Mesenchymal Stem Cells Supernatant Research Articles

Annexin A1 Is a Physiological Modulator of Neutrophil Maturation and Recirculation Acting on the CXCR4/CXCL12 Pathway.

Neutrophil production and traffic in the body compartments is finely controlled, and the strong evidences support the role of CXCL12/CXCR4 pathway on neutrophil trafficking to and from the bone marrow (BM). We recently showed that the glucocorticoid-regulated protein, Annexin A1 (AnxA1) modulates neutrophil homeostasis and here we address the effects of AnxA1 on steady-state neutrophil maturation and trafficking. For this purpose, AnxA1(-/-) and Balb/C wild-type mice (WT) were donors of BM granulocytes and mesenchymal stem cells and blood neutrophils. In vivo treatments with the pharmacological AnxA1 mimetic peptide (Ac2-26) or the formyl peptide receptor (FPR) antagonist (Boc-2) were used to elucidate the pathway of AnxA1 action, and with the cytosolic glucocorticoid antagonist receptor RU 38486. Accelerated maturation of BM granulocytes was detected in AnxA1(-/-) and Boc2-treated WT mice, and was reversed by treatment with Ac2-26 in AnxA1(-/-) mice. AnxA1 and FPR2 were constitutively expressed in bone marrow granulocytes, and their expressions were reduced by treatment with RU38486. Higher numbers of CXCR4(+) neutrophils were detected in the circulation of AnxA1(-/-) or Boc2-treated WT mice, and values were rescued in Ac2-26-treated AnxA1(-/-) mice. Although circulating neutrophils of AnxA1(-/-) animals were CXCR4(+) , they presented reduced CXCL12-induced chemotaxis. Moreover, levels of CXCL12 were reduced in the bone marrow perfusate and in the mesenchymal stem cell supernatant from AnxA1(-/-) mice, and in vivo and in vitro CXCL12 expression was re-established after Ac2-26 treatment. Collectively, these data highlight AnxA1 as a novel determinant of neutrophil maturation and the mechanisms behind blood neutrophil homing to BM via the CXCL12/CXCR4 pathway. J. Cell. Physiol. 231: 2418-2427, 2016. © 2016 Wiley Periodicals, Inc.

Effects of bone marrow mesenchymal stem cells supernatant on hippocampal GFAP expression and learning and memory in rats with neonatal hypoxic ischemic brain damage

Objective To analyze the expression of glial fibrillary acidic protein(GFAP) in hippocampal of rats with hypoxic-ischemic brain damage(HIBD) after injection of bone marrow stromal cells(BMSCs) supernatant into the lateral ventricle of the neonate rats, assess the ability of learning and memory, and explore the mechanism. Methods 7-day-old SD rats were randomly divided into sham operation group, model group, cells and supernatant group, with 20 rats in each group. The HIBD model was established via the ligation of left carotid arteries followed by 2-hour hypoxia. One week later, rats in control group were injected with 0.01 mol/L PBS 2 μl via the left ventricle. Meanwhile, the cell and the supernatant groups were injected with BMSCs and supernatant harvested from BMSC culture, respectively, via the same route.In the sham operation group, the left carotid arteries were separated but not ligated, and no hypoxia treatment was imposed on this group. They also received 0.01 mol/L PBS injection 2 μl per weak post surgery. 8 weeks later, Morris water maze test was performed to assess the learning and memory, and the expression of GFAP in the dentate gyrus of rats was measured by immunohistochemistry. Results Morris water maze showed that the searching time of the probe trail(T1: (15.40±2.80) s) and reversal probe trail(T2: (16.45±1.16) s )of the model group was shorter than that of the sham operation group(T1: (19.96±2.57)s, T2: (2: 25.32±2.54)s, P 0.05). The integral optical density(IOD) value of GFAP positive cells was higher in the model group than that in the sham operation group(15.26±1.49 vs 12.82±2.56, P 0.05). Conclusion BMSC supernatant injected through lateral ventricle can improve the HIBD rats' abilities of learning and memory. The mechanism might be that MSCs secrete some cytokines to promote central nervous system repair. Key words: Bone marrow mesenchymal stem cells; Supernatant; Hypoxic ischemic brain damage; Glial fibrillary acidic protein; Memory

Micro-vesicles derived from bone marrow stem cells protect the kidney both in vivo and in vitro by microRNA-dependent repairing.

Micro-vesicles (MVs) from bone mesenchymal stem cells (MSCs) have been shown to contribute to the recovery of damaged kidney. The aims of the present study are to investigate the biological effects and repair mechanisms of MVs. Micro-vesicles were obtained from MSC supernatants. In vitro, the proximal tubular epithelial cells (HK-2) were treated with transforming growth factor (TGF-β1). The expressions of E-cadherin and α-smooth muscle actin (α-SMA) were evaluated. In vitro, the mice were divided into: control, unilateral ureteral obstruction (UUO), UUO+MSC, and UUO+MV group. MVs and MSCs were injected after surgery. The mice were killed 7/14 days after surgery and handled for further tests. The micro-RNA expressions were labeled using the miRCURY Hy3/Hy5 Power labeling kit and hybridized on the miRCURY LNA Array. In vitro, MV reversed transforming growth factor-β1 (TGF-β1)-induced morphological changes, and firmed the expression of E-cadherin and reduced the secretion of α-SMA in HK2 cells. In vivo, the level of blood urea nitrogen (BUN) in the MV and MSC group was lower than the UUO (P < 0.01). The Scr level decreased after 7 days of MV treatment (P < 0.05). Administration of MSC and MV reduced Scr level at day 14 (P < 0.05). The level of serum UA decreased with MV administration (day 7,14, P < 0.01). Herein, a total of 503 expressed miRNAs were detected, of which, 266 were in MSC, including 237 in MVs. Micro-vesicles (MVs) protect kidneys both in vivo and vitro, and MVs are superior to MSCs in some respects. MVs can be a potential therapy in treatment of kidney diseases.

The Effect of Granulocyte Colony–Stimulating Factor on Immune-Modulatory Cytokines in the Bone Marrow Microenvironment and Mesenchymal Stem Cells of Healthy Donors

Granulocyte colony stimulating factor (G-CSF) is sometimes administered to donors before bone marrow (BM) harvest. G-CSF-primed (G-BM) and unprimed BM (U-BM)-derived mesenchymal stem cells (MSC) were obtained from 16 healthy donors and were expanded invitro. Their proliferative characteristics, morphology, and differentiation capacity were examined. Supernatants of the second passage of MSCs were evaluated for transforming growth factor β1, hepatocyte growth factor, and prostaglandin E2 (PGE2) levels and compared with controls. The analyses of cytokines in the G-BM- and U-BM-derived MSCs supernatants revealed that PGE2 levels were significantly lower in the G-CSF-primed samples. These cytokines were also measured in BM plasma. The level of hepatocyte growth factor in G-BM plasma was significantly increased. The current study is the first to show the effects of G-CSF on the BM microenvironment of healthy human donors. The preliminary data suggest that G-BM- and U-BM-derived MSCs have similar morphologic/phenotypic properties and differentiation capacity but differ in their secretory capacity. Significant changes in cytokine levels of BM plasma in G-CSF-primed donors were also demonstrated. These findings suggest that BM MSCs and changes in the BM microenvironment may contribute to the effects of G-CSF on inflammation and immunomodulation.

Proangiogenic compositions of microvesicles derived from human umbilical cord mesenchymal stem cells.

Introduction & ObjectiveMicrovesicles (MVs) derived from mesenchymal stem cells (MSCs) have been shown to promote angiogenesis. This study was aimed to shed a light on the mechanisms by analyzing the angiogenesis-promoting compositions of MSC-MVs. Also we try to figure out the impact of hypoxia on angiogenesis.MethodsMVs were isolated from the culture supernatants of MSCs under hypoxia/normoxia and serum-deprivation condition. The morphological features of MVs were revealed by an electron microscope and the origin of the MVs was identified by a bead-bound assay. An antibody array was used to analyze the expression of angiogenic cytokines from MVs and the parent MSCs as well. The major candidate factors were screened and the results were validated by immune blotting.ResultsMSC-MVs were around 80 nm in diameter. They expressed CD29, CD44, and CD73, but not CD31 and CD45. Antibody array showed that both MSCs and MVs expressed many angiogenesis-promoting biomolecules, including interleukin-6 (IL-6), basic fibroblast growth factors (bFGF), and recptor of urokinase-type plasminogen activator (UPAR). MSC-MVs contained angiogenin, vascular endothelial growth factor (VEGF), monocyte chemotactic protein-1 (MCP-1) and the receptor-2 for vascular endothelial growth factor at higher levels than the parent MSCs. Under hypoxic condition most cytokines were expressed in greater quantity than normoxic in MSCs while in MVs there was no significant difference between hypoxic and normoxic conditions except UPAR, Angiogenin, VEGF, IGF, Tie-2/TEK, and IL-6 which were higher in MVs under hypoxic conditions than those in normoxic condition.ConclusionUpon serum-deprivation condition, MSCs could secrete MVs that contain a variety of factors contributing to their angiogenesis-promoting function. And among them, Angiogenin, VEGF, MCP-1, VEGF R2 might be of greater importance than the other cytokines. Also UPAR, Angiogenin, VEGF, IGF, Tie-2/TEK, IL-6 might be responsible for hypoxia-augmented proangiogenic effects of MVs.

Immunomodulatory effects of mice mesenchymal stem cells on maturation and activation of dendritic cells.

Mesenchymal stem cells (MSCs) possess a wide range of immunomodulatory functions mostly in immune cells including dendritic cells (DCs). DCs are the key cells in the immune response and play an important role in initiating cell-mediated immunity. To evaluate the immunomodulatory effects of MSCs supernatant on maturation and function of DCs. Bone marrow derived mice MSCs were isolated and cultured. Twenty-four, forty-eight and seventy-two hours after passage 6, supernatants were collected and MSCs were assessed by flow cytometric analysis for the expression of CD34, CD44, CD45 and SCA-1. Splenic DCs were isolated using MACS and then co-cultured with MSCs supernatant. Expression of CD86, CD40 and MHC-II on DCs were also evaluated by flow cytometry. H3-thymidine incorporation by proliferating T cells was determined in two separate MLR assay settings. In one setting, DCs were co-cultured with T cells in the presence of MSCs supernatant, and in the other setting DCs were treated with MSCs supernatant and then were co-cultured with T cells. Production of IL-12, IL-6 and IL-10 cytokines was measured in the supernatant of DCs treated with MSCs supernatant. We also measured IFN-γ and IL-4 levels in MLR supernatant. The results showed that 72h MSCs supernatant could decrease the expression of MHC-II and CD86. The T cell proliferation was inhibited in the presence of MSCs supernatant and MSCs supernatant treated DCs as demonstrated by MLR assay. A significant increase in IL-4 level and a non significant decrease in IFN-γ level in MLR supernatant was observed. However, IL-6, IL-10 and IL-12 production did not change significantly. MSCs supernatant has a time dependent effect on the maturation of DCs. Also, it could alter cytokine production from responding T cells toward Th2. Generally, the findings of this study supported the immunomodulatory effect of MSCs supernatant on DCs maturation and function.

A comparative study of mesenchymal stem cell transplantation with its paracrine effect on control of hyperglycemia in type 1 diabetic rats.

BackgroundMany studies suggested mesenchymal stem cells (MSCs) transplantation as a new approach to control hyperglycemia in type 1 diabetes mellitus through differentiation mechanism. In contrary others believed that therapeutic properties of MSCs is depends on paracrine mechanisms even if they were not engrafted. This study aimed to compare these two approaches in control of hyperglycemia in STZ-induced diabetic rats.MethodsAnimals were divided into five groups: normal; diabetic control; diabetic received MSCs; diabetic received supernatant of MSCs; diabetic received co-administration of MSCs with supernatant. Blood glucose, insulin levels and body weight of animals were monitored during experiment. Immunohistochemical and immunofluorescence analysis were performed to monitor functionality and migration of labeled-MSCs to pancreas.ResultsFirst administration of MSCs within the first 3 weeks could not reduce blood glucose, but second administration significantly reduced blood glucose after week four compared to diabetic controls. Daily injection of supernatant could not reduce blood glucose as efficient as MSCs. Interestingly; Co-administration of MSCs with supernatant significantly reduced blood glucose more than other treated groups. Insulin levels and body weight were significantly increased in MSCs + supernatant-treated animals compared to other groups. Immunohistological analysis showed an increase in number and size of islets per section respectively in supernatant, MSCs and MSCs + supernatant-treated groups.ConclusionPresent study exhibited that repeated-injection of MSCs reduced blood glucose and increased serum insulin levels in recipient rats. Injection of supernatant could not reverse hyperglycemia as efficient as MSCs. Interestingly; co-administration of MSCs with supernatant could reverse hyperglycemia more than either group alone.

Tolerogenic and Regenerative Effects of Extracellular Vesicles Released From Bone Marrow-Derived Mesenchymal Stem Cells in Kidney Transplantation.

Mesenchymal stem cells (MSC) exert immunomodulatory and regenerative effects by releasing paracrine mediators including extracellular vesicles (EV), nanoparticles involved in cell-to-cell communication through transfer of proteins, lipids, mRNA and microRNA. The aim of the study was the evaluation of MSC-derived EV tolerogenic and regenerative effects in kidney transplantation (KT), including T regulatory cell (Treg) generation and inhibition of kidney tubular epithelial cell (TEC) apoptosis observed in T cell-mediated rejection (TCMR). EV were characterized for size, protein and RNA content after MSC supernatant ultracentrifugation. The effects of EV were studied on: 1) T cells isolated from peripheral blood of KT recipients and co-cultured with B cells purified by spleen of matched donors; 2) human TEC cultured in an inflammatory microenvironment (perforin, granzyme B, Fas-Ligand, TNF-alpha, IFN-gamma) typical of TCMR. As detected by Nanosight, MSC-derived EVs sized 60-150 nm and expressed on their surface (FACS) molecules of the integrin family essential for their internalization into target cells. EV carried different microRNA and mRNA including the immunoregulatory Foxp3, Tim-1 and thrombospondin-1 similarly to cells from which they originated. EV were internalized in T cells inhibiting proliferation induced by PHA/ionomycin or by co-culture with matched-donor spleen-derived B cells. EVs horizontally transferred to T cells Foxp3 mRNA inducing a Treg phenotype. Tregs generated by EV stimulation abated T cell proliferation. MSC-EV potentially induced a regulatory phenotype also in B cells through increased expression of Tim-1. EV were internalized in TEC inhibiting functional alterations and apoptosis induced by the TCMR-like microenvironment. In particular, EV preserved the expression of molecules of fully differentiated TEC (megalin, ZO-1, AQP-2, solute carriers). All these effects were decreased after treatment with RNase, the enzyme able to degrade EV-carried RNA. MSC-derived EVs exert tolerogenic and regenerative effects in KT by inhibiting T cell proliferation, by inducing T and B cell regulatory phenotypes and by protecting TEC from immune-mediated injury. These actions are related to transfer of specific RNA from EV to target cells.

AB0131 Abnormality of Mesenchymal Stem Cells in Patients with Rheumatoid Arthritis

BackgroundIt has been shown that human umbilical cord mesenchymal stem cells (MSCs) could provide some benefits for the treatment of rheumatoid arthritis (RA) patients with refractory disease.ObjectivesThe aim of this...

Calcitriol modulates the effects of the supernatants of bone-marrow–derived mesenchymal stem cells on neutrophil functions

Mesenchymal stem cells (MSCs) in bone marrow form a niche that has inevitable interactions with neutrophils. Moreover, previous documents have shown that calcitriol has an important role in regulating the cell growth of MSCs. This study set out to investigate the effects of calcitriol on the interaction between bone-marrow-derived MSCs and neutrophils. MSCs were isolated from the bone marrow of rats and pulsed with different concentrations of calcitriol (50, 100, and 200 nM) for different periods of time (24, 48, and 72 h). As the next step, the supernatants of MSCs cocultured with neutrophils for 4 h and neutrophil functions were evaluated. The results showed that the supernatants of MSCs treated with calcitriol could significantly increase the phagocytosis of Staphylococcus aureus by neutrophils and, conversely, decrease the respiratory burst intensity of neutrophils. Moreover, treatment of MSCs with calcitriol can cause a significant decrease in the percentage of neutrophil apoptosis. These findings were concurrent with a significant increase in IL-6 levels in the supernatant of calcitriol-treated MSCs. Consequently, the supernatant of bone-marrow-derived MSCs was pulsed with calcitriol, and the exertion of a protective role against potentially harmful reactive oxygen species production preserved phagocytosis and the survival rate of neutrophils.

Stem cell therapy for the treatment of parasitic infections: is it far away?

Stem cell therapy is an interventional treatment that introduces new cells into damaged tissues, which help in treating many diseases and injuries. It has been proved that stem cell therapy is effective for the treatment of cancers, diabetes mellitus, Parkinson's disease, Huntington's disease, cardiovascular diseases, neurological disorders, and many other diseases. Recently, stem cell therapy has been introduced to treat parasitic infections. The culture supernatant of mesenchymal stem cells (MSCs) is found to inhibit activation and proliferation of macrophages induced by the soluble egg antigen of Schistosoma japonicum, and MSC treatment relieves S. japonicum-induced liver injury and fibrosis in mouse models. In addition, transplantation of MSCs into naïve mice is able to confer host resistance against malaria, and MSCs are reported to play an important role in host protective immune responses against malaria by modulating regulatory T cells. In mouse models of Chagas disease, bone marrow mononuclear cell has been shown effective in reducing inflammation and fibrosis in mice infected with Trypanosoma cruzi, and transplantation of the bone marrow mononuclear cells prevents and reverses the right ventricular dilatation induced by T. cruzi infection in mice. Preliminary clinical trials demonstrate that transplantation of bone marrow derived-cells may become an important therapeutic modality in the management of end-stage heart diseases associated with Chagas disease. Based on these exciting results, it is considered by stating that it is firmly believed that, within the next few years, we will be able to find the best animal models and the appropriate stem cell type, stem cell number, injection route, and disease state that will result in possible benefits for the patients with parasitic infections, and stem cell therapy, although at an initial stage currently, will become a real therapeutic option for parasitic diseases.

Abnormalities in cytokine secretion from mesenchymal stem cells in psoriatic skin lesions

Psoriasis is a chronic inflammatory and proliferative skin disease associated with immune abnormalities. In our preliminary studies, it was found that bone marrow mesenchymal stem cells (BMSCs) were abnormal in patients with psoriasis. Mesenchymal stem cells (MSCs) are not only present in the bone marrow, but can also be separated from the skin.We reasoned that an investigation into the biological characteristics of MSCs in psoriatic skin lesions could more realistically reflect the actual conditions for the pathogenesis of psoriasis. To reveal whether MSCs in psoriatic skin lesions are abnormal. We obtained MSCs from psoriatic skin lesions and healthy human skin and identified these cells using flow cytometry and a cell differentiation assay; cytokine concentrations in the culture supernatants were measured with the ELISA assay. We compared cytokine concentrations in culture supernatants of MSCs derived from psoriatic skin lesions as well as those from healthy human skin. Compared with healthy control skintissue, in psoriatic skin lesions, MSC secretion of EGF, SCF, and IL-11 increased; secretion of bFGF, IL-3, IL-6, IL-8, and HGF decreased (p<0.05); and secretion of VEGF, M-CSF, G-CSF, GM-CSF, LIF, IL-1, IL-7, IL-10, and TNF-α showed no significant difference (p>0.05). Surface markers and the differentiation capacity of cells from the two sources were similar. This study demonstrated that MSCs derived from psoriatic skin lesions had abnormalities, which may be one of the pathogenic mechanisms of psoriasis.

Alterations of connexin 43 expression and SDF-1α secretion of bone marrow mesenchymal stem cells co-cultured with myeloma cells

To construct a co- culture system of mesenchymal stem cells (MSC) and multiple myeloma (MM) cells and investigate the alterations of connexin 43 (CX43) expression and stromal derived growth factor (SDF)- 1α secretion of MSC. CX43 expression and SDF- 1α secretion of MM cell lines (RPMI8226) and human primary MM cells were analyzed by western blot and immunofluorescence. Western blot, RT- PCR and immunofluorescence were employed to detect the alterations of CX43 expression and distribution in MSC directly and indirectly co-cultured with myeloma cells. Lucifer yellow dye spread was utilized to evaluate gap junctional intercellular communication (GJIC) between co- cultured MSC. Transwell was applied to study the transmigration of RPMI8266 induced by MSC under the condition of 18α- glycyrrhetinic acid (18α-GA). The level of SDF- 1α was detected by EILSA. RPMI8266, U266 and one-third primary MM cells expressed CX43 at low or moderate levels. CX43 wasn't expressed in XG- 4 and XG- 7 cells but highly expressed in MSC. The expressions of CX43 mRNA of MSC were up- regulated after directly and indirectly co- cultured with RPMI8226, 1.36 and 2.10 times that of MSC cultured alone respectively. Western blot analysis showed that CX43 protein expression of MSC was also up-regulated, mainly distributed in cytoplasm. Lucifer yellow dye spread showed that GJIC was up-regulated in MSC. SDF-1α concentration in supernatant of MSC directly and indirectly co-cultured with RPMI8226 were (373.02±10.11)pg/ml and (309.71±10.71)pg/ml respectively, which were higher than that of MSC cultured alone (237.84±9.23)pg/ml (P<0.01), and could be inhibited by 18α-GA [(237.84±9.23)pg/ml and (94.31±6.44)pg/ml] respectively (P<0.01). 18α-GA could inhibit the transmigration of RPMI8226 induced by MSC, decrease from (8.00±0.67)% to (4.82±0.19)%. CX43 expression of MSC was up-regulated after directly and indirectly co-cultured with MM cells, which could improve the level of SDF-1α secretion of MSC. GJ inhibitor could downregulate SDF-1α secretion of MSC and inhibit the transmigration of MM cells induced by MSC.

Mesenchymal stem cells contribute to endogenous FVIII:c production

Besides the liver, it has been difficult to identify which organ(s) and/or cellular component(s) contribute significantly to the production of human FVIII:c (FVIII). Thus far, only endothelial cells have been shown to constitute a robust extrahepatic source of FVIII, possibly explaining both the diverse presence of FVIII mRNA in the body, and the observed increase in FVIII levels during liver failure. Here, we investigate whether human mesenchymal stem cells (MSC), ubiquitously present in different organs, could also contribute to FVIII production. MSC isolated from human lung, liver, brain, and bone marrow expressed FVIII message as determined by quantitative-RT-PCR. Using an antibody specific for FVIII, confocal microscopy, and umbilical cord-derived endothelial cells (HUVEC) as a negative control, we demonstrated that, in MSC, FVIII protein was not stored in granules; rather, it localized to the perinuclear region. Furthermore, functional FVIII was detected in MSC supernatants and cell lysates by aPTT and chromogenic assays. These results demonstrate that MSC can contribute at low levels to the functional FVIII pool, and advance the understanding of the physiology of FVIII production and secretion.

Complement C3a signaling mediates production of angiogenic factors in mesenchymal stem cells

A major portion of the beneficial effect of mesenchymal stem cells (MSC) is due to the production of trophic and angiogenic factors by these cells, and one of the efforts to improve the therapeutic efficacy of these cells lies in enhancing this capacity. Since there is complement activation in all areas of tissue injury, and both C3a and C5a activate MSC, it was asked whether stimulation with C3a or C5a would upregulate the production of trophic factors by MSC. C3a caused significant up-regulation of various angiogenic factors, including VEGF, CXCL8/IL-8 and IL-6. In contrast there was no detectable production of the pro-inflammatory cytokines TNF-α and IL-1β in spite of nuclear translocation of NFκB. Although C5a also caused moderate up-regulation of angiogenic factors, the effect was borderline significant. Furthermore the production of angiogenic factors induced by C3a was of physiological relevance: Supernatants of MSCs cultured under serum-free conditions induced minimal tube formation of HUVECs as an in vitro measure of angiogenesis; tube formation was considerably enhanced, when supernatants from C3a-stimulated MSC were used, while C3a itself had no direct angiogenic effect on HUVECs. The signaling cascade responsible for the production of angiogenic factors by C3a or C5a could be defined as activation of the rho cascade which was necessary for nuclear translocation of NFκB p65 and of phospho-ERK1/2. Although rho was only transiently activated, inhibition of the rho or “downstream of it” of the NFκB pathway, prevented C3a-and C5a-induced up-regulation of angiogenic factors.

Induction of Thymic Stromal Lymphopoietin in Mesenchymal Stem Cells by Interaction with Myeloma Cells

AbstractAbstract 1825Multiple myeloma (MM) is a B-cell neoplasm characterized by clonal proliferation of malignant plasma cells in the bone marrow (BM). The development of new drugs has increased survival time, but the disease is still incurable. Recently, the BM microenvironment has been reported to play an important role in proliferation and survival of MM cells, suggesting that clarification of the interaction between BM stromal cells and MM cells may provide insights to allow the development of novel treatment strategies for MM.As the signal between MM cells and the BM microenvironment should be bidirectional, it is possible that MM cells produce signals that alter the BM microenvironment to one appropriate for their survival. To validate this hypothesis, we examined the changes in gene expression profile in human BM-derived mesenchymal stem cells (MSC) by co-culture with MM cell line OPM-2. cDNA array analysis indicated that 2 days of co-culture with OPM-2 induced multiple factors in MSC, which are known to have positive effects on survival of MM cells, such as interleukin-6, insulin-like growth factor-1, stromal cell-derived factor 1, vascular cell adhesion molecule 1 (VCAM1), and intercellular adhesion molecule 1 (ICAM1). Other than known factors, thymic stromal lymphopoietin (TSLP), which is a cytokine involved in progression of pancreatic and breast cancer through induction of T helper 2 cell responses, was found to be upregulated by co-culture (ratio 2.22). To confirm the results of cDNA array analysis, MSC co-cultured with OPM-2 or KMS-34 were tested for expression and secretion of TSLP by real-time PCR and ELISA. Real-time PCR revealed that the expression of TSLP in co-cultured MSC was two to three times higher than that in MSC monocultures. Significantly elevated levels of TSLP secretion from MSC after stimulation with PMA/ionomycin were observed by co-culture with OPM-2 or KMS-34 compared with monoculture (201.3 ± 5.0 pg/ml, 176.1 ± 13.3 pg/ml, and 96.6 ± 5.5 pg/ml, respectively; P < 0.01) as measured by ELISA. TSLP did not exhibit any effect on proliferation or drug resistance of MM cells independent of the level of TSLP receptor expression, therefore we evaluated the immunomodulatory effects of TSLP secreted by MSC. Monocyte-derived dendritic cells (MDDC) were first treated with the supernatant of either MSC or MSC co-cultured with KMS-34. Then, allogeneic naïve CD4+ T cells were co-cultured with pretreated MDDC. MDDC treated with the supernatant of co-cultured MSCs expanded IL-13+ CD4+ T cells to a greater extent than MDDC treated with the supernatant of MSC monocultures (10.3 ± 3.7 %, 5.9 ± 4.2 %, respectively; P = 0.02), and addition of anti-TSLP neutralizing antibodies to the supernatant decreased the expansion of IL-13+ CD4+ T cells (10.3 ± 3.7 %, 7.1 ± 2.6 %, respectively; P = 0.04).These results suggested that MM cells act to form more advantageous BM microenvironment for their survival through induction of pleiotropic molecules in MSC involved in not only proliferation and survival of MM cells, but also modifying immunological status. Disclosures:No relevant conflicts of interest to declare.

167 MSC-Concentrated Supernatant: A Novel Therapeutical Approach in Inflammation-Induced Preterm Brain Injury?

Background and aimsPreterm brain injury of premature born infants is a main cause of disability and represents an enormous individual, familial and social burden. Up to 50% of these children...

Mesenchymal Stem Cells in Inflammation Microenvironment Accelerates Hepatocellular Carcinoma Metastasis by Inducing Epithelial-Mesenchymal Transition

In response to inflammation, mesenchymal stem cells (MSCs) are known to migrate to tissue injury sites to participate in immune modulation, tissue remodeling and wound healing. Tumors apply persistent mechanical and pathological stress to tissues and causes continual infiltration of MSCs. Here, we demonstrate that MSCs promote human hepatocellular carcinoma (HCC) metastasis under the influence of inflammation. The metastasis promoting effect could be imitated with the supernatant of MSCs pretreated with IFNγ and TNFα. Interestingly, treatment of HCC cells with the supernatant leads to epithelial-mesenchymal transition (EMT), an effect related to the production of TGFβ by cytokines stimulated MSCs. Importantly, the levels of MSCs expressing SSEA4 in clinical HCC samples significantly correlated with poor prognosis of HCC, and EMT of HCC was strongly associated with a shorter cancer-free interval (CFI) and a worse overall survival (OS). Therefore, our results suggest that MSCs in tumor inflammatory microenvironment could promote tumor metastasis through TGFβ-induced EMT.

Mesenchymal Stem Cells Contribute to the Abnormal Bone Marrow Microenvironment in Patients with Chronic Idiopathic Neutropenia by Overproduction of Transforming Growth Factor-β1

Chronic idiopathic neutropenia (CIN) is a granulopoiesis disorder associated with an inhibitory bone marrow (BM) microenvironment consisting of activated T-lymphocytes and pro-inflammatory mediators. In this study, we investigated the possible involvement of BM mesenchymal stem cells (MSCs) in the pathophysiology of CIN by assessing the frequency and function of BM MSCs in terms of the proliferative/clonogenic characteristics, the differentiation capacity, the potential to produce pro-inflammatory cytokines, and the ability to suppress T-cell proliferation. The frequency, differentiation capacity toward adipocytes, chondrocytes, or osteoblasts, and immunosuppressive potential to inhibit mitogen-induced T-cell proliferation did not differ significantly between patient (n = 14) and normal (n = 21) MSCs. Tumor necrosis factor-α, interleukin-1β, and interleukin-6 levels in MSC supernatants did not differ significantly between patients and controls; however, transforming growth factor (TGF)-β1 levels were significantly elevated in patients, particularly in those displaying the -509C/T TGF-β1 polymorphism. Patient MSCs displayed defective proliferative/clonogenic potential, which could not be attributed to altered cellular survival characteristics or to increased TGF-β1 production as TGF-β1 neutralization did not restore the impaired colony formation by patient MSCs. We conclude that although BM MSCs do not exert a significant role in the immune deregulation associated with CIN, they contribute to the inhibitory microenvironment by overproducing TGF-β1, at least in patients displaying the -509C/T polymorphism.

Endogenous Expression of Functional Factor VIII by Human Mesenchymal Stem Cells In Culture

AbstractAbstract 2216The liver is considered to be the primary site of factor VIII (FVIII) production in the body; however, evidence is mounting that suggests there are secondary sites in which considerable synthesis of FVIII takes place. Studies of FVIII mRNA expression in various human tissues have revealed that FVIII message can be found throughout the body. Additionally, acute liver failure correlates with an increase in circulating FVIII levels. Some reports have identified endothelial cells as a significant extra-hepatic source of FVIII, possibly explaining both the widespread presence of FVIII mRNA and the increase in FVIII levels upon liver failure. However, the possibility exists that other cell types present throughout the body also produce FVIII and contribute to circulating FVIII levels. Mesenchymal Stem Cells (MSCs) represent a potential alternative; they are a diverse group of stromal cells which can be found in the perivascular regions of multiple tissues throughout the body. Previous studies demonstrated that MSCs are capable of efficiently producing and secreting high levels of FVIII in vitro when transduced with FVIII-encoding viral vectors, but to date, the innate ability of MSCs to produce FVIII has not been explored. As such, we investigated the potential for MSCs to produce endogenous FVIII message and secrete functional protein. MSCs isolated based on Stro-1 positivity from human lung, liver, brain, and bone marrow (BM) were grown in cell culture and assayed for production of FVIII message by both microarray analysis and RT-PCR. Microarray data showed that there were significant amounts of FVIII message in all four cell types tested and that the amount of message in BM MSCs was three-fold higher than each of the other three cell types. RT-PCR analysis confirmed the presence of FVIII message in all four MSC populations. Secretion of functional FVIII protein was subsequently measured using a chromogenic assay. MSC culture supernatants were collected for either 24 or 48 hours, and FVIII activity was determined using pooled normal human plasma as a control to create a standard curve. FVIII activity in the supernatants of MSCs was in the range of 0.6 to 2.0 mU/1×10^6 cells/ 24hr. Moreover, MSCs continued to express and produce FVIII during time in culture until our last evaluation at passage 20, indicating that there is an innate ability of these cells to continually produce FVIII. Taken together, these data demonstrate that human MSCs are capable of producing and secreting functional FVIII in vitro, and given their widespread location throughout the body, this finding raises the possibility that, in vivo, these cells might significantly contribute to the total FVIII pool. This is the first report, to our knowledge, that implicates MSCs as a potential endogenous source for circulating FVIII. Further studies of in vivo FVIII expression by MSCs are warranted and may provide a clearer understanding of extra-hepatic FVIII production in the body while aiding in the discovery of novel therapies for hemophilia A. Disclosures:No relevant conflicts of interest to declare.