Phosphatidylserine-mediated uptake of extracellular vesicles by hepatocytes ameliorates liver ischemia-reperfusion injury.

Compelling evidence suggests that mesenchymal stem cell-derived extracellular vesicles (MSC-EVs) promote regeneration in animal models of liver injury by delivering signaling molecules. However, their target cells and uptake mechanism remain elusive. In this study, MSC-EVs were intravenously administered in a mouse model of liver ischemia-reperfusion injury (IRI). Our results revealed that MSC-EVs exhibit enhanced liver targeting in IRI mice, and injured hepatocytes display a greater capacity for MSC-EV uptake. We found that phosphatidylserine (PS) displayed on the exterior of injured hepatocytes promotes MSC-EV internalization, possibly by binding to MFGE8, a protein expressed on the MSC-EV membrane. Furthermore, the therapeutic effect of MSC-EVs on liver IRI is highly dependent on this PS-mediated uptake pathway. Our findings provide evidence that MSC-EVs preferentially target injured hepatocytes, relying on a PS-dependent uptake route to exert hepatoprotective effects, which are critical for the future design of EV-based therapeutic strategies for liver IRI.

The sustained PGE2 release matrix improves neovascularization and skeletal muscle regeneration in a hindlimb ischemia model.

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 E2 (PGE2), a lipid signaling molecule, has significant therapeutic potential for tissue repair and regeneration. Though PGE2 has been well reported in tissue regeneration, the application of PGE2 is hampered by its short half-life in vivo and the lack of a viable system for sustained release of PGE2. RESULTS: In this study, we designed and synthesized a new PGE2 release matrix by chemically bonding PGE2 to collagen. Our results revealed that the PGE2 matrix effectively extends the half-life of PGE2 in vitro and in vivo. Moreover, the PGE2 matrix markedly improved neovascularization by increasing angiogenesis, as confirmed by bioluminescence imaging (BLI). Furthermore, the PGE2 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 PGE2 to collagen for sustained release and may facilitate the development of PGE2-based therapies to significantly improve tissue regeneration.

In vivo two-photon microscopy reveals the contribution of Sox9+ cell to kidney regeneration in a mouse model with extracellular vesicle treatment.

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-CreERT2; R26mTmG 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.

High-efficient generation of natural killer cells from peripheral blood with preferable cell vitality and enhanced cytotoxicity by combination of IL-2, IL-15 and IL-18.

Natural killer (NK) cells are pivotal effector lymphocytes characterized for the innate immune response to pathogenic microorganism and tumor cells without priming and sensitization. Despite emerging knowledge has highlighted the rosy prospects in tumor immunosurveillance, yet the large-scale clinical application of NK cell-based therapy is hindered largely attributes to the defects in generating sufficient and high-quality cellular products. Herein, on the basis of 16 kinds of candidate combinations, we investigated the feasibility of cytokine cocktail-based strategy for convenient and standardized NK cell cultivation as well as the multifaceted characteristics and cytotoxicity against tumor cells. Our results revealed that joint utilization of Interleukin (IL)-2, IL-15, IL-18 manifested the optimal facilitation upon the ex vivo expansion and proportion of NK cells in peripheral blood mononuclear cells (PBMCs). Meanwhile, the obtained NK cell population expressed high levels of activating molecules (CD16 and NKG2D) and exhibited splendid cytotoxicity against K562 cell line. Collectively, with the aid of cytokine-based programming, we established an alternative strategy for facilitating the large-scale persistence and activation of NK cells from peripheral blood, which would benefit the NK cell- and chimeric antigen receptor-modified NK (CAR-NK) cell-based autologous or allogeneic tumor immunotherapy.

Multifaceted modifications for a cell size-based circulating tumor cell scope technique hold the prospect for large-scale application in general populations.

Circulating tumor cells (CTCs) indicate the diagnosis and prognosis of cancer patients, together with benefiting individual treatment and anticancer drug development. However, their large-scale application in general population still requires systematically multifaceted modifications for currently proprietary new technologies based on filtration. We primitively utilized a cell size-based platform to evaluate the recovery efficiency of spiked abnormal cell lines and analyzed circulating abnormal cells (CACs). To dissect the subpopulations of CACs, we conducted immunofluorescent (IF) staining with a combination of unique biomarkers of CTCs and circulating endothelial cells (CECs). Furthermore, we improved the CTC screening system by assessing the feasibility of transferring CTCs for automatic IF analysis, together with simulating and optimizing the circumstances for long-term CTC storage and transportation. We detected CACs in 15 HD candidates with CTC characteristics such as abnormally large cytomorphology, high nuclear-cytoplasmic ratio, and positive for panCK or VIM staining. Thereafter, we improved accuracy of the platform by distinguishing CTCs from CECs, which satisfied the elementary requirement for small-scale CTC screening in HD candidates. Finally, large-scale CTC screening in general population was available after multifaceted modifications including automatic analysis by transferring CTCs on slides, choosing the appropriate blood-collecting tube, optimizing the conditions for long-term CTC storage and transportation, and evaluating the potential effect on the CTC phenotype. Hence, we systematically modified the scope of technique parameters, improved the accuracy of early cancer detection, and made it realizable for large-scale CTC or CEC screening in general population.

Twist1 promotes dendritic cell-mediated antitumor immunity.

Dendritic cells (DCs) play a central role in autoimmunity, immune homeostasis, and presentation of tumor antigens to T cells in order to prime antitumor responses. The number of tumor-infiltrating DCs is associated with survival and prognosis in cancer. Twist1 is a well-known regulator of tumor initiation and promotion, but whether and how DC-derived Twist1 regulates antitumor responses remains poorly understood. Here, we generated a mouse line with Twist1 conditionally depleted in DCs and found that Twist1-deficiency in DCs did not affect the DCs and T cell homeostasis under steady-state conditions; however, in melanoma models, the proportion of conventional DCs (cDCs) in draining lymph nodes (DLNs) was significantly decreased. Accordingly, a decreased ratio and number of tumor-infiltrating cDCs were observed, which reduced the recruitment of tumor-infiltrating T cells. Furthermore, production of IFN-γ, a crucial antitumor factor, by T cells, was dramatically decreased, which can further dampen the T cell antitumor functions. Collectively, our data indicate that Twist1 in DCs regulates antitumor functions by maintain the number of tumor-infiltrating DCs and T cells, and their antitumor activity.

Combination of umbilical cord mesenchymal stem cells and standard immunosuppressive regimen for pediatric patients with severe aplastic anemia.

BACKGROUND: Defects of bone marrow mesenchymal stem cells (BM-MSCs) in proliferation and differentiation are involved in the pathophysiology of aplastic anemia (AA). Infusion of umbilical cord mesenchymal stem cells (UC-MSCs) may improve the efficacy of immunosuppressive therapy (IST) in childhood severe aplastic anemia (SAA). METHODS: We conducted an investigator-initiated, open-label, and prospective phase IV trial to evaluate the safety and efficacy of combination of allogenic UC-MSCs and standard IST for pediatric patients with newly diagnosed SAA. In mesenchymal stem cells (MSC) group, UC-MSCs were injected intravenously at a dose of 1 × 106/kg per week starting on the 14th day after administration of rabbit antithymocyte globulin (ATG), for a total of 3 weeks. The clinical outcomes and adverse events of patients with UC-MSCs infusion were assessed when compared with a concurrent control group in which patients received standard IST alone. RESULTS: Nine patients with a median age of 4 years were enrolled as the group with MSC, while the data of another 9 childhood SAA were analysed as the controls. Four (44%) patients in MSC group developed anaphylactic reactions which were associated with rabbit ATG. When compared with the controls, neither the improvement of blood cell counts, nor the change of T-lymphocytes after IST reached statistical significance in MSC group (both p > 0.05) and there were one (11%) patient in MSC group and two (22%) patients in the controls achieved partial response (PR) at 90 days after IST. After a median follow-up of 48 months, there was no clone evolution occurring in both groups. The 4-year estimated overall survival (OS) rate in two groups were both 88.9% ± 10.5%, while the 4-year estimated failure-free survival (FFS) rate in MSC group was lower than that in the controls (38.1% ± 17.2% vs. 66.7% ± 15.7%, p = 0.153). CONCLUSIONS: Concomitant use of IST and UC-MSCs in SAA children is safe but may not necessarily improve the early response rate and long-term outcomes. This clinical trial was registered at ClinicalTrials.gov, identifier: NCT02218437 (registered October 2013).

Lkb1 in dendritic cells restricts CD8+Foxp3+regulatory T cells expansion in vivo.

Liver kinase B1 (Lkb1) in dendritic cells (DCs) plays a key role in maintaining immunity homeostasis and adaptive immunity by controlling the CD4+Foxp3+T regulatory cell (CD4+Tregs) pool and T cells activation. However, the function of Lkb1 in DCs for the regulation of CD8+Foxp3+T regulatory cells (CD8+Tregs) has not been addressed. Herein, we found that Lkb1-deficient DCs could lead to excessive CD8+Tregs expansion in multiple organs. We found that OX40 expression was significantly higher in Lkb1-deficient DCs compared with that in wild-type (WT) mice, suggesting a potential pathway of CD8+Treg expansion. Moreover, we found that CD8+Tregs from mice with conditional deletion Lkb1 in DCs (KO) displayed an activated phenotype and expressed higher levels of specific markers, including ICOS and CD103. Interestingly, compared with the WT mice without lipopolysaccharide(LPS) treatment, we found that CD8+Tregs population increased in the WT mice with LPS treatment which can selectively delete Lkb1 protein in DCs. However, there was no significant difference in CD8+Tregs population in the KO mice between LPS treatment group and non-LPS treatment. Collectively, our findings identified Lkb1 in DCs as a crucial regulator of CD8+Treg expansion.

Exosomes from mesenchymal stromal cells enhance imatinib-induced apoptosis in human leukemia cells via activation of caspase signaling pathway.

BACKGROUND AIMS: Imatinib (IM), a tyrosine kinase inhibitor targeting the BCR-ABL oncoprotein, remains a major therapeutic strategy for patients with chronic myelogenous leukemia (CML). However, IM resistance is still a challenge in the treatment of CML. Recently, it was reported that exosomes (Exo) were involved in drug resistance. Therefore, the present study investigated whether Exo secreted by human umbilical cord mesenchymal stromal cells (hUC-MSC-Exo) affected the sensitivity of K562 cells to IM. METHODS: hUC-MSC-Exo were isolated and identified. K562 cells were then treated or not with IM (1 µmol/L) in combination with hUC-MSC-Exo (50 µg/mL). Cell viability and apoptosis were determined by cell counting kit 8 (CCK-8) and annexin V/propidium iodide (PI) double staining, respectively. Apoptotic proteins, caspase and their cleaved forms were detected by Western blot. RESULTS: It was shown that hUC-MSC-Exo alone had no effect on cell viability and apoptosis of K562 cells. However, hUC-MSC-Exo promoted IM-induced cell viability inhibition and apoptosis. Moreover, hUC-MSC-Exo enhanced the increased Bax expression and the decreased Bcl-2 expression that were induced by IM. Compared with IM alone, caspase-9 and caspase-3 were further activated by combination of hUC-MSC-Exo with IM. Finally, the effects of hUC-MSC-Exo on K562 cells could be reversed by pretreatment of K562 cells with caspase inhibitor Z-VAD-FMK (30 µmol/L) DISCUSSION: These results indicate that hUC-MSC-Exo enhanced the sensitivity of K562 cells to IM via activation of caspase signaling pathway. Therefore, combining IM with hUC-MSC-Exo could be a promising approach to improve the efficacy of CML treatment.

Molecular Imaging of Inducible VEGF Expression and Tumor Progression in a Breast Cancer Model.

BACKGROUND: Tumor derived vascular endothelial growth factor (VEGF) can stimulate proliferation and migration of endothelial cells and recruit endothelial progenitor cells into tumors for vascular formation via a paracrine manner. Now increasing evidence suggests that VEGF also serves as an autocrine factor promoting cell survival and tumor angiogenesis. Real time visualization of VEGF activity in the early stages of tumor formation using molecular imaging will provide unprecedented insight into the biological processes of cancer. METHODS: The mouse breast cancer cell line 4T1 was transfected with an inducible, bidirectional tetracycline (Bi-Tet) promoter driving VEGF and renilla luciferase (Rluc). This was used to quantitatively image conditional switching of VEGF by bioluminescence imaging (BLI) under the control of systemic administration of doxycycline. Simultaneously, 4T1 cells were labelled with the double fusion reporter gene (Fluc-eGFP) to establish a breast cancer model. RESULTS: We found that inducible VEGF could promote proliferation and attenuate apoptosis due to oxidative stress in an autocrine manner in vitro. In vivo studies revealed that induction of VEGF expression during early tumor development not only dramatically enhanced tumor growth but also increased tumor angiogenesis as visualized by BLI. Finally, immunohistochemistry staining confirmed that inducing VEGF expression promoted cell survival and tumor neovascularization. CONCLUSION: Together the inducible bidirectional tetracycline (Bi-Tet) co-expression system combined with the dual bioluminescence imaging (BLI) system provides a platform to investigate a target gene's role in the pathologic process of cancer and facilitates noninvasive monitoring of biological responses in real time.

[Human Umbilical Cord-derived Mesenchymal Stem Cells Secrete Interleukin-6 to Influence Differentiation of Leukemic Cells].

OBJECTIVE: To investigate the effect of human umbilical cord-derived mesenchymal stem cells (UC-MSC) on the differentiation of leukemic cells. METHODS: The co-culture system of UC-MSC with acute promyelocytic leukemic cell line NB4 cells was constructed in vitro,and the differentiation status of the leukemic cells was assessed by cell morphology,nitroblue tetrazolium reduction test,and cell surface differentiation marker CD11b. RESULTS: UC-MSC induced the granulocytic differentiation of NB4 cells. When UC-MSC and a small dose of all-trans retinoic acid were applied together,the differentiation-inducing effect was enhanced in an additive manner. Interleukin (IL)-6Ra neutralization attenuated differentiation and exogenous IL-6-induced differentiation of leukemic cells. CONCLUSION: UC-MSC can promotd granulocytic differentiation of acute promyelocytic leukemia cells by way of IL-6 and presented additive effect when combined with a small dose of all-trans retinoic acid.

Tsc1 is a Critical Regulator of Macrophage Survival and Function.

BACKGROUND/AIMS: Tuberous sclerosis complex 1 (Tsc1) has been shown to regulate M1/M2 polarization of macrophages, but the precise roles of Tsc1 in the function and stability of macrophages are not fully understood. Here we show that Tsc1 is required for regulating the survival, migration and phagocytosis of macrophages. METHODS: Mice with Tsc1 homozygous deletion in myeloid cells (LysMCreTsc1(flox/flox); Tsc1 KO) were obtained by crossing Tsc1(flox/flox) mice with mice expressing Cre recombinase under the control of Lysozyme promoter (LysMCre). The apoptosis and growth of macrophages were determined by flow cytometry and Real-time PCR (RT-PCR). The phagocytosis was determined using a Vybrant™ phagocytosis assay kit. The migration of macrophages was determined using transwell migration assay. RESULTS: Peritoneal macrophages of Tsc1 KO mice exhibited increased apoptosis and enlarged cell size. Both M1 and M2 phenotypes in Tsc1-deficient macrophages were elevated in steady-state as well as in inflammatory conditions. Tsc1-deficient macrophages demonstrated impaired migration and reduced expression of chemokine receptors including CCR2 and CCR5. Phagocytosis activity and ROS production were enhanced in Tsc1-deficient macrophages. Furthermore, pharmacological inhibition of the mammalian target of rapamycin complex 1 (mTORC1) partially reversed the aberrance of Tsc1-deficient macrophages. CONCLUSION: Tsc1 plays a critical role in regulating macrophage survival, function and polarization via inhibition of mTORC1 activity.

Inhibition of Notch Signaling Promotes the Adipogenic Differentiation of Mesenchymal Stem Cells Through Autophagy Activation and PTEN-PI3K/AKT/mTOR Pathway.

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.

Aberrant expression and significance of OCT-4A transcription factor in leukemia cells.

OBJECTIVE: To determine the contribution of the OCT-4 to the pathogenesis of leukemia. METHODS: Bone marrow (BM) samples obtained from 72 patients with leukemia, and 18 normal healthy subjects were assayed for their OCT-4 expression using both flow cytometry and RT-PCR. RESULTS: OCT-4 expression in BM nucleated cells of acute leukemia patients (n=33) was significantly higher than that of complete remission and chronic phase leukemia patients (n=39, p<0.001) and healthy donors (n=18, p<0.001). OCT-4 expression had a significant positive relation with CD34 expression (n=43, r=0.721, p<0.001) and the proportion of naive cells (n=60, r=0.687, p<0.001). In addition, the results of QRT-PCR detection showed that the OCT-4A had increased expression in BM nucleated cells in the patients with acute leukemia (n=33, median 16.585, range 0.38-169.62) compared to that in leukemia patients with chronic phase and in complete remission (n=39, median 3.34, range 0.04-44.49, p<0.001) and that of normal controls (n=18, median 2.89, range 0.18-16.23, p<0.001). CONCLUSION: OCT-4A expression was significantly increased in the BM nucleated cells of patients with acute leukemia, indicating that OCT-4A may play an important role in the pathogenesis of leukemia and may serve as a molecular target for the development of novel diagnostic and treatment strategies in leukemia.

Transfer of the IL-37b gene elicits anti-tumor responses in mice bearing 4T1 breast cancer.

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.

Down-regulation of lipocalin 2 suppresses the growth of human lung adenocarcinoma through oxidative stress involving Nrf2/HO-1 signaling.

Lipocalin 2 (LCN2), a multifunctional secretory protein known as neutrophil gelatinase-associated lipocalin (NGAL), is expressed in a variety of cancers. However, little is known about the biological functions of NGAL in the development of lung adenocarcinoma. In the present study, we primarily found that NGAL expression was up-regulated in human lung adenocarcinoma tissues. Additionally, depletion of NGAL expression decreased the ability of cell proliferation and induced cell apoptosis. Furthermore, with the addition of N-acetylcysteine, a scavenger of reactive oxygen species (ROS), it was found that NGAL depletion was sufficient to cause apoptosis of lung adenocarcinoma cells by generating ROS through the inhibition of the nuclear factor E2-related factor 2/heme oxygenase-1 anti-oxidant pathway. Finally, the effect of NGAL down-regulation on the growth of human lung adenocarcinoma was determined in BALB/c nude mice. These findings demonstrate that NGAL may be a potential therapy target for patients with lung adenocarcinoma.

IL-37b suppresses T cell priming by modulating dendritic cell maturation and cytokine production via dampening ERK/NF-κB/S6K signalings.

Interleukin 37b (IL-37b) plays a key role in suppressing immune responses, partially by modulating the function of dendritic cells (DCs). However, the precise mechanisms are still largely unknown. Here, we investigated the effects of IL-37b on DC maturation and T cell responses induced by DCs, and explored the involved signaling pathways. It was found that IL-37b down-regulated the expressions of co-stimulatory molecules CD80 and CD86 on DCs in vitro. At the same time, the expressions of pro-inflammatory cytokines, such as TNF-α and IL-6, were suppressed, while the expression of the T cell inhibitory cytokine TGF-ß was increased in IL-37b-treated DCs. In addition, the activation effect of DCs on T cells was impaired by IL-37b. We further revealed that extracellular single-regulated kinase (ERK), nuclear factor-κB (NF-κB), and mTOR-S6K signaling pathways were involved in the inhibition of DCs induced by IL-37b. This was confirmed by the similarly suppressive effect of chemical inhibitors against NF-κB, ERK, and S6K on the expressions of IL-6 and TNF-α in DCs. In conclusion, these results demonstrated that IL-37b suppressed DC maturation and immunostimulatory capacity in T cell priming by involving in ERK, NF-κB, and S6K-based inhibitory signaling pathways.

Programming of the development of tumor-promoting neutrophils by mesenchymal stromal cells.

BACKGROUND/AIMS: Neutrophils obtain immunosuppressive function during tumor development, yet the mechanisms are largely unknown. This study explored whether and how mesenchymal stromal cells (MSCs), the key component of tumor microenvironment, regulate the suppressive function of neutrophils. METHODS: Immunosuppressive function of neutrophils was evaluated by T cell proliferation assay and 4T1 breast tumor model; molecular mechanisms were explored by transcriptional profiling, Real-time RT-PCR, arginase activity assay, and iNOS inhibition experiments. RESULTS: After being cocultured with MSCs primed by TNF-α (TNF-MSCs), CD11b(+)Ly6G(+) neutrophils isolated from bone marrow of normal mice or spleen of tumor-bearing mice obtained immunosuppressive function to inhibit T cell proliferation in vitro, and to enhance 4T1 tumor progression in vivo. Moreover, arginase activity and expression of iNOS, saa3, some cytokines and chemokines and their receptors, were upregulated in neutrophils after co-culture with TNF-MSCs. Inhibition of iNOS activity attenuated the suppressive effect of TNF-MSC pre-cocultured neutrophils on T cell proliferation. CONCLUSION: MSCs program neutrophils into an immunosuppressive and tumor-promoting phenotype.

Decreased intracellular pH induced by cariporide differentially contributes to human umbilical cord-derived mesenchymal stem cells differentiation.

BACKGROUND/AIMS: Na+/H+ exchanger 1 (NHE1) is an important regulator of intracellular pH (pHi). High pHi is required for cell proliferation and differentiation. Our previous study has proven that the pHi of mesenchymal stem cells is higher than that of normal differentiated cells and similar to tumor cells. NHE1 is highly expressed in both mesenchymal stem cells and tumor cells. Targeted inhibition of NHE1 could induce differentiation of K562 leukemia cells. In the present paper we explored whether inhibition of NHE1 could induce differentiation of mesenchymal stem cells. METHODS: MSCs were obtained from human umbilical cord and both the surface phenotype and functional characteristics were analyzed. Selective NHE1 inhibitor cariporide was used to treat human umbilical cord-derived mesenchymal stem cells (hUC-MSCs). The pHi and the differentiation of hUC-MSCs were compared upon cariporide treatment. The putative signaling pathway involved was also explored. RESULTS: The pHi of hUC-MSCs was decreased upon cariporide treatment. Cariporide up-regulated the osteogenic differentiation of hUC-MSCs while the adipogenic differentiation was not affected. For osteogenic differentiation, ß-catenin expression was up-regulated upon cariporide treatment. CONCLUSION: Decreased pHi induced by cariporide differentially contributes to hUC-MSCs differentiation.

Biomimetic Supramolecular Assembly with IGF-1C Delivery Ameliorates Inflammatory Bowel Disease (IBD) by Restoring Intestinal Barrier Integrity.

The management of dysfunctional intestinal epithelium by promoting mucosal healing and modulating the gut microbiota represents a novel therapeutic strategy for inflammatory bowel disease (IBD). As a convenient and well-tolerated method of drug delivery, intrarectal administration may represent a viable alternative to oral administration for the treatment of IBD. Here, a biomimetic supramolecular assembly of hyaluronic acid (HA) and ß-cyclodextrin (HA-ß-CD) for the delivery of the C domain peptide of insulin-like growth factor-1 (IGF-1C), which gradually releases IGF-1C, is developed. It is identified that the supramolecular assembly of HA-ß-CD enhances the stability and prolongs the release of IGF-1C. Furthermore, this biomimetic supramolecular assembly potently inhibits the inflammatory response, thereby restoring intestinal barrier integrity. Following HA-ß-CD-IGF-1C administration, 16S rDNA sequencing reveals a significant increase in the abundance of the probiotic Akkermansia, suggesting enhanced intestinal microbiome homeostasis. In conclusion, the findings demonstrate the promise of the HA-based mimicking peptide delivery platform as a therapeutic approach for IBD. This biomimetic supramolecular assembly effectively ameliorates intestinal barrier function and intestinal microbiome homeostasis, suggesting its potential for treating IBD.