCAM-1 can be used as a marker of MSC subpopulation with superior angiogenic potential., Methods: MSCs were isolated from placenta chorionic villi (CV). The VCAM-1(+/-) CV-MSCs population were separated by Flow Cytometry and subjected to a comparative analysis for their angiogenic properties including angiogenic genes expression, vasculo-angiogenic abilities on Matrigel in vitro and in vivo, angiogenic paracrine activities, cytokine array, and therapeutic angiogenesis in vascular ischemic diseases., Results: Angiogenic genes, including HGF, ANG, IL8, IL6, VEGF-A, TGFβ, MMP2 and bFGF, were up-regulated in VCAM-1(+)CV-MSCs. Consistently, angiogenic cytokines especially HGF, IL8, angiogenin, angiopoitin-2, μPAR, CXCL1, IL-1β, IL-1α, CSF2, CSF3, MCP-3, CTACK, and OPG were found to be significantly increased in VCAM-1(+) CV-MSCs. Moreover, VCAM-1(+)CV-MSCs showed remarkable vasculo-angiogenic abilities by angiogenesis analysis with Matrigel in vitro and in vivo and the conditioned medium of VCAM-1(+) CV-MSCs exerted markedly pro-proliferative and pro-migratory effects on endothelial cells compared to VCAM-1(-)CV-MSCs. Finally, transplantation of VCAM-1(+)CV-MSCs into the ischemic hind limb of BALB/c nude mice resulted in a significantly functional improvement in comparison with VCAM-1(-)CV-MSCs transplantation., Conclusions: VCAM-1(+)CV-MSCs possessed a favorable angiogenic paracrine activity and displayed therapeutic efficacy on hindlimb ischemia. Our results suggested that VCAM-1(+)CV-MSCs may represent an important subpopulation of MSC for efficient therapeutic angiogenesis.

Mesenchymal stem cells (MSCs) have shown their therapeutic potency for treatment of cardiovascular diseases owing to their low immunogenicity, ease of isolation and expansion, and multipotency. As multipotent progenitors, MSCs have revealed their ability to differentiate into various cell types and could promote endogenous angiogenesis via microenvironmental modulation. Studies on cardiovascular diseases have demonstrated that transplanted MSCs could engraft at the injured sites and differentiate into cardiomyocytes and endothelial cells as well. Accordingly, several clinical trials using MSCs have been performed and revealed that MSCs may improve relevant clinical parameters in patients with vascular diseases. To fully comprehend the characteristics of MSCs, understanding their intrinsic property and associated modulations in tuning their behaviors as well as functions is indispensable for future clinical translation of MSC therapy. This review will focus on recent progresses on endothelial differentiation and potential clinical application of MSCs, with emphasis on therapeutic angiogenesis for treatment of cardiovascular diseases.

Human umbilical cord mesenchymal stromal cells (hUC-MSCs) are currently being used as novel therapeutic agents in numerous clinical trials. Previous works have shown that hUC-MSCs possess profound immunomodulatory capacities through IL-1 stimulation produced by peripheral blood mononuclear cells (PBMCs), their main cellular partner in most pathophysiological and therapeutic situations. The present study was designed to explore the role of TNF-α in these interactions. In these experiments, we demonstrated that TNF-α originated from PBMCs under the influence of IL-1. We also showed that TNF-α acted differently depending upon the concentrations reached. At low concentrations it clearly contributed to IL-6 and monocyte chemotactic protein 1 (MCP-1) production. At high concentrations, used alone or in association with the TNF-related apoptosis-inducing ligand, TNF-α also stimulated hUC-MSC IL-6 but, more intensely, MCP-1 production. This stimulation was associated but independent of apoptosis induction in a process involving Inhibitor of Apoptosis Proteins. Interferon gamma (IFN-γ), tested to stimulate PBMC and tissue activation, amplified IL-6 and MCP-1 production and cell death by, apparently, a different process involving necrosis. Our findings bring new insights into the complex interactions between hUC-MSCs and PBMCs, involving cytokines, chemokines and cell death, and are of fundamental importance for tissue homeostasis.

Aim: IL-37b has shown anti-cancer activities in addition to its anti-inflammatory properties. In this study, we investigated the effects of IL-37b on breast carcinoma growth in mice and to determine the involvement of T cell activation in the effects., Methods: IL-37b gene was transferred into mouse breast carcinoma cell line 4T1 (4T1-IL37b cells), the expression of secretory IL-37b by the cells was detected, and the effects of IL-37b expression on the cell proliferation in vitro was evaluated. After injection of 4T1 cells or 4T1-IL37b cells into immunocompetent BALB/c mice, immunodeficient BALB/c nude mice and NOD-SCID mice, the tumor growth and survival rate were measured. The proliferation of T cells in vitro was also detected., Results: IL-37b was detected in the supernatants of 4T1-IL37b cells with a concentration of 12.02 ± 0.875 ng/mL. IL-37b expression did not affect 4T1 cell proliferation in vitro. BALB/c mice inoculated with 4T1-IL37b cells showed significant retardation of tumor growth. BALB/c mice inoculated with both 4T1 cells and mitomycin C-treated 4T1-IL37b cells also showed significant retardation of tumor growth. But the anti-cancer activity of IL-37b was abrogated in BALB/c nude mice and NOD-SCID mice inoculated with 4T1-IL37b cells. Recombinant IL-37b slightly promoted CD4(+) T cell proliferation without affecting CD8(+) T cell proliferation., Conclusion: IL-37b exerts anti-4T1 breast carcinoma effects in vivo by modulating the tumor microenvironment and influencing T cell activation.

Background: The Notch signaling pathway is implicated in a broad range of developmental processes, including cell fate decisions. This study was designed to determine the role of Notch signaling in adipogenic differentiation of human bone marrow derived MSCs (BM-MSCs)., Methods: The Notch signaling was inhibited by the γ-secretase inhibitor N-[N-(3,5-difluor- ophenacetyl-L-alanyl)]-S-phenylglycine t-butylester (DAPT). The markers involving adipogenic differentiation of MSCs, the relative pathway PTEN-PI3K/Akt/mTOR and autophagy activation were then analyzed. Furthermore, the autophagy inhibitor chloroquine (CQ) and 3-methyladenine (3-MA) were used to study the role of autophagy in the DAPT-induced the adipogenic differentiation of MSCs., Results: We first confirmed the down -regulation of Notch gene expression during MSCs adipocyte differentiation, and showed that the inhibition of Notch signaling significantly enhanced adipogenic differentiation of MSCs. Furthermore, Notch inhibitor DAPT induced early autophagy by acting on PTEN-PI3K/Akt/mTOR pathway. The autophagy inhibitor CQ and 3-MA dramatically abolished the effects of DAPT-induced autophagy and adipogenic differentiation of MSCs., Conclusion: Our results indicate that inhibition of Notch signaling could promote MSCs adipogenesis mediated by autophagy involving PTEN-PI3K/Akt/mTOR pathway. Notch signaling could be a novel target for regulating the adipogenic differentiation of MSCs., (© 2015 S. Karger AG, Basel.)

Introduction: Mesenchymal stem cells (MSCs) are widely investigated in clinical researches to treat various diseases. Classic culture medium for MSCs, even for clinical use, contains fetal bovine serum. The serum-containing medium (SCM) seems a major obstacle for MSCs-related therapies due to the risk of contamination of infectious pathogens. Some studies showed that MSCs could be expanded in serum free medium (SFM); however, whether SFM would change the biological characteristics and safety issues of MSCs has not been well answered., Methods: Human umbilical cord mesenchymal stem cells (hUC-MSCs) were cultured in a chemical defined serum free medium. Growth, multipotency, surface antigen expression, telomerase, immunosuppressive ability, gene expression profile and genomic stability of hUC-MSCs cultured in SFM and SCM were analyzed and compared side by side., Results: hUC-MSCs propagated more slowly and senesce ultimately in SFM. SFM-expanded hUC-MSCs were different from SCM-expanded hUC-MSCs in growth rate, telomerase, gene expression profile. However, SFM-expanded hUC-MSCs maintained multipotency and the profile of surface antigen which were used to define human MSCs. Both SFM- and SCM-expanded hUC-MSCs gained copy number variation (CNV) in long-term in vitro culture., Conclusion: hUC-MCSs could be expanded in SFM safely to obtain enough cells for clinical application, meeting the basic criteria for human mesenchymal stem cells. hUC-MSCs cultured in SFM were distinct from hUC-MSCs cultured in SCM, yet they remained therapeutic potentials for future regenerative medicine.

Objective: To observe the effect of transfecting the gene human insulin-like growth factor (hIGF)-1 into human umbilical cord blood mesenchymal stem cells (hUCB-MSCs) via non-viral vector., Methods: This study was performed in the Affiliated Hospital of Qingdao University, Qingdao, China from June 2012 to May 2013. Twelve hUCB samples were harvested, and isolated in lymphocyte separation medium, and then cultured. Surface antigen expression in MSCs was detected by flow cytometry. Recombinant plasmid pIRES2-enhanced green fluorescent protein (EGFP)-hIGF-1 was transfected into MSCs by X-treme GENE HP DNA transfection reagent. Then, EGFP was observed with reverse fluorescent microscope at different time points. Enzyme-linked immunosorbent assay was used to determine the hIGF-1 protein concentration in supernatants. Immunofluorescence microscopy and reverse transcription polymerase chain reaction were used to detect the expression of hIGF-1 in the hUCB-MSCs. Expression of type II collagen was detected by immunohistochemistry staining., Results: Transfection efficiency was 28.74 +/- 7.31%. The cluster of differentiation (CD)90, CD105, and CD146 expression increased CD34, CD45, and anti-HLA-DR expression decreased. Results of immunofluorescence microscopy and RT-PCR confirmed expression of the hIGF-1 gene. The hIGF-1 protein concentration in the supernatants showed a peak level at 34.63 +/- 1.61 ng/ml 48 hours after transfection. Immunohistochemical analysis of transfected hUCB-MSCs proved that type II collagen could be expressed positively., Conclusion: Human IGF-1 gene can be transfected into hUCB-MSCs, and expressed at a high level with subsequent expression of type II collagen.

We examined if transplantation of combined haploidentical hematopoietic stem cells (HSC) and mesenchymal stem cells (MSC) affected graft failure and graft-versus-host disease (GVHD) in patients with severe aplastic anemia (SAA). Patients with SAA-I (N = 17) received haploidentical HSCT plus MSC infusion. Stem cell grafts used a combination of granulocyte colony-stimulating factor (G-CSF)-primed bone marrow and G-CSF-mobilized peripheral blood stem cells of haploidentical donors and the culture-expanded third-party donor-derived umbilical cord MSCs (UC-MSCs), respectively. Reduced intensity conditioning consisted of fludarabine (30 mg/m2·d)+cyclosphamide (500 mg/m2·d)+anti-human thymocyte IgG. Transplant recipients also received cyclosporin A, mycophenolatemofetil, and CD25 monoclonal antibody. A total of 16 patients achieved hematopoietic reconstitution. The median mononuclear cell and CD34 count was 9.3×10(8)/kg and 4.5×10(6)/kg. Median time to ANC was >0.5×10(9)/L and PLT count >20×10(9)/L were 12 and 14 days, respectively. Grade III-IV acute GVHD was seen in 23.5% of the cases, while moderate and severe chronic GVHD were seen in 14.2% of the cases. The 3-month and 6-month survival rates for all patients were 88.2% and 76.5%, respectively; mean survival time was 56.5 months. Combined transplantation of haploidentical HSCs and MSCs on SAA without an HLA-identical sibling donor was safe, effectively reduced the incidence of severe GVHD, and improved patient survival.

Background: Mesenchymal stem cells are capable of self-renewal and multi-lineage differentiation. They are used extensively to treat several diseases. Traditionally, mesenchymal stem cells are cultured in serum-containing media, typically supplemented with fetal bovine serum (FBS). However, the variability of FBS is likely to skew experimental results. Although serum-free media used to expand mesenchymal stem cells has facilitated remarkable achievements, immunomodulation of these cells in under serum-free conditions is poorly understood. We hypothesized that mesenchymal stem cells expanded in serum-free media will retain powerful immunoregulatory functions in vitro and in vivo., Design and Methods: Immunosuppressive activity and the immunomodulatory cytokines produced by mesenchymal stem cells in serum-free media were characterized in vitro. Immunomodulation by serum-free mesenchymal stem cell expansion in monocrotaline-induced pulmonary hypertension was explored in vivo., Results: Similar to cells in serum-containing media, mesenchymal stem cells expanded in serum-free media inhibited proliferation and apoptosis of CD4(+)T cells. They also exhibited strong immunosuppressive activities and secreted high levels of immunomodulatory cytokines such as PGE2, IDO1, COX2, IL-6, and IL-1β, but not HGF. On the other hand, growth of mesenchymal stem cells in serum-free media attenuated pulmonary vascular remodeling and inhibited mRNA expression of proinflammatory cytokines TNF-α, IFN-γ, IL-6, IL-1β, and IL-18., Conclusions: Mesenchymal stem cells in serum-free media maintained powerful immunomodulatory function in vitro and in vivo; serum-free media may replace serum-containing media for basic research and clinical applications., (© 2014 S. Karger AG, Basel.)

Cultured human umbilical cord mesenchymal stem cells (hUC-MSCs) are being tested in several clinical trials and encouraging outcomes have been observed. To determine whether in vitro expansion influences the genomic stability of hUC-MSCs, we maintained nine hUC-MSC clones in long-term culture and comparatively analyzed them at early and late passages. All of the clones senesced in culture, exhibiting decreased telomerase activity and shortened telomeres. Two clones showed no DNA copy number variations (CNVs) at passage 30 (P30). Seven clones had ≥1 CNVs at P30 compared with P3, and one of these clones appeared trisomic chromosome 10 at the late passage. No tumor developed in immunodeficient mice injected with hUC-MSCs, regardless of whether the cells had CNVs at the late passage. mRNA-Seq analysis indicated that pathways of cell cycle control and DNA damage response were downregulated during in vitro culture in hUC-MSC clones that showed genomic instability, but the same pathways were upregulated in the clones with good genomic stability. These results demonstrated that hUC-MSCs can be cultured for many passages and attain a large number of cells, but most of the cultured hUC-MSCs develop genomic alterations. Although hUC-MSCs with genomic alterations do not undergo malignant transformation, periodic genomic monitoring and donor management focusing on genomic stability are recommended before these cells are used for clinical applications.

Granulocyte colony-stimulating factor (G-CSF) has exhibited efficacy at preventing the progression of pulmonary hypertension (PH); however, the exact mechanism is not completely clear. The aim of the present study was to assess whether this protective effect was mediated by the upregulation of circulating endothelial progenitor cells (EPCs) via the nitric oxide (NO) system. PH was induced in male Sprague-Dawley (SD) rats by the administration of a single subcutaneous injection of monocrotaline (MCT). The rats were treated with recombinant human G-CSF (rhG-CSF, 50 μg/kg/day) by subcutaneous injection from day five to day seven subsequent to the injection of MCT. Nω-nitro-L-arginine methyl ester (L-NAME, 4 mg/kg/day) was intragastrically administered in addition to rhG-CSF as a negative intervention. The changes in hemodynamics and histology, the number and function of circulating EPCs and the concentration of plasma NO were evaluated. With the occurrence of PH in the rat model, the number and function of circulating EPCs were demonstrated to be markedly downregulated. Moreover, a reduced plasma concentration of NO was observed, which was positively correlated with the number of circulating EPCs. Administration of rhG-CSF elevated the plasma level of NO, upregulated the number and function of circulating EPCs and effectively improved pulmonary hemodynamics and vascular reconstruction. Furthermore, the positive correlation between the levels of plasma NO and circulating EPCs was also observed in the rhG-CSF treatment group. However, the protective effect of rhG-CSF in PH was attenuated by L-NAME, which mediated the downregulation of NO and the EPCs. Thus, the present study suggests that G-CSF may attenuate the progression of MCT-induced PH by improving vascular injury repair mechanisms via the NO-mediated upregulation of EPCs.

Mesenchymal stem cells (MSCs) are being explored extensively as a promising treatment for autoimmune diseases. We have recently reported that MSCs could ameliorate experimental autoimmune uveoretinitis (EAU) in rats. In this study, we examined further the effects of MSCs on the dynamics of T cell subsets in both eye and spleen and their cytokine production during the course of EAU. We focused on when and where the MSCs had inhibitory effects on T helper type 1 (Th1) and Th17 cells and how long the inhibitory effect lasted, in order to provide more mechanistic evidence for MSCs on the treatment of uveitis. Compared to the control group, administration of MSCs decreased the production of Th1 and Th17 cytokines significantly, while the production of Th2 and regulatory T cell (T(reg)) cytokines [interleukin (IL)-10 and transforming growth factor (TGF)-β] was elevated during the entire course of EAU. Correspondingly, the dynamic levels of IL-17 in the aqueous humour (AqH) were reduced in MSC-treated rats. Moreover, the ratio of Th17/T(reg) cells in both spleen and eye was decreased. These results provide powerful evidence that MSCs can regulate negatively both Th1 and Th17 responses and restore the balance of Th17/T(regs) in the whole course of EAU, which is important for the regression of the disease., (© 2013 British Society for Immunology.)

Mesenchymal stem cells (MSCs) reside in almost all of the body tissues, where they undergo self-renewal and multi-lineage differentiation. MSCs derived from different tissues share many similarities but also show some differences in term of biological properties. We aim to search for significant differences among various sources of MSCs and to explore their implications in physiopathology and clinical translation. We compared the phenotype and biological properties among different MSCs isolated from human term placental chorionic villi (CV), umbilical cord (UC), adult bone marrow (BM) and adipose (AD). We found that CD106 (VCAM-1) was expressed highest on the CV-MSCs, moderately on BM-MSCs, lightly on UC-MSCs and absent on AD-MSCs. CV-MSCs also showed unique immune-associated gene expression and immunomodulation. We thus separated CD106(+)cells and CD106(-)cells from CV-MSCs and compared their biological activities. Both two subpopulations were capable of osteogenic and adipogenic differentiation while CD106(+)CV-MSCs were more effective to modulate T helper subsets but possessed decreased colony formation capacity. In addition, CD106(+)CV-MSCs expressed more cytokines than CD106(-)CV-MSCs. These data demonstrate that CD106 identifies a subpopulation of CV-MSCs with unique immunoregulatory activity and reveal a previously unrecognized mechanism underlying immunomodulation of MSCs.

Background: CD151 is highly expressed in breast cancer cells and has been shown to accelerate breast cancer by enhancing cell growth and motility, but its regulation is poorly understood. To explore post-translation regulation of CD151, for example microRNAs, will be of great importance to claim the mechanism., Methods: A luciferase reporter assay was used to determine whether CD151 was a target of miR-124. The levels of CD151 mRNA were detected by real-time PCR and CD151 protein expression was measured by western blot and flow cytometry. The effects of miR-124 expression on growth, apoptosis, cell cycle and motility of breast cancer cells were determined., Results: We discovered that miR-124 directly targets the 3' untranslated region (3'-UTR) of CD151 mRNAs and suppresses its mRNA expression and protein translation. Both siRNA of CD151 and miR-124 mimics could significantly inhibit proliferation of breast cancer cell lines via cell cycle arrest but does not induce apoptosis. Meanwhile, miR-124 mimics significantly inhibited the motility of breast cancer cells., Conclusion: miR-124 plays a critical role in inhibiting the invasive and metastatic potential of breast cancer cells, probably by directly targeting the CD151 genes. Our findings highlight an important role of miR-124 in the regulation of invasion and metastasis by breast cancer cells and suggest a potential application for miR-124 in breast cancer treatment., (Copyright © 2013 S. Karger AG, Basel.)

Objective: To validate a novel method of expanding late endothelial progenitor cells (EPC) in vitro., Methods: We cultured mononuclear cells (MNC) from human peripheral blood on the plate with the feeder layer cells, i.e. irradiated late EPC or human umbilical vein endothelial cells. After 21 days, the numbers of late EPC colonies were counted separately. And the surface antigen of late EPC was detected by fluorescence-activated cell sorter (FACS) and their in vitro ability of forming vascular structure examined by matrigel., Results: Both colony numbers of late EPC with feeder layer cell culturing were over 20 times than those without (40.0 ± 3.9, 39.3 ± 3.1 vs 2.0 ± 1.3, both P < 0.05). And the former's late EPC colony appeared earlier. The late EPC expressed CD31, CD34, eNOS, Flt-1, P1H12, Sendo, VE cadherin and CD117. And vascular structures were discerned., Conclusions: The method of feeder layer cells may vastly expand the quantity of late EPC. And microenvironment plays an important role in its expansion.

Aplastic anemia (AA) is a marrow failure syndrome mediated by aberrant T-cell subsets. Mesenchymal stem cells (MSCs) play an important role in maintaining immune homeostasis through modulating a variety of immune cells. However, little is known about the immunomodulation potential of bone marrow MSCs (BM-MSCs) in AA. Here, we reported that BM-MSCs from AA patients were reduced in suppressing the proliferation and clonogenic potential of CD4(+) T cells and the production of tumor necrosis factor-α (TNF-α) and interferon-γ (IFN-γ), which was associated with decreased prostaglandin E2 (PGE2). Meanwhile, BM-MSCs from AA patients were defective to promote CD4(+)CD25(+)FOXP3(+) regulatory T cells expansion through reduced transforming growth factor-β (TGF-β). No significant difference between AA and normal BM-MSCs was observed in affecting the production of interleukins (IL)-4, IL-10 and IL-17. Our data indicate that BM-MSCs were impaired in maintaining the immune homeostasis associated with CD4(+) T cells, which might aggravate the marrow failure in AA.