Showing papers on "Hepatocyte growth factor published in 2016"

Scatter factor induces blood vessel formation in vivo (hepatocyte growth factor/angiogenesis/endothelium/psoriasis/plasninogen activator)

Abstract: Scatter factor (also known as hepatocyte growth factor) is a glycoprotein secreted by stromal cells that stimulates cell motility and proliferation. In vitro, scatter factor stimulates vascular endothelial cell migration, proliferation, and organization into capillary-like tubes. Using two different in vivo assays, we showed that physiologic quantities of purified native mouse scatter factor and recombinant human hepato- cyte growth factor induce angiogenesis (the formation of new blood vessels). The angiogenic activity was blocked by specific anti-scatter factor antibodies. Scatter factor induced cultured microvascular endothelial cells to accumulate and secrete sig- nificantly increased quantities of urokinase, an enzyme asso- ciated with development of an invasive endothelial phenotype during angiogenesis. We further showed that immunoreactive scatter factor is present surrounding sites of blood vessel formation in psoriatic skin. These rmdings suggest that scatter factor may act as a paracrine mediator in pathologic angio- genesis associated with human inflammatory disease.

3D spheroid culture enhances survival and therapeutic capacities of MSCs injected into ischemic kidney.

Abstract: Three-dimensional (3D) cell culture has been reported to increase the therapeutic potentials of mesenchymal stem cells (MSCs). In this study, we aimed to investigate the therapeutic effects of 3D spheroids of human adipose-derived MSCs for acute kidney injury (AKI). In vitro studies indicated that 3D spheroids of MSCs produced higher levels of extracellular matrix proteins (including collagen I, fibronectin and laminin), and exhibited stronger anti-apoptotic and anti-oxidative capacities than two-dimensional (2D) cultured cells. Furthermore, 3D culture increased the paracrine secretion of cytokines by MSCs, including angiogenic factors (VEGF and basic fibroblast growth factor), anti-apoptotic factors (epidermal growth factor and hepatocyte growth factor), the anti-oxidative factor insulin-like growth factor and the anti-inflammatory protein tumour necrosis factor-alpha stimulated gene/protein 6. Consistent with in vitro experiments, 3D spheroids of MSCs showed enhanced survival and paracrine effects in vivo. More importantly, when injected into the kidney of model rats with ischemia-reperfusion (I/R)-induced AKI, 3D spheroids were more beneficial in protecting the I/R kidney against apoptosis, reducing tissue damage, promoting vascularization and ameliorating renal function compared with 2D cultured cells. Therefore, the 3D culture strategy improved the therapeutic effects of MSCs, and might be promising for AKI treatment.

Umbilical Cord-derived Mesenchymal Stem Cells Instruct Monocytes Towards an IL10-producing Phenotype by Secreting IL6 and HGF.

Abstract: Human UC-MSCs are regarded as an attractive alternative to BM-MSCs for clinical applications due to their easy preparation, higher proliferation and lower immunogenicity. However, the mechanisms underlying immune suppression by UC-MSCs are still unclear. We studied the mechanism of inhibition by UC-MSCs during the differentiation of monocytes into DCs and focused on the specific source and the role of the involved cytokines. We found that UC-MSCs suppressed monocyte differentiation into DCs and instructed monocytes towards other cell types, with clear decreases in the expression of co-stimulatory molecules, in the secretion of inflammatory factors and in allostimulatory capacity. IL6, HGF and IL10 might be involved in this process because they were detected at higher levels in a coculture system. UC-MSCs produce IL-6 and HGF, and neutralization of IL-6 and HGF reversed the suppressive effect of UC-MSCs. IL10 was not produced by UC-MSCs but was exclusively produced by monocytes after exposure to UC-MSCs, IL-6 or HGF. In summary, we found that the UC-MSC-mediated inhibitory effect was dependent on IL6 and HGF secreted by UC-MSCs and that this effect induced monocyte-derived cells to produce IL10, which might indirectly strengthen the suppressive effect of UC-MSCs.

First-in-human study and clinical case reports of the alveolar bone regeneration with the secretome from human mesenchymal stem cells

Abstract: Secreted growth factors and cytokines in the conditioned medium from bone marrow-derived mesenchymal stem cells (MSC-CM) have several effects on cell behavior. Our previous studies revealed that MSC-CM enhances bone regeneration by increasing cell mobilization, angiogenesis, and osteogenesis in vitro and in vivo. This clinical study was undertaken to evaluate the safety and use of MSC-CM for alveolar bone regeneration in eight patients who were diagnosed as needing bone augmentation prior to dental implant placement. The protocol of this clinical study was approved by the ethics committee of Nagoya University Hospital. MSC-CM was prepared from conditioned medium from commercially available human bone marrow-derived MSCs. Patients were treated with beta-tricalcuim phosphate (β-TCP) or an atelocollagen sponge soaked with MSC-CM. Clinical and radiographic assessments were performed during the follow-up period. Histological assessments were also performed in some cases. Clinical and histological data from patients who underwent the SFE procedure without MSC-CM were also used retrospectively as reference controls. MSC-CM contained several cytokines such as insulin-like growth factor-1, vascular endothelial growth factor, transforming growth factor-β1, and hepatocyte growth factor in relatively low amounts. No systemic or local complications were reported throughout the study. Radiographic evaluation revealed early bone formation in all cases. Histological evaluation also supported the radiographic findings. Furthermore, infiltration of inflammatory cells was scarce throughout the specimens. MSC-CM was used safely and with less inflammatory signs and appears to have great osteogenic potential for regenerative medicine of bone. This is the first in-human clinical study of alveolar bone regeneration using MSC-CM.

Elevated hepatocyte growth factor expression as an autocrine c‐Met activation mechanism in acquired resistance to sorafenib in hepatocellular carcinoma cells

Abstract: Hepatocellular carcinoma (HCC) is the most common type of primary liver cancer and the third leading cause of cancer-related deaths worldwide. Limitations in HCC treatment result due to poor prognosis and resistance against traditional radiotherapy and chemotherapies. The multikinase inhibitor sorafenib is the only FDA approved drug available for advanced HCC patients, and development of second-line treatment options for patients who cannot tolerate or develop resistance to sorafenib is an urgent medical need. In this study, we established sorafenib-resistant cells from Huh7 and Mahlavu cell lines by long-term sorafenib exposure. Sorafenib-resistant HCC cells acquired spindle-shape morphology, upregulated mesenchymal markers, and showed significant increase in both migration and invasion abilities compared to their parental counterparts. Moreover, after long-term sorafenib treatment, HCC cells showed induction of hepatocyte growth factor (HGF) synthesis and secretion along with increased levels of c-Met kinase and its active phosphorylated form, indicating autocrine activation of HGF/c-Met signaling. Importantly, the combined treatment of the resistant cells with c-Met kinase inhibitor SU11274 and HGF neutralizing antibody significantly reversed the increased invasion ability of the cells. The combined treatment also significantly augmented sorafenib-induced apoptosis, suggesting restoration of sorafenib sensitivity. These results describe, for the first time, compensatory upregulation of HGF synthesis leading to autocrine activation of HGF/c-Met signaling as a novel cellular strategy in the acquisition of sorafenib resistance. Therefore, we suggest that combinatorial therapeutic strategies with HGF and c-Met inhibitors comprise promising candidates for overcoming sorafenib resistance.

Hepatic Radiofrequency Ablation–induced Stimulation of Distant Tumor Growth Is Suppressed by c-Met Inhibition

Abstract: Radiofrequency ablation of normal liver, which simulates complete clinical ablation of a focal tumor by creating an ablative margin, can stimulate distant tumor growth in two c-Met–positive tumor lines (and not in a matched c-Met–negative cell line), is driven by a combination of periablational tissue reactions and systemic in-tumor effects that are in part mediated by hepatocyte growth factor/c-Met pathway and vascular endothelial growth factor (VEGF) and can be blocked with c-Met and VEGF receptor inhibitors given in a short window after ablation.

Comparative Analysis of Human Mesenchymal Stem Cells from Umbilical Cord, Dental Pulp, and Menstrual Blood as Sources for Cell Therapy

Abstract: Although mesenchymal stem cells (MSCs) based therapy has been considered as a promising tool for tissue repair and regeneration, the optimal cell source remains unknown. Umbilical cord (UC), dental pulp (DP), and menstrual blood (MB) are easily accessible sources, which make them attractive candidates for MSCs. The goal of this study was to compare the biological characteristics, including morphology, proliferation, antiapoptosis, multilineage differentiation capacity, and immunophenotype of UC-, DP-, and MB-MSCs in order to provide a theoretical basis for clinical selection and application of these cells. As a result, all UC-, DP-, and MB-MSCs have self-renewal capacity and multipotentiality. However, the UC-MSCs seemed to have higher cell proliferation ability, while DP-MSCs may have significant advantages for osteogenic differentiation, lower cell apoptosis, and senescence. These differences may be associated with the different expression level of cytokines, including vascular endothelial growth factor, fibroblast growth factor, keratinocyte growth factor, and hepatocyte growth factor in each of the MSCs. Comprehensively, our results suggest DP-MSCs may be a desired source for clinical applications of cell therapy.

Priming Dental Pulp Stem Cells With Fibroblast Growth Factor-2 Increases Angiogenesis of Implanted Tissue-Engineered Constructs Through Hepatocyte Growth Factor and Vascular Endothelial Growth Factor Secretion

Abstract: Tissue engineering strategies based on implanting cellularized biomaterials are promising therapeutic approaches for the reconstruction of large tissue defects. A major hurdle for the reliable establishment of such therapeutic approaches is the lack of rapid blood perfusion of the tissue construct to provide oxygen and nutrients. Numerous sources of mesenchymal stem cells (MSCs) displaying angiogenic potential have been characterized in the past years, including the adult dental pulp. Establishment of efficient strategies for improving angiogenesis in tissue constructs is nevertheless still an important challenge. Hypoxia was proposed as a priming treatment owing to its capacity to enhance the angiogenic potential of stem cells through vascular endothelial growth factor (VEGF) release. The present study aimed to characterize additional key factors regulating the angiogenic capacity of such MSCs, namely, dental pulp stem cells derived from deciduous teeth (SHED). We identified fibroblast growth factor-2 (FGF-2) as a potent inducer of the release of VEGF and hepatocyte growth factor (HGF) by SHED. We found that FGF-2 limited hypoxia-induced downregulation of HGF release. Using three-dimensional culture models of angiogenesis, we demonstrated that VEGF and HGF were both responsible for the high angiogenic potential of SHED through direct targeting of endothelial cells. In addition, FGF-2 treatment increased the fraction of Stro-1+/CD146+ progenitor cells. We then applied in vitro FGF-2 priming to SHED before encapsulation in hydrogels and in vivo subcutaneous implantation. Our results showed that FGF-2 priming is more efficient than hypoxia at increasing SHED-induced vascularization compared with nonprimed controls. Altogether, these data demonstrate that FGF-2 priming enhances the angiogenic potential of SHED through the secretion of both HGF and VEGF.

Oligonucleotide‑Based Mimetics of Hepatocyte Growth Factor

Abstract: Oligonucleotide-based hepatocyte growth factor (HGF) mimetics are described. A DNA aptamer to Met, a cognate receptor for HGF, was shown to induce Met activation when used in dimer form. The most potent aptamer dimer, ss-0, which was composed solely of 100-mer single-stranded DNA, exhibited nanomolar potency. Aptamer ss-0 reproduced HGF-induced cellular behaviors, including migration and proliferation. The present work sheds light on oligonucleotides as a novel chemical entity for the design of growth factor mimetics.

Hepatocyte growth factor inhibition: a novel therapeutic approach in pancreatic cancer

Abstract: Pancreatic stellate cells (PSCs, which produce the stroma of pancreatic cancer (PC)) interact with cancer cells to facilitate PC growth. A candidate growth factor pathway that may mediate this interaction is the HGF–c-MET pathway. Effects of HGF inhibition (using a neutralising antibody AMG102) alone or in combination with gemcitabine were assessed (i) in vivo using an orthotopic model of PC, and (ii) in vitro using cultured PC cells (AsPC-1) and human PSCs. We have shown that human PSCs (hPSCs) secrete HGF but do not express the receptor c-MET, which is present predominantly on cancer cells. HGF inhibition was as effective as standard chemotherapy in inhibiting local tumour growth but was significantly more effective than gemcitabine in reducing tumour angiogenesis and metastasis. HGF inhibition has resulted in reduced metastasis; however, interestingly this antimetastatic effect was lost when combined with gemcitabine. This suggests that gemcitabine treatment selects out a subpopulation of cancer cells with increased epithelial–mesenchymal transition (EMT) and stem-cell characteristics, as supported by our findings of increased expression of EMT and stem-cell markers in tumour sections from our animal model. In vitro studies showed that hPSC secretions induced proliferation and migration, but inhibited apoptosis, of cancer cells. These effects were countered by pretreatment of hPSC secretions with a HGF-neutralising antibody but not by gemcitabine, indicating a key role for HGF in PSC–PC interactions. Our studies suggest that targeted therapy to inhibit stromal–tumour interactions mediated by the HGF–c-MET pathway may represent a novel therapeutic approach in PC that will require careful modelling for optimal integration with existing treatment modalities.

Long-Duration Three-Dimensional Spheroid Culture Promotes Angiogenic Activities of Adipose-Derived Mesenchymal Stem Cells.

Abstract: Mesenchymal stem cells (MSCs) offer significant therapeutic promise for various regenerative therapies. However, MSC-based therapy for injury exhibits low efficacy due to the pathological environment in target tissues and the differences between in vitro and in vivo conditions. To address this issue, we developed adipose-derived MSC spheroids as a novel delivery method to preserve the stem cell microenvironment. MSC spheroids were generated by suspension culture for 3 days, and their sizes increased in a time-dependent manner. After re-attachment of MSC spheroids to the plastic dish, their adhesion capacity and morphology were not altered. MSC spheroids showed enhanced production of hypoxia-induced angiogenic cytokines such as vascular endothelial growth factor (VEGF), stromal cell derived factor (SDF), and hepatocyte growth factor (HGF). In addition, spheroid culture promoted the preservation of extracellular matrix (ECM) components, such as laminin and fibronectin, in a culture time- and spheroid size-dependent manner. Furthermore, phosphorylation of AKT, a cell survival signal, was significantly higher and the expression of pro-apoptotic molecules, poly (ADP ribose) polymerase-1 (PARP-1) and cleaved caspase-3, was markedly lower in the spheroids than in MSCs in monolayers. In the murine hindlimb ischemia model, transplanted MSC spheroids showed better proliferation than MSCs in monolayer. These findings suggest that MSC spheroids promote MSC bioactivities via secretion of angiogenic cytokines, preservation of ECM components, and regulation of apoptotic signals. Therefore, MSC spheroid-based cell therapy may serve as a simple and effective strategy for regenerative medicine.

Hepatocyte Growth Factor Is Required for Mesenchymal Stromal Cell Protection Against Bleomycin-Induced Pulmonary Fibrosis

Abstract: The incidence of idiopathic pulmonary fibrosis is on the rise and existing treatments have failed to halt or reverse disease progression. Mesenchymal stromal cells (MSCs) have potent cytoprotective effects, can promote tissue repair, and have demonstrated efficacy in a range of fibrotic lung dis- eases; however, the exact mechanisms of action remain to be elucidated. Chemical antagonists and short hairpin RNA knockdown were used to identify the mechanisms of action used by MSCs in pro- motingwound healing,proliferation,andinhibiting apoptosis. Usingthe bleomycininducedfibrosis model, the protective effects of early or late MSC administration were examined. The role for he- patocyte growth factor (HGF) in MSC protection against bleomycin lung injury was examined using HGF knockdown MSC. Terminal deoxynucleotidyl transferase (TdT) dUTP nick-end labeling assay was performed on ex vivo lung sections to examine the effects of MSC on apoptosis. MSC condi- tioned media (CM) enhanced wound closure and inhibited apoptosis of pulmonary cells in vitro. HGF was required for MSC CM enhancement of epithelial cell proliferation and inhibition of apo- ptosis. In contrast, MSC required COX-2 for CM to inhibit fibroblast proliferation. In a murine model, early administration of MSC protected against bleomycin induced lung fibrosis and correlated with reduced levels of the proinflammatory cytokine interleukin-1 b , reduced levels of apoptosis, and significantly increased levels of HGF. These protective effects were in part mediated by MSC derived HGFasHGFknockdown MSC were unable toprotect againstfibrosisinvivo.Thesefindingsdelineate the mechanisms of MSC protection in a preclinical model of fibrotic lung disease.

HGF/Met Signaling in Head and Neck Cancer: Impact on the Tumor Microenvironment.

Abstract: Studies to date have revealed several major molecular alterations that contribute to head and neck squamous cell carcinoma (HNSCC) initiation, progression, metastatic spread, and therapeutic failure. The EGFR is the only FDA-approved therapeutic target, yet responses to cetuximab have been limited. Activation and cross-talk of cellular receptors and consequent activation of different signaling pathways contribute to limited activity of blockade of a single pathway. The hepatocyte growth factor (HGF) receptor, Met, has been implicated in HNSCC tumorigenesis and EGFR inhibitor resistance. HGF, the sole ligand of Met, is overexpressed in the tumor microenvironment. The role of HGF/Met signaling in proliferation, metastasis, and angiogenesis has been investigated in HNSCC, leading to clinical trials with various Met inhibitors and HGF antibodies. However, the role of the HGF/Met signaling axis in mediating the tumor microenvironment has been relatively understudied in HNSCC. In this review, we discuss the functional roles of Met and HGF in HNSCC with a focus on the tumor microenvironment and the immune system. Clin Cancer Res; 22(16); 4005-13. ©2016 AACR.

The hepatocyte growth factor-expressing character is required for mesenchymal stem cells to protect the lung injured by lipopolysaccharide in vivo.

Abstract: Acute respiratory distress syndrome (ARDS) is a life-threatening condition in critically ill patients. Recently, we have found that mesenchymal stem cells (MSC) improved the permeability of human lung microvascular endothelial cells by secreting hepatocyte growth factor (HGF) in vitro. However, the properties and functions of MSC may change under complex circumstances in vivo. Here, we sought to determine the role of the HGF-expressing character of MSC in the therapeutic effects of MSC on ARDS in vivo. MSC with HGF gene knockdown (MSC-ShHGF) were constructed using lentiviral transduction. The HGF mRNA and protein levels in MSC-ShHGF were detected using quantitative real-time polymerase chain reaction and Western blotting analysis, respectively. HGF levels in the MSC culture medium were measured by enzyme-linked immunosorbent assay (ELISA). Rats with ARDS induced by lipopolysaccharide received MSC infusion via the tail vein. After 1, 6, and 24 h, rats were sacrificed. MSC retention in the lung was assessed by immunohistochemical assay. The lung wet weight to body weight ratio (LWW/BW) and Evans blue dye extravasation were obtained to reflect lung permeability. The VE-cadherin was detected with inmmunofluorescence, and the lung endothelial cell apoptosis was assessed by TUNEL assay. The severity of lung injury was evaluated using histopathology. The cytokines and HGF levels in the lung were measured by ELISA. MSC-ShHGF with markedly lower HGF expression were successfully constructed. Treatment with MSC or MSC carrying green fluorescent protein (MSC-GFP) maintained HGF expression at relatively high levels in the lung at 24 h. MSC or MSC-GFP decreased the LWW/BW and the Evans Blue Dye extravasation, protected adherens junction VE-cadherin, and reduced the lung endothelial cell apoptosis. Furthermore, MSC or MSC-GFP reduced the inflammation and alleviated lung injury based on histopathology. However, HGF gene knockdown significantly decreased the HGF levels without any changes in the MSC retention in the lung, and diminished the protective effects of MSC on the injured lung, indicating the therapeutic effects of MSC on ARDS were partly associated with the HGF-expressing character of MSC. MSC restores lung permeability and lung injury in part by maintaining HGF levels in the lung and the HGF-expressing character is required for MSC to protect the injured lung.

Alterations in acute myeloid leukaemia bone marrow stromal cell exosome content coincide with gains in tyrosine kinase inhibitor resistance.

Abstract: Bone marrow stromal cells (BMSC) in acute myeloid leukaemia (AML) contribute to extrinsic drug resistance, generally attributed to cell–cell contact or secreted cytokines (Jacamo et al, 2014). However, a recent report indicates that stromal protection may also occur via other soluble factors (Yang et al, 2014). Extracellular vesicles, such as exosomes, traffic protein and RNA between cells (Valadi et al, 2007). Stromal exosomes were recently shown to confer a proliferative advantage to multiple myeloma cells via transfer of microRNA (miRNA) (Roccaro et al, 2013). The functional impact of stromal exosomes in AML has not been studied. We therefore hypothesized that AML stroma release exosomes that protect leukaemia cells and traffic a distinct subset of miRNA and cytokines. We isolated stromal cells from the marrow aspirates of 20 patients (AML-BMSC; see Table SI for patient characteristics) and five healthy controls (N-BMSC) according to a previously published protocol (Tyner et al, 2013). The cells were fibroblastic, adherent and expressed CD90, but not CD45 epitopes (Fig 1A). Reverse transcription polymerase chain reaction (RT-PCR) confirmed the expression of canonical stromal transcripts in both populations (Fig 1B) (Boxall & Jones, 2012). However, quantitative analysis revealed altered expression in AML-BMSC for CXCL12, KITLG and CXCL1, as well as for genes previously reported in modified stroma in myelodysplastic syndrome (IGFBP4, ANGPTL4) (Fig 1C) (Medyouf et al, 2014). The contribution of stromal-derived exosomes has been established in other malignancies (Roccaro et al, 2013). We isolated vesicles using ultracentrifugation and then used vesicle tracking analysis, electron microscopy and Western blotting to demonstrate a vesicle population conforming in size, morphology and tetraspanin membrane proteins to exosomes (Fig 1D) (Valadi et al, 2007). Fig 1 AML-BMSCs have altered gene expression profiles and release exosomes that are enriched in select mi-RNA. (A) Light micrograph and immunophenotype of primary AML-BMSCs and N-BMSCs showing adherent, fibroblastic morphology and positivity for CD90 and negativity ... Exosome biogenesis allows for selective incorporation of different RNA species (Valadi et al, 2007). We determined the spectrum of BMSC exosome RNA using a Bioanalyser (Agilent, Santa Clara, CA, USA) and observed a relatively greater abundance of small RNAs compared with the parent cell (Fig 1D). Increased levels of MIR155 and MIR375 can independently identify AML patients at high risk for recurrence, with MIR155 contributing to the pathogenesis of several other haematological malignancies (Marcucci et al, 2013; Wang et al, 2013). Reasoning that stromal cell reprogramming in AML leads to unique exosomal miRNA incorporation, we used quantitative RT-PCR to compare the MIR155 and MIR375 levels in stromal cells from 12 patients (4 normal, 8 AML) and the exosomes released during the culture period. Strikingly, all eight AML-BMSC samples showed a statistically significant fold increase in incorporation of MIR155 and MIR375 in the exosomes relative to their parent cells, while no such increase was observed for N-BMSCs (Fig 1E). The comparative enrichment in AML-BMSC exosomes persisted even after 10 passages in tissue culture devoid of leukaemia cells, suggesting a durable change in stromal cell biology (Fig 1E). We then directly compared miRNA content between exosome isolates, independent of the parent cell content, and found MIR155, but not MIR375, was consistently elevated in AML-BMSC exosomes (Fig 1F). Taken together, the data indicate that exosomes released from AML-BMSCs are selectively enriched for miRNA that signify disease risk status in AML. Cytokines and growth factors contribute to leukaemia niche function (Yang et al, 2014). We reasoned that exosome- associated and directly secreted cytokine alterations from AML-BMSCs exist. In a multiplex screening approach using bead-based technology (Luminex, Austin, TX, USA) and enzyme-linked immuno-sorbent-assay (ELISA) kits (Life Technologies, Carlsbad, CA, USA), we studied the concentration of cytokines in the exosomes and in the vesicle-depleted soluble protein fraction of conditioned media from AML-BMSCs (n = 10) and N-BMSCs (n = 3). Exosomal cytokine concentrations ranged from 1·6 pg/ml (B-FGF) to >10 000 pg/ml [interleukin (IL) 8], while other cytokines were undetected in exosomes or in the soluble protein fraction from BMSCs (IL2, IL5, IL17A, TNF, IFNG). We found statistically significant enrichment of epidermal growth factor (P < 0·05), as well as a relative depletion of hepatocyte growth factor (P = 0·08) in exosomes from AML-BMSC, changes that were not observed in the soluble protein fraction when compared to N-BMSCs (Fig 2A). Concentrations of other cytokines, whether exosome-associated or freely secreted, did not differ between the two populations. We separately analysed TGFB1, given its correlation with treatment response in AML (Hong et al, 2014). We found TGFB1 at concentrations ranging from 200 to 2000 pg/ml in exosomes from 10/10 AML-BMSC samples, but below the level of assay detection in exosomes from N-BMSCs (Fig 2B). Taken together, our results suggest that stromal cells in AML modify their exosome-associated cytokine concentrations in a manner independent from the directly secreted fraction. Fig 2 Exosomes from AML-BMSCs have altered cytokine levels and confer chemo-protection to AML cells: (A) Box-and-Whisker plot of a multiplex protein assay displaying the relative concentration of 19 cytokines in exosomes and in the vesicle-depleted soluble ... Our findings, along with existing reports on general stromal protection in AML, led us to hypothesize that stromal exosomes alter chemo-resistance in AML cells. Representing a standard component of AML therapy, we treated MOLM-14 FLT3 internal tandem duplication (FLT3-ITD+) AML cells with the nucleoside analogue cytarabine after exposure to exosomes from AML-BMSC, N-BMSC or control media (Fig 2C). The data provide the first evidence, to our knowledge, that exosomes from both AML patients and controls (Total n = 10, 6 AML-BMSC, 4 N-BMSC) protect AML cells from cytarabine. Further, when AML cells were treated with the FLT3 inhibitor AC220 after exposure, only AML-BMSC exosomes significantly protected AML cells (n = 6), while N-BMSC exosomes provided no such protective effect (n = 3) (Fig 2C). Our data in aggregate suggest that stromal cells in AML patients undergo modification that includes alterations in function as well as the protein and RNA present in the exosomes they release. We report a first demonstration that stromal cells in AML release exosomes enriched for known clinical risk factors, including TGFB1, MIR155 and MIR375. Finally, our data add stromal exosome trafficking as a candidate mechanism for extrinsic chemo-resistance within the niche in AML, with differential protection against kinase pathway inhibition observed only by AML-BMSC exosomes. Such unique protection could occur, for example, by exosomal miRNA-directed down-regulation of promoters of apoptosis or cell differentiation, thereby releasing the leukaemia cell from kinase pathway dependence. Future studies in this area may uncover new mediators of such resistance.

Hypoxic Conditioned Medium From Human Adipose-Derived Stem Cells Promotes Mouse Liver Regeneration Through JAK/STAT3 Signaling

Abstract: UNLABELLED Adipose-derived stem cells (ASCs) mainly exert their function by secreting materials that are collectively termed the secretome. Despite recent attention to the secretome as an alternative to stem cell therapy, the culture conditions for generating optimal secretome contents have not been determined. Therefore, we investigated the role of hypoxic-conditioned media (HCM) from ASCs. Normoxic-conditioned media (NCM) and HCM were obtained after culturing ASCs in 20% O2 or 1% O2 for 24 hours, respectively. Subsequently, partially hepatectomized mice were infused with saline, control medium, NCM, or HCM, and then sera and liver specimens were obtained for analyses. Hypoxia (1% O2) significantly increased mRNA expression of mediators from ASCs, including interleukin-6 (IL-6), tumor necrosis factor α (TNF-α), hepatocyte growth factor (HGF), and vascular endothelial growth factor (VEGF). HCM infusion significantly increased the number of Ki67-positive cells in the liver (p < .05). HCM infusion significantly increased phospho-signal transducer and activator of transcription 3 (STAT3) and decreased suppressor of cytokine signaling 3 (SOCS3) expression in the liver (p < .05). To determine the role of IL-6 in liver regeneration, we then performed IL-6 RNA interference study. Conditioned media (CM) obtained from ASCs, which were transfected with either siIL-6 or siControl, were administered to partially hepatectomized mice. The siIL-6 CM groups exhibited lower liver proliferation (Ki67-positive cells) and markers of regeneration (protein expression of proliferating cell nuclear antigen, p-STAT3, HGF, and VEGF and liver weights) than the siControl CM groups (p < .05). Taken together, hypoxic preconditioning of ASCs increased expression of mediators promoting anti-inflammatory and regenerative responses. The liver regenerative effects of HCM appear to be mediated by persistent and uninhibited expression of STAT3 in the liver, which results from decreased expression of SOCS3. SIGNIFICANCE In this study, it was found that treatment with the medium from hypoxic-preconditioned adipose-derived stem cells (ASCs) increased the viability of hepatotoxic hepatocytes and enhance liver regeneration in partially hepatectomized mice. In addition, the researchers first revealed that the hepatoprotective effects of hypoxic-conditioned media are mediated by persistent and uninhibited expression of signal transducer and activator of transcription 3 in the liver, which result from a decreased expression of suppressor of cytokine signaling 3. Therefore, the hypoxic preconditioning of ASCs is expected to play a crucial role in regenerative medicine by optimizing the production of a highly effective secretome from ASCs.

PDGFRα plays a crucial role in connective tissue remodeling.

Abstract: Platelet derived growth factor (PDGF) plays a pivotal role in the remodeling of connective tissues. Emerging data indicate the distinctive role of PDGF receptor-α (PDGFRα) in this process. In the present study, the Pdgfra gene was systemically inactivated in adult mouse (α-KO mouse), and the role of PDGFRα was examined in the subcutaneously implanted sponge matrices. PDGFRα expressed in the fibroblasts of Pdgfra-preserving control mice (Flox mice), was significantly reduced in the sponges in α-KO mice. Neovascularized areas were largely suppressed in the α-KO mice than in the Flox mice, whereas the other parameters related to the blood vessels and endothelial cells were similar. The deposition of collagen and fibronectin and the expression of collagen 1a1 and 3a1 genes were significantly reduced in α-KO mice. There was a significantly decrease in the number and dividing fibroblasts in the α-KO mice, and those of macrophages were similar between the two genotypes. Hepatocyte growth factor (Hgf) gene expression was suppressed in Pdgfra-inactivated fibroblasts and connective tissue. The findings implicate the role of PDGFRα-dependent ECM and HGF production in fibroblasts that promotes the remodeling of connective tissue and suggest that PDGFRα may be a relevant target to regulate connective tissue remodeling.

Human mesenchymal stromal cells exert HGF dependent cytoprotective effects in a human relevant pre-clinical model of COPD.

Abstract: Bone-marrow derived mesenchymal stromal cells (MSCs) have potent immunomodulatory and tissue reparative properties, which may be beneficial in the treatment of inflammatory diseases such as COPD. This study examined the mechanisms by which human MSCs protect against elastase induced emphysema. Using a novel human relevant pre-clinical model of emphysema the efficacy of human MSC therapy and optimal cell dose were investigated. Protective effects were examined in the lung through histological examination. Further in vivo experiments examined the reparative abilities of MSCs after tissue damage was established and the role played by soluble factors secreted by MSCs. The mechanism of MSC action was determined in using shRNA gene knockdown. Human MSC therapy and MSC conditioned media exerted significant cytoprotective effects when administered early at the onset of the disease. These protective effects were due to significant anti-inflammatory, anti-fibrotic and anti-apoptotic mechanisms, mediated in part through MSC production of hepatocyte growth factor (HGF). When MSC administration was delayed, significant protection of the lung architecture was observed but this was less extensive. MSC cell therapy was more effective than MSC conditioned medium in this emphysema model.

Multifaceted Therapeutic Benefits of Factors Derived From Dental Pulp Stem Cells for Mouse Liver Fibrosis

Abstract: Chronic liver injury from various causes often results in liver fibrosis (LF). Although the liver possesses endogenous tissue-repairing activities, these can be overcome by sustained inflammation and excessive fibrotic scar formation. Advanced LF leads to irreversible cirrhosis and subsequent liver failure and/or hepatic cancer. Here, using the mouse carbon tetrachloride (CCl4)-induced LF model, we showed that a single intravenous administration of stem cells derived from human exfoliated deciduous teeth (SHEDs) or of SHED-derived serum-free conditioned medium (SHED-CM) resulted in fibrotic scar resolution. SHED-CM suppressed the gene expression of proinflammatory mediators, such as TNF-α, IL-1β, and iNOS, and eliminated activated hepatic stellate cells by inducing their apoptosis, but protected parenchymal hepatocytes from undergoing apoptosis. In addition, SHED-CM induced tissue-repairing macrophages that expressed high levels of the profibrinolytic factor, matrix metalloproteinase 13. Furthermore, SHED-CM suppressed the CCl4-induced apoptosis of primary cultured hepatocytes. SHED-CM contained a high level of hepatocyte growth factor (HGF). Notably, HGF-depleted SHED-CM (dHGF-CM) did not suppress the proinflammatory response or resolve fibrotic scarring. Furthermore, SHED-CM, but not dHGF-CM, inhibited CCl4-induced hepatocyte apoptosis. These results suggest that HGF plays a central role in the SHED-CM-mediated resolution of LF. Taken together, our findings suggest that SHED-CM provides multifaceted therapeutic benefits for the treatment of LF. Significance This study demonstrated that a single intravenous administration of stem cells from human exfoliated deciduous teeth (SHEDs) or of the serum-free conditioned medium (CM) derived from SHEDs markedly improved mouse liver fibrosis (LF). SHED-CM suppressed chronic inflammation, eliminated activated hepatic stellate cells by inducing their apoptosis, protected hepatocytes from undergoing apoptosis, and induced differentiation of tissue-repairing macrophages expressing high levels of the profibrinolytic factor matrix metalloproteinase 13. Furthermore, hepatocyte growth factor played a central role in the SHED-CM-mediated resolution of LF. This is the first report demonstrating the multifaceted therapeutic benefits of secreted factors derived from SHEDs for LF.